Thus, conflicting signals in a data set may not be the result of sampling or modeling errors, but due to the fact that hybridization has played a significant role in the evolutionary history of the species under consideration. Assuming that the initial set of gene trees is correct, a basic problem for biologists is to compute this minimum number of hybridization events to explain this set. In this paper, we describe a new reduction-based algorithm for computing the minimum number, when the initial data set consists of two trees. Although the two-tree problem is NP-hard, our algorithm always gives the exact solution and runs efficiently on many real biological problems. Previous algorithms for the two-tree problem either solve a restricted version of the problem or give an answer with no guarantee of the closeness to the exact solution. We illustrate our algorithm on a grass data set. This new algorithm is freely available for application at either  For many groups of taxa , this is an appropriate representation. However, because of non-tree-like evolutionary processes such as hybridization, horizontal gene transfer, and recombination, not all groups of taxa are suited to this type of representation. Collectively referred to as reticulation events, these processes result in species that are a mixture of DNA regions derived from different ancestors.In the following, we restrict our attention to hybridization. During such an event, two lineages recombine to create a new species which may have the same number of chromosomes as its parents (diploid hybridization) or the sum of all parental chromosomes (polyploid hybridization). Eukaryotes, whose evolutionary past contains hybridization, include certain groups of plants, birds, and fish see .The effect of hybridization in evolution has been recognized for quite some time. For example, since 1930’s, botanists have suggested that the morphological variation in the New Zealand flora is due to hybridization . HoweverFormalized mathematically, this fundamental problem is NP-hard even when the initial collection consists of two rooted binary phylogenetic trees . ConsequIn this paper, we describe a new, and recently implemented, exact algorithm for solving the two-tree problem (with no restrictions) based on three reductions that preserve the amount of hybridization. All of these reductions make use of the similarities between the two trees. It has been recently shown that two of the reductions are enough to guarantee that the algorithm is fixed-parameter tractable, where the parameter is the smallest number of hybridizations to explain the initial two trees . This mehttp://www.bi.uni-duesseldorf.de/~linzand http://www.math.canterbury.ac.nz/~cas83. As the implementation requires the two input trees to be given in a special type of string format, the interested reader can also download two sample trees and a short manual describing how to use the software. The program output contains the simplified trees after applying the three reductions (see Section 2.1) and the minimum number of hybridization events to explain the two initial trees.The new algorithm described in this paper has been implemented in Perl and is available for application at Poaceae) data set which consists of sequence data for six genetic loci and six corresponding gene trees. Each of the 15 different pairs of trees are considered.The notation and terminology in this paper follows Full details of the algorithm described in this paper can be found in 2.rooted binary phylogenetic X-tree X and whose root has degree two while all other interior vertices have degree three. A cluster of X that contains precisely the elements that are descendants of some vertex of We begin with a formal description of the two-tree problem. A rooted acyclic digraph is a digraph with no directed cycles. Each such digraph has a distinguished vertex ρ whose in-degree is zero and has the property that there is a directed path from ρ to every other vertex. For a vertex υ in a digraph, we will denote the in-degree of υ (the number of edges directed into υ) by d −(υ) and the out-degree of υ (the number of edges directed out of υ) by d +(υ). A hybridization network X is a rooted acyclic digraph with root ρ, in whichX is the set of vertices of out-degree zero,d +(ρ) ≥2, andd −(υ) ∈{1,2}, and no vertex υ has d −(υ) = 1 and d +(υ) = 1.for all other vertices υ, A To illustrate these concepts, two rooted binary phylogenetic trees X-trees, hybridization networks on X can be used to represent the ancestral history of a collection of present-day species that includes hybridization. The set X represents the collection of present-day species. Vertices of in-degree two represent an exchange of genetic information between the hypothetical ancestors. These vertices are called as hybridization vertices. To quantify the number of hybridization events, the hybridization number of a hybridization network h data set. This data set was provided by the Poaceae family is among the six families with the highest number of natural hybrids. Therefore, it is more likely that the conflicting signals in the data are due to hybridization rather than other factors and so it is an appropriate data set for our purposes.In this section, we describe the application of HPoaceae data set consists of sequence data for six different genetic loci: internal transcribed spacer of ribosomal DNA (ITS); NADH dehydrogenase, subunit F (ndhF); phytochrome B (phyB); ribulose 1,5-biphosphate carboxylase/oxygenase, large subunit (rbcL); RNA polymerase II, subunit β” (rpoC2); and granule bound starch synthase I (waxy). A summary describing the sequence origin, the number of sequences, and the alignment length for each gene in the data set is given in The ybridNumber to each of the 15 different pairwise combinations of gene trees, where, for each combination, we restricted the gene trees to taxa common to both. The size of the overlapping taxa set for each combination is given in the second column of For each loci, a rooted binary phylogenetic tree was reconstructed using the fastDNAmL program . These gybrid-Number that highlights the extent to which the reductions incorporated in HybridNumber can reduce the size of the problem instance. This application involves the two phylogenetic trees of the chloroplast sequence phytochrome B (phyB) and the nuclear sequence of the internal transcribed spacer of ribosomal DNA (ITS) which have an overlapping taxa set of 30 present-day species , the algorithm performs two further iterations. At the end of these two iterations, we obtain two more pairs of trees as indicated by the square brackets B and C in In the next step, the cluster reduction rule divides the problem into two smaller problems by searching for a minimal cluster of size at least two that is common to both trees in The final step in the algorithm is to exhaustively find the hybridization number of the three pairs of non-identical trees in This is remarkably quick given that the two initial trees contain 30 taxa and the hybridization number is 8. As a comparison, we tried finding the hybridization number of these two trees without the three reductions. After 1 week, the algorithm was still running!ybridNumber calculates the hybridization number within a couple of minutes. Furthermore, the hybridization numbers of all but three pairs are found within a time span of 2 days. The successfully completed pairs contained up to 40 taxa and have hybridization numbers as high as 14. Those three pairs of trees for which the running time is given as 2 days in In 4.Due to reticulate evolution, phylogenetic gene trees reconstructed for different genetic loci often reveal conflicting tree topologies, because processes like hybridization, horizontal gene transfer, and recombination are not tree-like. The extent to which such events occur is of increasing interest for many evolutionary studies.ybridNumber is not a heuristic, and its solution is not restricted in any way. Calculating this minimum number is computationally a hard problem, and so if the initial two gene trees only share a few similarities, then in many cases the exact calculation of the hybridization number is computationally infeasible. However, if the two gene trees share a number of common features—pendant subtrees, chains, or clusters—which is likely for many biological examples, the new algorithm performs remarkably well and the hybridization number can be found in reasonable time.In this paper, we have described a newly implemented algorithm to calculate exactly the minimum number of hybridization events that explains two phylogenetic gene trees. Unlike previous algorithms, HybridNumber calculates a lower bound for the number of hybridization events to explain the differences between two phylogenetic gene trees (assuming that hybridization is the only cause of incongruence between the two trees). It is possible that the real number of hybridization events that happened during the evolution of the collection of present-day species under consideration is underestimated. Indeed, it is possible that some hybridization events are never recognized. Nevertheless, the algorithm provides an important first step towards an understanding of the extent to which hybridization has influenced evolution.Note that HX-trees Of course, in addition to computing the hybridization number of two rooted phylogenetic Finally, it is clear that extending this work to allow for more than two trees in the input is important. Such extensions are discussed in the corresponding author’s PhD thesis.


Varicellovirus of the Herpesviridae subfamily Alphaherpesvirinae includes a cluster of viruses antigenically and genetically related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5 (BoHV-5), bubaline herpesvirus 1 (BuHV-1), caprine herpesvirus 1 (CpHV-1), cervid herpesviruses 1 (CvHV-1) and 2 (CvHV-2) and elk herpesvirus 1 (ElkHV-1). Considering the serological relationship between these ruminant alphaherpesviruses, several surveys have studied the occurrence of BoHV-1 related virus infection in wild and domestic ruminant species. In this way, a recent investigation has indicated, in Belgium, a high increase in the serological prevalence of BoHV-1 related virus infection in free-ranging red deer population. In this context, it has been decided to investigate the presence of an alphaherpesvirus spreading in the Belgian free-ranging red deer population.The genus The current study reports the first isolation in a free-ranging red deer of a BoHV-1 closely related virus. The isolate was antigenically, genomically and genetically characterised by comparison with several ruminant alphaherpesvirus. Immunofluorescence assays revealed the isolate was antigenically distinct from bovine and caprine alphaherpesviruses. Similarly, BamHI and BstEII restriction analyses demonstrated the genomic difference between the isolate and the other ruminant alphaherpesviruses. Next, the sequencing of selected parts of UL27 and US8 genes showed a high degree of homologies between each BoHV-1 related ruminant alphaherpesvirus and the isolate. Besides the close relationship between all ruminant alphaherpesviruses, the phylogenetic analysis revealed that the isolate clustered with CvHV-1.The first isolation of a virus closely related to BoHV-1 in a free-ranging red deer is reported. Data demonstrate that a CvHV-1 strain, named Anlier, circulates in wild red deer in continental Europe. Anlier strain show consistent differences with the virus isolated from Scottish farmed red deer. All together, these results improve our understanding of ruminant alphaherpesviruses. Herpesviridae includes nearly two hundred viruses isolated from various hosts including molluscs, fishes, amphibians, reptiles, birds, mammals and at least one invertebrate. Based on biological and molecular properties, the family has been divided into three subfamilies of viruses, Alpha-, Beta- and Gammaherpesvirinae, which have co-evolved with different host species. Illustrating the concept, several ruminant alphaherpesviruses form a cluster of antigenically and genetically related viruses , BoHV-5 N569 UL27 [AF078726.2], CvHV-1 Banffshire 82 UL27 [AF078729.2] and CvHV-2 Salla 82 UL27 [AF078727.2].The sequences reported in this paper have been deposited in the GenBank database under the accession numbers The author(s) declares that there are no competing interests.JT and ET designed the experiments, analysed the data and drafted the manuscript together. JT performed the experiments. JT and FG collected red deer samples. FW participated to the sequence alignment and constructed phylogenetic trees. SB and AL helped to draft the manuscript. All authors read and approved the final manuscript.


The neuroinvasive property of several alpha-herpesviruses underlies an uncommon infectious process that includes the establishment of life-long latent infections in sensory neurons of the peripheral nervous system. Several herpesvirus proteins are required for replication and dissemination within the nervous system, indicating that exploiting the nervous system as a niche for productive infection requires a specialized set of functions encoded by the virus. Whether initial entry into the nervous system from peripheral tissues also requires specialized viral functions is not known. Here we show that a conserved deubiquitinase domain embedded within a pseudorabies virus structural protein, pUL36, is essential for initial neural invasion, but is subsequently dispensable for transmission within and between neurons of the mammalian nervous system. These findings indicate that the deubiquitinase contributes to neurovirulence by participating in a previously unrecognized initial step in neuroinvasion. Subsets of herpesviruses, such as herpes simplex virus and pseudorabies virus, are neuroinvasive pathogens. Upon infection, these viruses efficiently target peripheral nervous system tissue and establish a life-long infection for which there is no cure. Very few pathogens are known that invade the nervous system proficiently, and the mechanism by which herpesviruses achieve neuroinvasion is largely unknown. In this study, we demonstrate that a viral protease plays a critical and specific role allowing the virus to cross the threshold of the nervous system, but is dispensable for subsequent replication and encephalitic spread within the brain. Neuroinvasive herpesviruses comprise a group of pathogens with individual members infecting different mammalian hosts. Four viruses of this group infect humans: herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella zoster virus (VZV) and simian herpes B virus (SHBV). The latter is associated with rare life-threatening zoonotic infections. Diseases associated with these viruses range from minor recurrent lesions to shingles, keratitis and encephalitis. All of these pathologies can occur as a consequence of viral dissemination into the nervous system. There are additional neuroinvasive herpesviruses that infect mammals other than humans. Among these, pseudorabies virus (PRV) has a broad host range and is commonly associated with severe encephalitic infections, making it a useful model for studies of neuroinvasion and pathogenesis [reviewed in 1]. In addition, the propensity of PRV to transmit neuron-to-neuron during encephalitic spread has led to its use as a self-amplifying tracer for mapping of the vertebrate neural circuitry [reviewed in 2]. PRV is closely related to VZV , and shares a common structure, similar genetic composition and a related infectious cycle with all neuroinvasive herpesviruses To date, mutant viruses displaying defects in neurotropism fall into two categories: neuron-specific replication mutants and axon transport mutants. Mutants of HSV-1 that fail to replicate in neurons include viruses lacking thymidine kinase activity or ICP34.5 Recently, an isolate of PRV mutated in a conserved deubiquitinase (DUB) domain of a viral structural protein, the pUL36 tegument protein, was reported to infect the nervous system with delayed kinetics following intranasal inoculation into mice A conserved cysteine residue in the pUL36 tegument protein that is critical for the proteolytic activity responsible for deubiquitination PRV is frequently used as a trans-synaptic neurotracer to map circuits within the mammalian nervous system [reviewed in 2]. In particular, neural circuits between the eye and brain have been extensively studied in rodents infected with PRV, which we used as the basis for our current studies of the C26A mutant virus Because anterograde spread of the C26A mutant of PRV was unexpected, and did not account for its severe attenuation, the brains of these animals were also examined for evidence of infection via retrograde autonomic circuits. Intravitreal injection into the eye can expose the iris and ciliary body to viral inoculum, which receive autonomic innervation. The time course of infection and retrograde spread of PRV in these circuits is somewhat variable due to the gelatinous matrix of the vitreous body . Nevertheless, in these experiments wild-type virus was reproducibly observed to have infected the Edinger-Westphal nucleus (EW) of the midbrain, preganglionic parasympathetic neurons that project to neurons in the ciliary ganglion via the oculomotor nerve, which in turn innervates the smooth muscles in the iris and ciliary body in the eye to mediate pupillary constriction and lens accommodation, respectively. In contrast, capsid fluorescence was never detected in the EW following C26A virus infection . Wild-tyTo determine if the C26A virus was capable of intracellular transport in both the retrograde and anterograde directions, we examined axonal transport in cultured dorsal root sensory neurons by tracking red-fluorescence emissions from capsid particles by time-lapse microscopy. In these assays, the C26A virus behaved similarly to the wild-type virus, having only small decreases in axon transport during both stages of infection . These fin vivo, stereotaxic injection of virus directly into the brain was employed. The suprachiasmatic nucleus (SCN), which receives axon projections from RGCs in the eye in vivo (from SCN neurons in the brain to RGCs in the eye to bipolar/amacrine cells in the eye).To further examine trans-synaptic retrograde transport The data up to this point indicated that the C26A virus was incapable of spread in a subset of neural circuits, but the basis for the selective loss of function could not strictly be attributed to an inability to transport in either retrograde or anterograde circuitry. Because the C26A defect was observed only in retrograde circuits innervating tissues in the anterior chamber of the eye (i.e. iris and ciliary body), which are less efficiently infected following intravitreal injections, animals were next injected in the anterior chamber of the eye directly. This infection route immediately exposes the iris and neighboring ciliary body to the viral inoculum and provides a more reliable infection of the EW with all neuroinvasive strains of PRV examined to date We next considered that the C26A virus typically propagated to titers 10-fold reduced relative to wild type . PrelimiSpread of C26A virus to the EW in the co-injection experiment indicated that PRV-152 had complemented the C26A defect. For complementation to occur, both viruses would have had to replicate in a common cell prior to entering the nervous system. This provided an incentive to examine the iris and ciliary body of the co-injected animals for co-infected cells . InfectiTo confirm that the C26A virus had a specific defect in neuroinvasion from tissues innervated by the PNS, PRV-152 and the C26A virus were independently injected into the skin and muscle of one eyelid each per animal; the smooth muscle of the eyelid receives both sympathetic and parasympathetic innervation To explain how the C26A virus spread retrogradely in one animal, we again considered the possibility that injection into the eyelid might have caused local tissue damage that allowed direct exposure of the inoculum to innervating axon projections. We expect that a similar event likely occurred in the one intravitreal injected animal in which C26A virus spread specifically to the oculomotor nucleus (see above). To determine if injection can, in some instances, allow for direct exposure of the inoculum to axons embedded within the peripheral tissue, the beta subunit of cholera toxin (CTB) conjugated to Alexa Fluor 594 was injected into the eyelid. CTB is a retrograde tracer, but unlike neuroinvasive herpesviruses which spread trans-synaptically, CTB labeling is restricted to the neuron cell body from which an axon projects. Furthermore, CTB must come into direct contact with the axon to label the distal neuronal cell body. Of 6 animals tested, only one showed CTB retrograde label into the brain. The absence of CNS labeling in the remaining five animals indicated that injection into the eyelid infrequently allowed for direct exposure to nerve endings. This finding was consistent with the conclusion that the C26A virus is specifically incapable of invading the mammalian nervous system from tissues innervated by the PNS, except in infrequent cases when nerve endings become directly exposed to the inoculum.Since its discovery four years ago in vivo, but largely failed to spread between the innervated non-neuronal tissue and the innervating neurons. Such a defect has never been observed for a neuroinvasive herpesvirus before, and reveals that a previously unrecognized barrier to viral dissemination into the nervous system is overcome by the amino-terminal deubiquitinase domain embedded in the pUL36 protein. The neuroinvasion defect was rescued by either co-infection with a virus encoding a wild-type pUL36 protein or by repairing the C26A mutation directly, confirming that the neuroinvasive defect mapped to the pUL36-encoded DUB.Our initial experiments produced results consistent with a selective loss of retrograde trafficking in the nervous system; however, retrograde transport of the C26A virus was unmasked by either infecting neurons directly in the retina or brain, or presumably by mechanical damage at the site of injection. An important insight was fortuitously made when the C26A mutant was co-infected with a virus encoding a wild-type pUL36 protein (PRV-Bartha), resulting in the rescue of the C26A defect and retrograde spread to the brain. This trans-complementation could only occur if the C26A virus remained competent to infect cells prior to neuroinvasion, which was subsequently confirmed by examining the eye for infection. Therefore, the C26A mutation resulted in a virus that replicated in non-neuronal cells both in culture and in the iris, and also replicated and spread in cultured neurons and neural circuits In contrast to the results presented here, a previous study that examined the role of the PRV DUB following intranasal infection reported only a general delay in neuroinvasion Identification of the molecular determinants that promote the neuroinvasive behavior of this class of herpesviruses not only provides insight into the underlying mechanisms of viral dissemination within host tissues, but also into the relationship between tissue tropism and pathogenesis. Yet, the majority of herpesviruses are not neuroinvasive pathogens, which argues that the pUL36 DUB may perform a more fundamental role during infection. Infection of cultured cells with DUB-mutant herpesviruses has revealed delays in viral assembly, propagation kinetics and cell-cell spread th of the protein's mass. In addition to pUL36 being a critical component of the herpesvirus structure that links the capsid to the tegument and envelope layers of the virion The pUL36 tegument protein is greater than 300 kDa in neuroinvasive herpesviruses such as HSV and PRV, with the amino-terminal DUB amounting to less than 1/10GCTCTGCGCTCCTCCCTCTCCTTAGGATGACGACGATAAGTAGGG5′- TCAGTATGACCCCGACCTGGGGCCCGGGTCGGGCGTCTCG and AGCCGAGACGCCCGACCCGGGCCCAACCAATTAACCAATTCTGATTAG5′- TCGTGAAGACCAGGCGCAGGAAGGAGAGGGAGGAGCGCAG. The primers encode the sequence to be mutated (bold) with surrounding homology to the UL36 gene, as well as homology to the pEPkan-S template plasmid (underlined). Transfection of the resultant infectious clone, pGS1652, into pig kidney epithelial (PK15) cells produced the PRV-GS1652 virus, which typically propagated to a titer of 1×107 plaque forming units (PFU) per ml. A revertant virus, PRV-GS1768, was constructed using the same methods. The primers used were TGCCTGCGCTCCTCCCTCTCCTTAGGATGACGACGATAAGTAGGG5′- TCAGTATGACCCCGACCTGGGGCCCGGGTCGGGCGTCTCG and GCACGAGACGCCCGACCCGGGCCCAACCAATTAACCAATTCTGATTAG5′- TCGTGAAGACCAGGCGCAGGAAGGAGAGGGAGGAGCGCAG. All mutations were confirmed by restriction digest analysis and sequencing.Two recombinant viruses were made from a derivative of the PRV (Becker strain) infectious clone, pBecker3, which was previously made to encode an mRFP1-VP26 (red-fluorescent capsid) fusion allele (pGS847) PRV152 is a derivative of the PRV-Bartha vaccine strain that encodes a GFP-expression cassette driven by the CMV immediate early promoter inserted in the gG locus, and was described previously Stocks of PRV-GS847 (WT), PRV-GS1652 (C26A), PRV-GS1768 (revertant) and PRV152 (Bartha-GFP) were harvested from infected PK15 cells grown in DMEM supplemented with 2% bovine growth supplement (Invitrogen). High titer virus stocks were prepared for a subset of animal studies as described previously Quantitation of viral propagation kinetics by single-step growth curve analysis was conducted as previously described, with the exception that Vero cells were substituted for PK15 cells Imaging of entry and egress axonal transport events was performed using primary cultured neurons from embryonic chicken (E8–E10) dorsal root ganglia, as previously described ad libitum. Animals were at least 14 weeks of age when used in experiments.Male Sprague-Dawley and Long Evans rats (Charles River Breeding Laboratories) were maintained under a light/dark cycle of 12 h light/12 h dark with food and water available 8 plaque-forming units/ml) or a unilateral anterior chamber injection of the same quantity of PRV-GS847, PRV-GS1652, PRV152 or a 1∶1 mixture of PRV-GS1652:PRV152, over a 1-min interval using a Nanoject II nanoinjector fitted with a glass micropipette . A fresh stock of virus was thawed for each injection. Animals were maintained in a biosafety level 2 facility for up to 11 days post-inoculation.Under isoflurane inhalation anesthetic (2.5–5%), animals received a unilateral intravitreal injection of 2 µl of either PRV-GS847 or PRV-GS1652 , animals were positioned in a Kopf stereotaxic apparatus . A craniotomy was performed, the dura was exposed and a zero depth reading was made prior to the dura mater being excised. A glass micropipette attached to a Nanoject II nanoinjector , angled laterally 10° to the vertical to avoid the midline third ventricle, was lowered into the hypothalamus to a region slightly dorsal to the suprachiasmatic nucleus (SCN) using coordinates determined empirically in test subjects. The micropipette was lowered 7.7–7.8 mm from the dura mater, thus avoiding involvement of the retinal axons in the optic chiasm, and approximately 200 nl (the Nanoject II was set to the 207 nl volume preset) of a 1×108 pfu/ml) and the upper eyelid of the other eye was injected with 2 µl of PRV152 (1×108 pfu/ml). In a separate set of experiments, animals received a unilateral upper eyelid injection of 2 µl of cholera toxin B subunit conjugated to Alexa Fluor 594  using a Nanoject II nanoinjector fitted with a glass micropipette .Under isoflurane inhalation anesthetic (2.5–5%), the skin of the upper eyelid of one eye was injected with 2 µl of PRV-GS1652 , and perfused transcardially with 0.9% saline followed by freshly prepared fixative consisting of 4% paraformaldehyde in phosphate buffer (0.1 M), pH 7.3. Brains were removed, stored in the same fixative containing 30% sucrose at 4°C overnight, and sectioned at 40 µm in the coronal plane on a sliding microtome equipped with a freezing stage . Sections were collected in phosphate buffer, mounted on subbed slides, blotted to remove excess buffer, and coverslipped with Vectashield mounting medium . Coverslips were sealed with fingernail polish to prevent dehydration, and slides were stored in the dark at 4°C. EGFP and mRFP1 fluorescence is stable under these conditions for several months with minimal quenching. Slides were examined using a Leica  DMRA light microscope equipped with epifluorescence and fitted with a microstepping servomotor in the z–axis. Images were captured using a Hamamatsu  C4742-95 CCD digital camera under epifluorescence using either EGFP optics  or mRFP optics  and deconvolved using Openlab fluorescence deconvolution software  running on an Apple Macintosh G4 platform. Digital images were pseudo-colored, and images were prepared using Adobe Photoshop version 6.0.1. Images were enhanced for brightness and contrast.


Worksite policy and environmental supports that promote physical activity, healthy eating, stress management, and preventive health screenings can contribute to the prevention of cardiovascular disease and lower employer costs. This study examines the availability of these four categories of supports in a statewide survey of New York State worksites.In 2002, we recruited a statewide sample of worksites in New York State with 75 or more employees to participate in a mailed survey assessing worksite policy and environmental supports for wellness and health promotion. The overall response rate was 34.8%. The analysis included data from 832 worksites.Worksite size was an independent predictor of health promotion supports with small (75–99 employees) and medium-small (100–199 employees) worksites reporting significantly fewer policy and environmental supports in all four categories than worksites with 300 or more employees. Worksites in which most employees were nonwhite reported fewer supports for physical activity, healthy eating, and stress management than worksites in which most employees were white. A wellness committee or wellness coordinator was associated with more health promotion supports, regardless of the size of the worksite or composition of its workforce.Worksites with fewer than 200 employees have an increased need for assistance in establishing environmental and policy supports promoting cardiovascular health. Worksites that have a wellness committee or coordinator are better able to establish and sustain supports with the potential to improve the health of their workers. Worksite health promotion programs have the potential to reach large segments of the adult population and allow control over interpersonal, environmental, and organizational factors that influence health behavior . They arOur primary goal was to examine how worksite programs promoting on-site preventive health screenings relate to efforts focused on policy and environmental changes that support physical activity, healthy eating, and stress management. Efforts to prevent cardiovascular disease through worksite health promotion have traditionally focused on establishing environmental and policy changes that promote physical activity, healthy eating, and tobacco control ,7. HowevA second goal was to examine the worksite and workforce characteristics associated with the availability of health promotion supports. Both national surveys and regional, state-based worksite surveys indicate that a worksite's size , its administrative support for employee wellness, and its industrial classification are associated with the availability of on-site health promotion activities -15. MoreWe used data from a statewide survey of New York State worksites. Since 1995, the New York State Department of Health (NYSDOH) has supported worksite health promotion programs emphasizing policies and environmental changes consistent with the social ecological model . We used Pearson correlation coefficients to convey the association between categories.Next, we used SUDAAN Release 7.5  to calculate the weighted estimates of the mean number of various categories of wellness supports by worksite characteristic. These estimates combined information about the sampling design and sample weights to generate accurate estimates of the means and 95% confidence intervals. The sample weights included information about the probability of selection, a nonresponse adjustment, and a poststratification adjustment.Finally, we used SUDAAN to construct linear models examining which worksite characteristics were independent predictors of categories of worksite supports for health promotion and cardiovascular health. The linear models used generalized estimating equations and calculated estimates of the regression coefficients using the Binder method ,25.23 = 7.7; P = .05).Worksite size and the presence of worksite administrative supports for wellness were associated with the availability of health promotion supports in the descriptive analyses and multivariate models. Small worksites (75–99 employees) reported significantly fewer healthy eating supports than medium-small (100–199 employees), medium-large (200–299 employees), and large (≥300 employees) worksites. Small worksites also reported fewer stress management supports than medium-large or large worksites and fewer physical activity supports and preventive health screenings than large worksites. Medium-small worksites resembled small worksites in having fewer supports for healthy eating, physical activity, and stress management than large worksites. Worksite size remained a significant and independent predictor of each of the four categories of supports in our multivariate models, with small and medium-small worksites reporting significantly fewer supports for all four categories than large worksites.Worksites with a wellness committee or wellness coordinator reported a greater number of all four categories of worksite health promotion supports than those without either of these administrative supports. Moreover, worksites having both types of administrative supports reported more supports for healthy eating and physical activity and more preventive health screenings than those having only a committee or a coordinator. The presence of one or more administrative supports for employee wellness was also associated with a greater number of all four categories of health promotion supports in our multivariate models, suggesting that the relationship could not be attributed to larger companies being more likely to have a wellness coordinator or committee.The racial and sex composition of the workforce and the extent to which the workforce was unionized were significantly associated with the number of health promotion supports available at a worksite in both the descriptive analyses and multivariate models. Worksites in which most workers were nonwhite had fewer supports for healthy eating, physical activity, and stress management. This association remained significant in our multivariate models. Worksites in which most workers were women had more environmental policy supports for physical activity, healthy eating, and stress management in our descriptive analyses, but only the association with healthy eating supports remained significant in the multivariate models. Both the descriptive and multivariate models demonstrated that worksites in which most of the workforce was unionized had more stress management supports. However, the multivariate models indicated worksites with a greater percentage of union members held fewer preventive health screenings during the previous year. The blue-collar status of the workforce was associated with the availability of physical activity supports in our descriptive analyses but was not associated with the availability of any of the categories of health promotion supports in the multivariate models.The findings from our statewide survey reinforce the need for additional efforts to promote worksite health promotion among New York State worksites. One strength of the study is that the findings are based on a statewide, population-based sample of New York State worksites. From 1985 through 1999, three national surveys of worksite health promotion activities and supports took place ,15,26. TOur primary goal was to examine how worksite programs promoting on-site preventive health screenings relate to efforts concentrating on policy and environmental changes that support physical activity, healthy eating, and stress management. Worksites holding on-site preventive health screenings during the previous year tended to have more policy and environmental supports for physical activity, healthy eating, and stress management. Moreover, the strength of the correlations among the four categories of supports was equivalent, implying preventive health screenings are incorporated into worksite health promotion programs much as traditional efforts to lower stress, increase physical activity, and encourage healthy eating are incorporated. This finding is consistent with a recent review concluding that certain company attributes — such as leadership support — provide an infrastructure that supports all types of worksite health promotion initiatives .A second goal was to examine the worksite and workforce characteristics associated with the availability of health promotion supports. We replicated the results of past studies demonstrating that smaller worksites have the fewest worksite health promotion supports -13. AlthOur analyses demonstrated that worksites with a wellness committee or wellness coordinator had more supports in all four support categories, regardless of the size or composition of the workforce. Furthermore, worksites with both a committee and coordinator had more supports than those with only one. The presence of two administrative supports may be robust predictors of supports because they encompass both employer and worker support for health promotion, both of which have been demonstrated to be critical to the success of worksite health promotion programs ,27. EstaThe racial and sex composition of the workforce and the extent to which a workforce was unionized were also associated with the type and extent of the health promotion supports. Our finding that worksites in which the workforce was mostly nonwhite had fewer healthy eating, physical activity, and stress management supports contributes to the body of literature indicating significant disparities in health and in the availability of health-related supports by race and ethnicity ,28. Our We found that greater union representation was associated with more stress management supports but fewer on-site preventive screenings in the past 12 months. A previous survey of New York State worksites, however, showed that greater union representation was associated with more supports for physical activity, screenings, and overall health promotion . One expOne limitation of our survey instrument was that its format led to missing data on workforce demographic characteristics. Although the missing data were not missing at random, the high rate of missing data on some items did not provide an alternative explanation for the findings reported. A second limitation is that the survey instrument included few questions related to the secondary prevention of cardiovascular disease and no questions related to stroke prevention. To address this limitation, the NYSDOH is repeating the statewide survey with a modified instrument that should provide a more comprehensive assessment. The follow-up survey will enable the NYSDOH Healthy Heart Program to determine changes in worksite health promotion supports that have occurred since this survey and will provide baseline data for tracking future worksite health promotion efforts related to blood pressure and cholesterol control and stroke prevention.


Cercopithecine herpesvirus 1 (B virus), an alphaherpesvirus endemic in Asian macaques, is closely related to herpes simplex virus (HSV). Most macaques carry B virus without overt signs of disease. However, zoonotic infection with B virus in humans usually results in fatal encephalomyelitis or severe neurologic impairment. Although the incidence of human infection with B virus is low, a death rate of >70% before the availability of antiviral therapy makes this virus a serious zoonotic threat. Knowledge of the clinical signs and risk factors for human B-virus disease allows early initiation of antiviral therapy and prevents severe disease or death.Nonhuman primates are widely used in biomedical research because of their genetic, anatomic, and physiologic similarities to humans. In this setting, human contact directly with macaques or with their tissues and fluids sometimes occurs.  Cercopithecine herpesvirus 1 (B virus) is known to be pathogenic for humans. Monkeys of the genus Macaca, which are widely used as animal models for biomedical research, naturally carry B virus. Infection in macaques is lifelong, with periodic, usually asymptomatic reactivation.Of the 35 herpesviruses identified in nonhuman primates, only Approximately 40 cases of zoonotic B-virus infection have been reported. Considering the number of people in contact with macaques, this number of cases is quite low. However, the death rate for B-virus infection before the availability of antiviral therapy is >70%. Neurologic sequelae are common in survivors. Treatment with antiviral medication may decrease the death rate, but rapid diagnosis and initiation of therapy are essential in controlling the spread of the virus in the central nervous system and limiting neurologic sequelae.Macaca mulatta) and died of progressive encephalomyelitis 15 days later. Two research groups obtained samples from patient W.B.: Gay and Holden and Sabin and Wright (The first documented case of human B-virus infection occurred in 1932 when a researcher (patient W.B.) was bitten on the hand by an apparently healthy rhesus macaque . This designation is based on virologic characteristics and serologic cross-reactivity with other members of the genus Simplexvirus, namely HSV type 1 (HSV-1), the causative agent of oral herpetic ulcers (cold sores) in humans and HSV type 2 (HSV-2), the agent of human genital herpes , as has been established for HSV. The infected cells are lysed, releasing virus to spread to other cells and sensory nerve endings, although direct entry into neurons without replication can occur (M. facicularis), two species used extensively in biomedical research. B virus has also been isolated from the stumptail (M. artoides), pig-tailed (M. nemestrina), Japanese (M. fuscata), bonnet (M. radiata), and Taiwan (M. cyclopis) macaque . As seen in humans with HSV, B-virus seropositivity increases with population age; seropositivity rates of 80% to 100% occur among most adult captive macaque populations , which indicates asymptomatic shedding of the virus. Lack of clinical signs of recurrent infection makes identification of shedding animals difficult. People working with these animals should consider every animal a potential source of B virus and use proper protective equipment and care when handling them (Most cases of human B-virus infection have involved direct contact with macaques, such as a bite, scratch, or mucosal contact with body fluid or tissue (Human B-virus disease generally occurs within 1 month of exposure (, antibody titers to HSV are not protective in human cases of B-virus exposure or infection (The possibility of asymptomatic or mild B-virus infection in humans has been suggested (With the discovery of simian immunodeficiency virus and its identification as a model for HIV infection, the number of macaques used in research has increased, as has the number of human B-virus cases. Guidelines for reducing and controlling exposure were first published in 1987 (http://www.cdc.gov/niosh/hid5.html, http://www.haz-map.com/Macaque.htm.The 2002 B Virus Working Group guidelines address issues to be considered in cases of possible exposure to or infection with the virus (According to the guidelines, the most important action in a case of potential exposure to B virus is to rapidly and thoroughly cleanse the wound or exposure site. HSV can enter sensory nerve endings within 5 minutes of exposure, and B virus is likely to infect just as rapidly. Bite wounds, scratches, or puncture wounds of nonmucosal surfaces should be cleansed with soap or detergent for at least 15 min (A physician with a patient who has potentially been exposed to B virus faces a conundrum. Before onset of neurologic symptoms, antiviral therapy is successful. However, few cases of potential exposure lead to infection. Prophylactic treatment is unnecessary in most cases of potential exposure because treatment can confound diagnostic testing by interfering with the humoral immune response (http://www.gsu.edu/~wwwvir/index.html.In addition to working closely with a physician trained to handle cases of B-virus exposure and infection, taking samples from the exposed person and the source animal is important for virus culture and serologic testing. A list of recommended swabs for virus culture and serum samples is available from: URL: Early suspicion and rapid diagnosis of B-virus infection are critical to the control of human infection. The extreme cross-reactivity of primate alphaherpesviruses has required the development of diagnostic methods that can differentiate between HSV and B-virus infection. Despite the inherent risk for exposure, direct culture of B virus has been the standard for diagnosis of infection. Culture of B virus requires a special containment facility since the virus is a biosafety level 4 pathogen (More recently, PCR methods have allowed direct demonstration of B-virus infection without the risk of working with virus cultures (The cases of human B-virus infection that have been described have all occurred in relation to contact with macaques in a biomedical research setting. However, this setting is not the only one in which humans have contact with macaques. The Woburn Safari Park in the U.K. recently culled all B-virus–positive macaques from its facility (In other situations, particularly when potentially seropositive macaques have been domesticated as pets, opportunities for exposure to B virus are frequent. One report documented many instances of potential exposures from bites, scratches, food sharing, close physical contact, and even shared chewing gum (In 1989, the National Institutes of Health’s National Center for Research Resources started funding specific pathogen-free macaque colony development. The timing and local nature of B-virus reactivation and shedding make detecting infection in an animal difficult. Therefore, serologic methods are used to screen and monitor animals for consideration as pathogen-free. Numerous negative serologic results are necessary to determine a macaque’s B-virus status. Although specific pathogen-free status reduces the likelihood of infection, this status does not eliminate the risk for infection entirely. Full protective equipment should be used for working with all macaques regardless of their pathogen status. While population numbers in these specific pathogen-free colonies are increasing, the demand for pathogen-free animals will continue to exceed the supply for some time (While antiviral therapy has substantially improved the survival rate for human B-virus infection, fatal cases still occur (A formalin-inactivated B-virus vaccine was developed and tested in the 1960s (The potential for fatal human infection with B virus is a constant concern because frequent exposures occur to humans in the course of caring for and using macaques in a research setting. Personal protective equipment and safe handling procedures have limited the incidence of human disease. However, little is known about the biology of B virus in the natural macaque host. A clear understanding of the real risk for B-virus shedding in its natural host will help identify opportunities to prevent or limit zoonotic B-virus disease.


Over the last ten years, miniaturized multiplexed immunoassays have become robust, reliable research tools that enable researchers to simultaneously determine a multitude of parameters. Among the numerous analytical protein arrays available, bead-based assay systems have evolved into a key technology that enables the quantitative protein profiling of biological samples whilst requiring only a minimal amount of sample material.A microfluidic bead-based immunoassay, µFBI, was developed to perform bead-based multiplexed sandwich immunoassays in a capillary. This setup allows the simultaneous detection of several parameters and only requires 200 ng of tissue lysate in a 1 µL assay volume. In addition, only 1 µL of detection antibodies and 1 µL of the reporter molecule Streptavidin-Phycoerythrin were required. The µFBI was used to compare the expression of seven receptor tyrosine kinases and their degree of tyrosine phosphorylation in breast cancer tissue and in normal tissue lysates. The total amount of HER-2, as well the degree of tyrosine phosphorylation was much higher in breast cancer tissue than in normal tissue. µFBI and a standard bead-based assay led to identical protein expression data. Moreover, it was possible to reduce the quantity of sample material required by a factor of 100 and the quantity of reagents by a factor of 30.The µFBI, microfluidic bead-based immunoassay, allows the analysis of multiple parameters from a very small amount of sample material, such as tumor biopsies or tissue sections. As early-stage tumor sample size is usually small, therefore it is only possible to obtain small amounts of material, for example, fine needle aspiration Multiplexed immunoassays based on protein microarray platforms have been broadly employed in the discovery and validation of disease-associated biomarkers as well as in clinical diagnostics research Multiplexed immunoassay can be performed in a capillary requiring only a minute amount of sample material. Phillips et al. (2007) analyzed the expression of twelve cytokines in dissected tissue lysates. The cytokines were captured by a mixture of immobilized capture antibodies, and subsequently labeled with a flurophore. Captured and labeled cytokines were separated by electrophoresis and the quantification of individual cytokine peaks was performed The schematic set up for µFBI is shown in The relevant parameters of the µFBI were optimized using an artificial capture assay consisting of an immobilized anti-biotin antibody and biotinylated PE as the analyte. The optimal number of antibody coupled beads was defined in a first experiment. 250, 500, 1000, or 2000 beads were pumped into the capillary and incubated for 5 min with Biotin-PE (100 ng/ml). The median fluorescent intensity (MFI) and the number of recovered beads were analyzed . The MFINext, the influence of the coating surface of capillary was investigated. Capillaries with four different physical surface properties  were tested for their performance of the µFBI assay . The higThirdly, the optimal assay volume  was determined using the Streptavidin - Biotin-PE assay. 1000 anti-biotin antibody coated beads were incubated with Biotin-PE (100 ng/ml) for 5 min by changing the flow rate from 0.1, 0.2, 0.4, 0.8, 1.6 up to 3.2 µl/min. The generated MFI and the number of recovered beads are given in The optimal incubation time was determined using the Streptavidin - Biotin-PE assay. The different incubation periods  of the anti-biotin antibody coated beads with 1 µL Biotin-PE (100 ng/ml) were achieved by adjusting the flow rate from 1 µl/min, down to 0.008 µl/min. The results revealed that the MFI value increased with longer incubation times whereas the number of recovered beads decreased below 50 when the incubation time was extended above 60 min . These dFinally, the performance of µFBI assay was evaluated by the quantitative analysis of different biotin-PE concentrations. Anti-biotin antibody coated beads were incubated with 1 µl of Biotin-PE using concentrations ranging from 0 to 5000 ng/ml for 5 min, respectively. As a negative control BSA coated beads of a differently color-coded bead type were used in the same assay. The data are shown in These sets of experiments allowed to define the number of beads (1000), the surface coating (phenyl-silicon), the analyte volume (1 µL), incubation time (60 min) and quantitation ability of the µFBI assay. In a next step, the µFBI was applied to the analysis of Receptor Tyrosine Kinases in tissue lysates.A commercially available bead-based sandwich immunoassay - WideScreenTM Receptor Tyrosine Kinase Assay (RTK) Kit - was selected to evaluate the performance of the µFBI. RTKs are critical regulators of numerous cell signaling pathways. Ligand binding to the extracellular domain of transmembrane RTKs triggers receptor dimerization and autophosphorylation of an intracellular kinase domain. This event ultimately triggers activation of downstream pathway proteins via phosphotyrosine-SH2 domain interactions. RTKs have been shown to be not only key regulators of normal cellular processes but also to have a critical role in the development and progression of many types of cancer Considering the relationship between the assay sensitivity and incubation time as well as influence of flow rate , the capMultiplexing of beads in the µFBI is not limited due to the very low sample volume of only 1 µL. In such a volume 100 different bead types can be incubated simultaneously and subsequently analyzed in a Luminex instrument. A set of 100 different bead types were incubated into the capillary for 1 h mimicking real incubation conditions. After this incubation time beads were pumped out of the capillary and counted in a Luminex 100 instrument. For each bead type a sufficient number of beads above 35 were counted which are sufficient to fulfill statistical requirements, when measuring MFIs .Several aspects have to be considered to perform a successful µFBI experiment. Firstly, bubbles in the capillary have to be avoided, as bubbles severely influence the accuracy of bead numbers or sample volume during the assay. According to our experience, this problem could be significantly minimized by tightly sealing the connection between the microfilter and capillary and a carefully operation. In addition the dead volume of beads or sample has to be controlled. In our experiment, the capillaries were washed thoroughly with washing buffer in between consecutive assay runs. After extensive washing no residual beads could be observed, which allowed us to reuse the setup. In the here presented experimental setup two capillaries were used allowing to perform two assays in parallel. To increase sample throughput the number of capillaries can be increased as described recently by Bainer et al. 6 carboxylated colour coded beads with an diameter of 5.6 µm were obtained from Luminex, Inc. , and were coupled with 25 µg (100 µg/ml) Bovine serum albumin ) or 25 µg (100 µg/ml) anti-biotin antibody  according to the manufacturer's protocol. The coupled beads were stored at 4°C in Roche buffer  with 0.05% sodium azide. Coupling controls were used to assess the immobilization efficiency by staining 1500 of each type of microspheres with 0, 0.1, 0.3, 1 and 3 ug/ml R-phycoerythrin conjugated goat anti-mouse IgG .2.5×10The µFBI set up consists of a syringe pump, incubation zone and two capillaries . The KDSThe WideScreen™ Receptor Tyrosine Kinase Assay Kit was obtained from EMD Chemicals Inc. . This kit enables the quantification of a set of key RTKs, including EGFR, IGFR, HGFR, PDGFRb, HER-2, VEGFR2, and Tie-2. Breast cancer tissue and normal tissue were kindly provided by Georg Sauer and Helmut Deissler, University hospital Ulm, Ulm Germany Before starting the assay, the capillaries and pumps were rinsed thoroughly to remove any remaining sample or beads with 20 µL 70% isopropanol . 1 µL diluted antibody-coupled beads, 1,000 for each analyte, were pumped into each capillary at a flow rate of 0.2 µl/min. The prepared tissue lysates (200 µg/ml) were then pumped into the capillary and incubated with the mixed antibody-coupled beads for 1 h at a flow rate of 0.016 µL/min. Subsequently, the mixed biotinylated detection antibodies  and Strepavidin-Phycoerythrin  were pumped into the capillary and incubated with the analytes captured on the beads for 1 h and 45 min, respectively. After the assay was finished, the beads were pumped out of the capillary in a volume of 5 µL and diluted into 100 µL with assay diluent (1X). Finally, the diluted beads were transferred into a 96-well ELISA microplate . Followed by incubation for 5 min on a thermomixer  at 650 rpm to mix the beads in solution, the resulting beads were submitted to a Luminex 100 IS system for fluorescent signal readout . The MFI value was extracted to perform data analysis.The assay was carried out according to the instructions given by the manufacturer for the WideScreen™ Receptor Tyrosine Kinase Assay Kit with minor modifications. In short, the MultiScreen HTS™ 96-well filter plate  was pre-wetted with assay diluent for 5 min, 100 µL/well. Then, 50 µl diluted (1X) capture beads were transferred to each well. After removing the excess solution by filtration, 100 µL of the same tissue lysates (200 µg/ml) were added to each well to incubate with the beads at 23°C for 1 h on a thermomixer (650 rpm). Subsequently, the beads were washed 3 times with 100 µL washing buffer in each well. Then, 30 µL mixed detection antibodies (1X), 60 µL assay diluent, 30 µL streptavidin-phycoerythrin (1X), 60 µL assay diluent were transferred to each well and incubated at 23°C for 1 h and 45 min on a thermomixer at 650 rpm, respectively. Followed by 3 washing steps, 120 µL assay diluent (1X) were transferred to each well and incubated for 5 min on a thermomixer (650 rpm). After these procedures, the beads were submitted to a Luminex 100 IS system for fluorescent signal readout .The graphs were drawn with Excel 2003  and origin 7.0 . The data of


To the Editor: Long incubation periods are an occasional feature of infection with rabies virus and should be considered in human cases of acute encephalitis in rabies-free countries where there has been a history of travel to rabies-endemic areas  corresponded to positions 71–376 of the Pasteur virus genome (NC_001542) encoding the first 102 aa of the viral nucleoprotein. Further sequences were obtained from published studies submitted to GenBank. Sequences were aligned and edited with DNAstar Lasergene version 7 suite of programs . Neighbor-joining and maximum likelihood analysis were undertaken by using PHYLIP version 3.5 as previously described (The phylogenetic tree produced by this investigation indicates that the 1990 sequence falls within the rabies virus clade and is clearly distinct from the 4ABLV sequences included . These fAlthough the case history could not provide evidence for interaction with a dog while her family was in Hong Kong Special Administrative Region, rabies was endemic within the colony at the time that the patient’s family was resident. From 1980 through 1984, 5 human cases were recorded (Such long incubation periods are rare for rabies virus infections. An earlier epidemiologic study of 177 cases in Amritsar, India, demonstrated that rabies developed within 6 months of exposure in 90% of human cases (


The data presented herein support the North American orthopoxviruses (NA OPXV) in a sister relationship to all other currently described Orthopoxvirus (OPXV) species. This phylogenetic analysis reaffirms the identification of the NA OPXV as close relatives of “Old World” (Eurasian and African) OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. The natural reservoir host(s) for many of the described OPXV species remains unknown although a clear virus-host association exists between the genus OPXV and several mammalian taxa. The hypothesized host associations and the deep divergence of the OPXV/NA OPXV clades depicted in this study may reflect the divergence patterns of the mammalian faunas of the Old and New World and reflect a more ancient presence of OPXV on what are now the American continents. Genes from the central region of the poxvirus genome are generally more conserved than genes from either end of the linear genome due to functional constraints imposed on viral replication abilities. The relatively slower evolution of these genes may more accurately reflect the deeper history among the poxvirus group, allowing for robust placement of the NA OPXV within Chordopoxvirinae. Sequence data for nine genes were compiled from three NA OPXV strains plus an additional 50 genomes collected from Genbank. The current, gene sequence based phylogenetic analysis reaffirms the identification of the NA OPXV as the nearest relatives of “Old World” OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. Additionally, the substantial genetic distances that separate the currently described NA OPXV species indicate that it is likely that many more undescribed OPXV/NA OPXV species may be circulating among wild animals in North America. Poxviridae family of viruses is divided into two sub-family groups based on the hosts they infect, vertebrates (Chordopoxvirinae) and invertebrates (Entomopoxvirinae). In general, poxviruses are complex double stranded-DNA viruses with a characteristic brick, or ovoid-shaped virion apparent through electron microscopy. The Chordopoxvirinae are universally distributed and capable of infecting a wide range of avian, reptilian and mammalian species. Some appear to have a limited host range, (Molluscum contagiosum and Variola virus) while others are found to infect multiple animal species. Many of these poxviruses can cause serious infections in livestock species, including sheep, goats, cattle, crocodiles, caimans, chickens, turkeys and ostriches. Consequently, these pathogens can have devastating effects on livestock production resulting in substantial economic losses. In addition, Chordopoxvirinae species frequently emerge as zoonoses in humans. Sources of human infection include international trade in exotic animals (e.g. Monkeypox virus), livestock management (e.g. Vaccinia virus and Orf virus), and even contact with domestic animals (e.g. Cowpox virus). When introduced into non-endemic regions, enzootic chordopoxviruses can threaten native and endemic species previously protected by isolation. Squirrelpox virus, presumed to have been imported to the British Isles with the North American grey squirrel (Sciurus carolinensis), is thought to have contributed to the rapid decline of red squirrels (Sciurus vulgaris) in many areas of the larger islands The Orthopoxvirus (OPXV), which includes Variola virus, the causative agent of smallpox, and Vaccinia virus, the principal source of smallpox vaccine. Additionally, several OPXV species are zoonotic, including Cowpox virus, Monkeypox virus, and some vaccinia-like virus species. The distribution of most described OPXV species is outside of North America. Those viruses presumed to be endemic to North America currently include only Raccoonpox virus, Skunkpox virus, and Volepox virus. The host range, geographic distribution, and precise taxonomic position of these North American orthopoxviruses (NA OPXV) remain largely undetermined.One of the best studied chordopoxvirus genera is Cowpox virus species are known to be carried, and hypothesized to be transmitted to humans and domestic animals, by rodent species including bank voles (Myodes glareolus) and striped field mice (Apodemus sylvaticus). Several other OPXV species are known to be associated with rodents, but the specific nature of these associations remains unknown. Ectromelia virus was described from captive colonies of lab mice (Mus spp) and some strains are highly lethal to this rodent species, however almost nothing is known regarding its natural distribution Monkeypox virus was initially isolated from captive non-human primates in Denmark Monkeypox virus was obtained from a Thomas's rope squirrel (Funisciurus anerythrus) collected in Zaire (currently Democratic Republic of Congo) in 1985 Taterapox virus was described in 1975 as an OPXV isolated from a “healthy” naked-soled gerbil (Tatera kempi) captured in Benin Oryzomys sp.) and found to belong to a larger clade of vaccinia-like viruses which cause disease in cattle and humans throughout the dairy producing regions of Brazil The natural reservoir host(s) for many of the described OPXV species remains unknown. However, the ecology and host systems of cowpox viruses have been extensively studied Volepox virus has been definitively associated with rodents, being isolated initially from a scab from a California vole . OPXV antibodies were found in several other geographically separated populations of this species over a period of two years, suggesting a long term association between the virus and host Peromyscus truei) in the same area of the initial isolation site near San Francisco Bay Raccoonpox virus and Skunkpox virus were originally each isolated from their namesake mammalian species. A wildlife study conducted across 35,000 acres of Aberdeen Proving Grounds, Maryland, reported the recovery of two Raccoonpox virus isolates and a seroconversion rate of 23% in wild raccoons found in the region Raccoonpox virus in a domestic cat Raccoonpox virus and raises the possibility of transmission of these poorly characterized viruses to domestic cats, as well as to other domestic or wild animals and perhaps even to humans. A limited infection was reported upon human exposure (needle stick) to a recombinant Raccoonpox virus vaccine vector, though natural human infection has yet to be reported Skunkpox virus was isolated from a noticeably ill and presumed (but not) rabid skunk in Colfax, Washington in 1978 and is the only NA OPXV isolated from an overtly ill animal. To further our understanding of the poxviruses circulating in North America, we compare nine genes from across the conserved central region of the poxvirus genome among the eight genera of Chordopoxvirinae. Skunkpox virus, Volepox virus and Raccoonpox virus are included in the analyses to establish the relationship of the North NA OPXV species relative to the currently recognized Orthopoxvirus family.Of the NA OPXV, only Raccoonpox virus (RACV) forms a monophyletic group with a Skunkpox virus/Volepox virus  sister grouping. The relative branch lengths between the NA OPXV and leading to other orthopoxviruses illustrates the considerable distance between the two clades (NA OPXV and other OPXV) and that the NA OPXV have much greater distances between species than is seen among the recognized African and Eurasian OPXV species. Outside of the OPXV clade a monophyletic Yatapoxvirus clade is sister to a larger non-OPXV clade. This non-OPXV/non-Yatapoxvirus clade consists of two clades. Deerpox virus (DPV) isolates are sister to a Capripoxvirus clade which is consistent with previous analyses Sheeppox virus (SPPV) sister to a monophyletic clade including Lumpy skin disease virus (LSDV) and Goatpox virus (GTPV). The second clade consists of Swinepox virus (SWPV), Rabbit fibroma virus (RFV), Myxoma virus (MYXV), Molluscum contagiosum virus (MOCV), Crocodilepox virus (CRV), Bovine papular stomatitis virus (BPSV) and Orf virus (ORFV) which form a ladder configuration beginning with LSDV as the first to split from the others and ending with BPSV and ORFV in a sister grouping.The phylogenetic tree derived from the selected genes was constructed by Bayesian inference and is shown in Ectromelia virus) are presented in Variola virus) (patristic distance  = 0.033874). Comparatively, the average patristic difference between ECTV_Mos and NA OPXV species (0.160133) is nearly five times the distance between ECTV_Mos and VARV_IND3_1967. Similarly, the two closest NA OPXV species, Volepox (VPXV) and Skunkpox virus (SKPV), lie at a patristic distance (0.070158) twice that of ECTV_Mos and VARV_IND3_1967. The results of the Shimodaira-Hasegawa test which analyzed the log likelihood scores of alternative tree topologies (with each NA OPXV constrained into the Old World OPXV clade) indicate that the original tree, with a monophyletic NA OPXV, has a significantly (P<.05) better topology.Pairwise genetic distances and branch lengths between NA OPXV species and their nearest Old World cousin, ECTV_Mos (Molluscum contagiosum (MOCV_st1), Crocodilepox virus (CRV_ZWE), and the parapoxviruses  are unusually GC-rich compared to the rest of the PoxviridaeLeporipoxvirus (43.6% and 39.5% for MYXV and RFV respectively) Molluscum contagiosum virus and Orf virus have been presented in a similar topology in previous studies Leporipoxvirus clade and moved Suipoxvirus from a sister position with Leporipoxvirus to a sister relationship with Deerpox virus. The large non-Orthopoxvirus clade is still formed by Yatapoxvirus, Leporipoxvirus, Deerpox virus and Capripoxvirus added in a stepwise manner from the deepest node. The relationship between Suipoxvirus, Deerpox virus and Capripoxvirus has been shown to be close in previous work Orthopoxvirus clade structure is presented in Bratke and McLysaght The genomes of Volepox virus, Cowpox virus, vaccinia-like viruses, and Taterapox virus are all believed to infect rodent species with little obvious disease in the host although, with the exception of Cowpox virus, only limited data are available Camelpox virus and Taterapox virus), or zoonotic (Monkeypox virus) OPXV species described as endemic to the African continent, and the genomic divergence between these viruses is relatively slight. Most of the African OPXV ecological surveillance studies have used serological assays that cross react between members of the OPXV genus and are not able to distinguish between exposures to different OPXV species. This, along with the low number of ecology studies relative to the size and ecological diversity of the African Continent, make it likely that there are several more African OPXV species that have not yet been described.Members of the OPXV genus infect several mammalian taxa as both enzootic and zoonotic pathogens, and in at least some cases are present asymptomatically. In terms of isolates obtained from wild animals, rodents are the most often associated mammalian taxon regarding the isolation of the currently described OPXV species. Raccoonpox virus and Volepox virus) have not been linked to any obviously pathogenic disease in animals or humans. Ecological studies of these two viruses indicate widespread and possibly long-term associations between the viruses and the mammal species from which they were isolated Skunkpox virus was isolated from a sick animal, it is not clear if the virus was the cause of the illness. The recent occurrence of Raccoonpox virus in a domestic cat in Canada illustrates the possibility of cross-species transmission of these viruses. Reports of Cowpox virus in domestic cats and humans have been occurring with increased frequency in Europe Cowpox virus infections may be attributable to the decrease in number of persons vaccinated for smallpox and the loss of cross-protection against other poxvirus species. These conditions also exist in North America and could lead to an increase in the transmission of NA OPXV to humans from the reservoir species or domestic animals.The topology and the relatively large patristic distances between NA OPXV and the other OPXV species indicate that the NA OPXV species are the most divergent members of OPXV currently described and are some of the least studied. All of these are mammal-associated and two of the three , also one of the larger genomes, is consistently sister to the remaining non-NA OPXV Orthopoxvirus group in this and other analyses It has been suggested that cowpox viruses represent the ancestral state of OPXV species in that they possess every gene present in every other OPXV Cowpox virus isolates  do not form a monophyletic clade. CPXV_GRI is most closely related to the vaccinia virus group, while CPXV_BR diverges from a deeper node on the tree. The phylogenetic and taxonomic incongruence of “cowpox virus”, despite some similar virological properties including chorioallantoic pock morphology, is likely the result of multiple OPXV species which cause a rash illness in cows. The traditional common names assigned to poxviruses are often confusing, as the disease presentation is superficially similar for many of the pathogenic species. Cows are susceptible to both Vaccinia virus and the multiple Cowpox virus clades. Lineages of these similar viruses may explain some of the historic confusion regarding cowpox, horsepox and vaccinia with regard to the condition and the causative viral agents Vaccinia virus. All of the vaccine and sylvan  isolates form a monophyletic clade which justifies the classification of all members of this clade under the species Vaccinia virus.As seen in previous studies Vaccinia virus, while initial studies demonstrated little to no hemagglutination inhibition (0 to 160) between vole sera and Vaccinia virus or Raccoonpox virusVaccinia virus, Variola virus and Cowpox virus) of the OPXV. Further studies are needed to clarify the classification of the group based on biologic criteria.The data presented herein support a monophyletic NA OPXV clade in a sister relationship to all other currently described OPXV species. The genus OPXV has been defined traditionally by some combination of: the presence of cross-neutralizing antibodies within members of the genus, genomic similarities, biologic phenotypes, similar immunodiffusion patterns, and hemagglutination inhibition activity. Hemagglutination-inhibition antibody titers were reported at 640 and 1280  for three samples (3.3%) of raccoon sera against The current, gene sequence based phylogenetic analysis reaffirms the identification of the NA OPXV as the nearest relatives of “Old World” OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. Two of the three NA OPXV species are known only from isolates obtained from carnivores. If these Carnivore species represent the natural host(s), then either these viruses have a very taxonomically broad host range or the origins of the genus OPXV could be much more ancient than previously thought, dating back to the common ancestor of the Rodentia and Carnivora. Given the relatively high genetic divergence within the NA OPXV clade it is conceivable that the hosts could be as taxonomically distant as rodents and carnivorans. However, it is also readily conceivable that the carnivoran species could have been infected by contact with rodents as with cowpox viruses in the Old World. Future ecological investigations are needed to refine the understanding of the ecological parameters which result in the maintenance of these NA OPXV viruses in the environment. Two scenarios could explain the observed relatively high genetic diversity within the NA OPXV. First, if one suggests that these are the only OPXV viruses currently native to North America, one must posit that there have been substantial virus extinction events during the course of NA OPXV evolution leaving behind a very small number of highly divergent relic species. Alternatively,the substantial genetic distances that separate the currently described NA OPXV species could indicate that many more undescribed extant OPXV/NA OPXV species may be circulating among wild animals in North America.All analyzed strains represent low passage (1–2 passages) isolates that are as close to the “wild” type viruses as possible. The isolates chosen for analysis were, when possible, from the original specimen described for the species. Genes from the central region of the poxvirus genome are generally more conserved than genes from either end of the linear genome due to functional constraints imposed on viral replication abilities. The relatively slower evolution of these genes may more accurately reflect the deeper history among the poxvirus group, allowing for robust placement of the NA OPXV within Chordopoxvirinae. Sequencing of three NA OPXV genomes was carried out using standard protocols of the Roche GS-20 pyrosequencing platform  and confirmatory Sanger sequencing. Sequence data for nine genes were compiled from these strains plus an additional 50 genomes collected from Genbank. A list of strains, isolates, and sources is available in http://toolkit.tuebingen.mpg.de/probcons). The RevTrans 1.4 Server (http://www.cbs.dtu.dk/services/RevTrans/) was then used to translate the amino acid alignment back into the original DNA sequence Nine coding sequences were selected from the central region of the genome: A7L, early transcription factor/VETF large subunit (82 kDa); A10L, major core protein; A24R, RNA polymerase 132; D1R, messenger RNA capping enzyme, large subunit; D5R, DNA independent NTPase (DNA replication); E6R, hypothetical protein; E9L, DNA polymerase; H4L, RNA polymerase associated protein; J6R, RNA polymerase 147. Gene designations refer to the VACV-COP genome. Seven of these coding regions make up a subset of the ‘core families’ used to estimate a poxvirus phylogeny by Bratke and McLysaght Phylogenetic analysis was carried out on the concatenated DNA alignments using Bayesian inference as executed by MrBayes using four “chains” run over five million generations In order to examine the validity of the monophyletic NA OPXV we constructed 3 constrained trees in which each of the NA OPXV species were separately excluded from the monophyletic cluster and forced into monophyly with the “Old World” OPXV see . The Shi


Drosophila melanogaster. To investigate the Drosophila early immune response to Drosophila C virus, we characterized the DCV infection-response transcriptome of Drosophila Schneider 2 (S2) cells at one hour post inoculation.The innate immune response like phagocytosis, encapsulation and antimicrobial peptide (AMP) production often occur in the early stage of host-pathogen interactions in Drosophila GeneChip (Affymetrix) assay. Then the results of signaling pathway and protein interaction about these genes were analyzed by MAS 3.0 software.The total RNA was extracted from treated S2 cells by using Trizol reagent and then analyzed by CapitalBio Corp for Most significantly affected genes (656 genes) by DCV infection were regulated as the same way in inactivated DCV treatment, but inactivated white spot syndrome virus (WSSV) showed a different transcriptome. DCV infection up-regulated the expression levels of 275 genes and down-regulated that of 442 genes significantly and some affected genes were related to phagocytosis. DCV infection activated the JAK/STAT pathway by 1 hour post incubation. The Imd pathway was activated and transcriptional induction of antimicrobial peptides (AMPs) from this pathway was enhanced by 1 hour post incubation. But the Toll pathway was not activated like Imd pathway and the expression levels of AMPs from this pathway was reduced. In addition, most pattern-recognition receptors were inhibited and the antiviral RNAi pathway was not activated in the early stage of DCV infection.In conclusion, the present study demonstrates that DCV infection may activate phagocytosis, JAK/STAT pathway and Imd pathway in the early host-virus interactions. These results indicate that DCV is capable of activating or inhibiting some immune responses in the host cells and these changes would be vital for virus entry and replication. Drosophila is governed by numerous signaling pathways that trigger antimicrobial peptide (AMP) production, phagocytosis, melanization, and encapsulation to limit infection after exposure to microbes [Drosophila C virus (DCV) is a non-occluded isometric virus which containing a positive sense RNA genome [D. melanogaster, making it an ideal model system for studying invertebrate host-virus interactions [Drosophila highlight the potential of the D. melanogaster model for studying antiviral innate immunity [Drosophila[Drosophila X virus replication in Drosophila and constitutive activation of the pathway resulted in decreased viral titer [Drosophila[Drosophila[D. melanogaster infected with the sigma virus (Rhabdoviridae) [Aedes aegypti[The innate immune response of microbes ,2. DrosoA genome ,4. DCV iractions . The mecimmunity . It is frosophila,8. The Tal titer . Recentlrosophila-12. The rosophila-13. Howeviridae) . And dens aegypti. So it iDrosophila S2 cells to DCV, inactivated DCV (inDCV), and inactivated WSSV (inWSSV). We selected inWSSV as a treatment but not WSSV because WSSV was not phagocytosed by S2 cells like DCV and inDCV and induced very complicated early response. We investigated the transcriptional profile of virus-challenged Drosophila S2 cells using oligonucleotide DNA microarrays to identify the Drosophila early immune response to DCV. This results of this study contribute to the understanding of early immunologic defense responses in invertebrate hosts to viral challenge, and this study paves the way for further experiments which investigate the roles of genes and pathways in antiviral immunity.White spot syndrome virus (WSSV) is a bacilliform, enveloped double stranded DNA virus that causes viral diseases in shrimp . To inveDrosophila S2 cells in early immune response to DCV to systematically dissect host functions important in responding to virus. Genome-wide analysis can be conducted easily in genetically tractable model hosts, such as D. melanogaster, and such analyses offer a new approach to identifying host genes required for host antiviral immunity. We conducted a genome-wide analysis of the Drosophila early immune response to DCV by using oligonucleotide microarrays. Our results show that DCV infection significantly up-regulated the expression levels of 275 genes and significantly down-regulated that of 442 genes at least 2-fold . Gene ontology and KEGG analyses revealed that most of these genes were related to cellular metabolism. The similar result was found in Aedes aegypti cells with dengue virus infection [P<0.01) in DCV treatment but not in the other two treatments. C-terminal Src kinase (Csk) is the major inhibitor of Src signaling, and it is linked with CG6410 (phosphatidylinositol binding), CG10479 (unknown function) and Cad96Ca (protein tyrosine kinase activity). In this study, Csk was up-regulated very significantly (P<0.01) in DCV treatment but not in the other two treatments. Csk functions with Src-family kinases to negative regulate cell proliferation and positive regulate apoptosis [We investigate ld Table . Some ofld Table A. In theld Table B. In addnfection . Among tnfection . βTub97Epoptosis -24. Endopoptosis ,26. So tDrosophila responsible for DCV infection, virus-induced RNA 1 (vir-1) is one of genes resulting from JAK/STAT signaling pathway which is not induced by pathogenic bacteria or fungi [Drosophila[P<0.01) in all three treatments, suggesting the involvement of JAK/STAT pathway in antiviral responses of Drosophila  in response to WSSV (a non-pathogenic virus of Drosophila) as it was in responses to DCV , and TepI was down-regulated very significantly (P<0.01) in pathogenic virus treatments (DCV and inDCV) but not in non-pathogenic virus treatment (inWSSV)  in all three treatments  activates the Toll pathway, and flies that are deficient for the Toll pathway transcription factor Dif are more susceptible to DXV infection [P<0.01), and Mkk4, ndl, 18w, Toll-8 and Pli were up-regulated significantly (P<0.01) in pathogenic virus treatments (DCV and inDCV) but not in non-pathogenic virus treatment (inWSSV)  in pathogenic virus treatments (DCV and inDCV) but was up-regulated significantly (P<0.01) in non-pathogenic virus treatment (inWSSV). Drosomycin (Drs) gene encodes a 44-residue inducible antifungal peptide, Drosomycin, in Drosophila melanogaster[Drosophila, DCV may inhibit the key factors of this pathway to evade humoral and cellular responses of host cells.Previously, it was shown that the nfection . Anothernfection . In this) Figure . The datanogaster. BecauseP<0.01) in all three treatments , and PGRP-LC was down-regulated in pathogenic virus treatments (DCV and inDCV) but not in non-pathogenic virus treatment (inWSSV) (Table E. coli infection [P<0.01) in all three treatments  in pathogenic virus treatments (DCV and inDCV) but not in non-pathogenic virus treatment (inWSSV)  Table . The NF-nfection . In thists Table . The res humoral . DENV inti cells ,17. In tV) Table . The datDrosophila and Ago2, Ars2, Dcr-2 and R2D2 as the core antiviral RNAi machinery [Drosophila, remained stable in all three experimental groups (data not shown). The antiviral RNAi pathway was not activated in S2 cells by 1 hour post incubation with DCV or inWSSV. Virus infection in Drosophila initiates a specific transcriptional response, including the induction of Vago, a recently identified antiviral molecule that is required to restrict viral replication in flies [P<0.01) in all three treatments  in pathogenic virus treatments (DCV and inDCV) but not in non-pathogenic virus treatment (inWSSV) (Table P<0.01) in all three treatments  by DCV infection only. TEM results also showed that no difference was found in DCV-infected S2 cells at one hour post infection, but DCV infection caused large harm to S2 cells at one day post infection  supplemented with 10% fetal bovine serum . DCV was inoculated in S2 cells at a multiplicity of infection (MOI) of 1 for 4 days and collected for purification as described before [7 copies/mL) were inoculated in S2 cells (1 × 106 cells/mL). After one hour, the S2 cells were collected and subjected to oligonucleotide microarray.d before . The WSSDrosophila GeneChip (Affymetrix) assay. And each treatment has 3 biological replicates that were measured by this way. Gene expression analysis was performed by using the Affymetrix  Drosophila GeneChip, using the laboratory methods in the Affymetrix GeneChip expression manual. Gene expression analysis was performed using triple arrays and triple independent mRNA samples for each treatment. Microarray data were analyzed by using Bio MAS (molecule annotation system) 3.0 software . Using the criterion of cutoff limitation as a fold change ≥ 2 or ≤0.5 and q-value ≤ 5%, differential expression genes were screened and clustered.The total RNA was extracted from treated S2 cells by using Trizol reagent  according to the manufacturer’s instructions. The total RNA samples were then analyzed by CapitalBio Corp for Through array analysis, the commonly altered genes were screened from DCV, and WSSV treatments. The selected genes were further analyzed in the context of Gene Ontology (GO) biological process and Kyoto Encyclopaedia of Genes and Genomes (KEGG) biological pathway. Then the results of signaling pathway and protein interaction about these genes were analyzed by MAS 3.0 software. To reveal the functions of predicted target genes, we used the ontology classification of genes based on gene annotation and summary information available through DAVID .The S2 cells were pelleted and fixed in the fixative containing 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 18 h at room temperature. Each sample was washed three times with 0.1 M sodium cacodylate buffer at room temperature. Then the sample was postfixed with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer with constant rotation for 1h, followed by washes three times using 0.1 M sodium cacodylate buffer at room temperature. The sample was stained with 2% uranyl acetate in 0.2 M sodium acetate buffer (pH 5.2) for 1 h at room temperature and subsequently washed three times with 0.2 M sodium acetate buffer at room temperature. The sample was dehydrated in an ascending acetone series  and then in 100% propylene oxide for 10 min with constant rotation at room temperature. After infiltration of sample with EMBED 812/Araldite 502 resin at room temperature, sections were prepared in a Reichert Ultracut OMU3 microtome  at 100 nm thickness, followed by staining with 2% uranyl acetate/70% methanol. The images were collected on a Hitachi 7650 transmission electron microscope  operating at 70 kV.P-values of less than 0.05 were considered significant and less than 0.01 were considered very significant.Statistical significance was determined by Student's t-test . The authors declare that they have no competing interests.ZF designed the experiments. ZF, DHJ and ZBN carried out the experiments. ZF, DHJ and ZBN analyzed the data, ZF wrote the paper. All authors read and approved the final manuscript.


Xlr3/4 of which three paralogs, Xlr3b/4b/4c, were maternally expressed and paternally silenced in mouse neonatal brain[Xlr locus using known autosomal imprinting mechanisms as a model.Genomic imprinting is the epigenetic process by which a subset of genes is expressed from only one allele based on the parent of origin. To date there are approximately 150 confirmed autosomal imprinted loci in the mammalian genome. The epigenetic hallmark of an autosomal imprinted locus is the presence of differential methylation at CpG islands otherwise known as a DMR. In 2005m) or paternal  X chromosome. To measure the difference in both primary transcript and mRNA levels of Xlr3b/4b/4c between these samples quantitative real time PCR was performed. To search for differential methylation, MeDIP followed by hybridization to an X chromosome tiling array was performed for a global view of methylation at the Xlr3/4 locus. For confirmation, targeted bisulfite sequencing was performed at CpG islands. Chromatin Immunoprecipitation (ChIP) followed by real time PCR was used to assess differential enrichment of H3K4me3, H3K27me3, H3K9me2, and H3K36me3 at Xlr3b. Furthermore, ChIP-Seq was performed to trace RNA Polymerase II (PolII) and H3K36me3 across the Xlr3/4 locus.Mice were generated that either carried the maternal (39, Xthe Xlr3/4 locus between 39, Xm and 39XP samples. Potential candidate regions for differential methylation according to MeDIP assays were disproven by bisulfite sequencing of CpG islands within those regions. Furthermore, all regions assayed at Xlr3b were hypermethylated on both alleles. We show that H3K4me3 and H3K27me3 are not enriched at either allele of Xlr3b while H3K9me2 is equally enriched on both. We show that the primary transcript of Xlr3b only shows imprinted expression at the 3’ end of the gene and this is confirmed by PolII enrichment indicating a stall on the paternal allele during transcriptional elongation. Lastly, H3K36me3 enrichment mirrors that of RNA PolII.We show that there is no differential methylation at Xlr3/4 locus and thus a novel mechanism must exist to regulate the imprint. The current model for this mechanism involves PolII stalling during transcriptional elongation along the paternal allele of Xlr3b. On the maternal allele PolII transcribes through the 3’end of the gene unimpeded. While the cause of the stall remains unknown it may involve H3K36me3 histone methyltransferases.We show that there is no DMR to govern gene expression at the