Sciurus vulgaris) following the introduction of the non-native gray squirrel(Sciurus carolinensis) to the United Kingdom. The red squirrel is a major UKconservation concern and understanding its continuing decline is important for any attempt tomitigate the decline. The red squirrel–gray squirrel system is also exemplary of theinterplay between infectious disease (apparent competition) and direct competition in driving thereplacement of a native by an invasive species. Time series data from Merseyside are presented onsquirrel abundance and squirrelpox disease (SQPx) incidence, to determine the effect of the pathogenand the non-native species on the native red squirrel populations. Analysis indicates that SQPx inred squirrels has a significant negative impact on squirrel densities and their population growthrate (PGR). There is little evidence for a direct gray squirrel impact; only gray squirrel presence(but not density) proved to influence red squirrel density, but not red squirrel PGR. The dynamicsof red SQPx cases are largely determined by previous red SQPx cases, although previous infection oflocal gray squirrels also feature, and thus, SQPV-infected gray squirrels are identified aspotentially initiating outbreaks of SQPx in red squirrels. Retrospective serology indicates thatapproximately 8% of red squirrels exposed to SQPV may survive infection during an epidemic.This study further highlights the UK red squirrel – gray squirrel system as a classic exampleof a native species population decline strongly facilitated by infectious disease introduced by anon-native species. It is therefore paramount that disease prevention and control measures areintegral in attempts to conserve red squirrels in the United Kingdom.Infectious disease introduced by non-native species is increasingly cited as a facilitator ofnative population declines, but direct evidence may be lacking due to inadequate population anddisease prevalence data surrounding an outbreak. Previous indirect evidence and theoretical modelssupport squirrelpox virus (SQPV) as being potentially involved in the decline of red squirrels( Sciurus carolinensis), the red squirrel (Sciurus vulgaris), andsquirrelpox virus (SQPV), for example, have generally assumed 100% mortality of red squirrelswhen exposed, and equal infection intensity between the two species, despite subsequent evidence tothe contrary  before,during, and after SQPx outbreaks have not previously been carried out. Hence, there is a dearth ofdata on the associations between SQPx incidence and variations in red and gray squirrel numbers, onpatterns of infection during an epidemic, and on survival and recovery within infected red squirrelpopulations. Here, we present data collected from 2002 to 2012 from before, during, and after anoutbreak of SQPx on the Sefton coast, Merseyside, UK. Data on deaths caused by SQPV in this systemare combined with line transect survey data to examine associations between SQPV infection andsubsequent variations in the abundance of red and gray squirrels. The following predictions aretested: 1) SQPV infection in red squirrels is associated with a reduction in red squirrel densityand population growth rate (PGR); 2) gray squirrel density has similar associations; and 3) redsquirrel SQPx cases are associated with previous gray squirrel SQPV infection locally. We alsoinvestigate whether red squirrels may survive epidemics of SQPx in their natural habitat to play arole in subsequent population recovery.2 (OSGrid Ref: 108:SD275080) , recording the number of squirrels of each speciesand their perpendicular distance from the transect line. Both transect position and surveyor (wherepossible) were kept constant throughout the course of the study.In 2002, the Lancashire Wildlife Trust established 24 line transects in the Sefton Coast area formonitoring squirrel abundance. Five additional transects where added in 2005 Fig. TransecFrom March 2003 to March 2012, red squirrel carcases found by the public or wildlife officerswere submitted for routine postmortem examination. Geographic location and date of discovery wererecorded. Bodies were either refrigerated at 4°C where necropsy was to be performed within48 h or stored at −20°C. Tissue samples were taken from all major organs, alongwith cutaneous samples taken from the face  and antebrachium (scent/sensorygland), areas known to harbor large amounts of SQPV in infected animals  according to the manufacturers' protocol andstored at −20°C.Squirrelpox virus (SQPV) infection of red squirrels was diagnosed initially by the presence ofgross lesions , and blood samples were taken fromthe greater saphenous vein using aseptic technique. The serum was separated by centrifugation withintwelve hours of collection and stored at −20°C. Dacron swabs  were used to swab the facial area and antebrachium gland.Squirrel serum was analyzed for SQPV antibodies using an enzyme-linked immunosorbent assay(ELISA) , and estimated graysquirrel densities from 0 to 541 squirrels/km2 . Estimated red squirrel PGR ranged from −0.84 to 0.66  and gray squirrel PGR from −0.66 to 0.57 . Overall, the red squirrel population on Merseyside was generallystable until a sudden, large decline of 87% between autumn 2007 and March 2009 , and one recorded gray squirrel presence only. Estimated red squirrel densities ranged from 0to 436 squirrels/kmA total of 448 red squirrel carcases were submitted during the course of the study. SQPVinfection was diagnosed in 187 individuals, of which 151 (81%) had died directly from theinfection and 33 (18%) had died from other causes but were PCR positive for SQPV. Threecarcases showed no lesions or overt cause of death but were SQPV PCR positive. Only individuals witha location record accurate to 50 m and a record of the date found were included in theregression analysis (148 of 187). These data are also summarized in Fig.Between March 2003 and October 2009, 309 gray squirrel carcases were submitted, of which52% (160) underwent PCR analysis for SQPV, and of these, 27% (43 of 160) werepositive. Eighty-one negative individuals and 31 positive individuals had spatial and temporalrecords appropriate (see above) to indicate gray SQPV status in the regression analysis. There wasno correlation between the overall SQPV infection prevalence in the two species.Abbreviations used throughout the analyses are summarized in TableWith transect as a random effect, the base model included year and season (October density wasgreater than in March). In the final model, red squirrel density was associated with gray squirrelpresence 24 months previously, gray squirrel PGR 12 months previously, red squirrelpoxcases 6 months previously, and contemporary gray squirrelpox cases, all of which had anegative influence . In the final model, red squirrel PGR was negativelyassociated with red squirrel density twelve months previously, red SQPx cases 6 monthspreviously, and the presence of SQPV in gray squirrels, but positively associated with gray squirrelPGR and with red squirrel density 2 years previously Table.With transect as a random effect, the base model included year and red squirrel density24 months previously. Red squirrel pox cases were positively associated with red SQPx cases 6and 12 months previously and with gray SQPV-positive squirrels 6 months previously,and negatively associated with red SQPx cases 18 and 24 months previously , both support a scenario in which the graysquirrel is responsible for initiating the infection in red squirrels. Prevalence of infection ingray squirrels was generally low, rates of viral shedding from infected gray squirrels are typicallyvery low  in gray squirrelsbeing associated with SQPV infection in native red squirrels, thus supporting our final prediction.Indeed, the indicated time lag of 2 months between infection in the two species, and thegreater coefficient displayed by the infected gray squirrels compared with the equivalent redsquirrel parameter  grays should be perhaps considered the minimum intervention necessary. Ifnot, red squirrels are unlikely to survive in sufficient numbers to recolonize an area. This isespecially important where populations are small and fragmented, where low numbers of individualssurvive, and where the absence of a surrounding uninfected population makes recolonizationunlikely.The success of any intervention will be dictated by the early detection of SQPV in a red squirrelpopulation. The first control measure conducted should be surveillance of both mortality and healthof remaining red squirrel populations. This can be by the form of passive carcase collection (as inthis study), but more active surveillance is recommended through more systemic surveys, whereresources allow. More intensive disease surveillance programs  should beadopted where resources allow, using strict biosecurity protocols.Once SQPV is confirmed in a population, the aim should be to limit the spread of disease as muchas possible with the removal of infected red squirrels in the vicinity. Methods on how this isperformed will be dependent on the availability of resources and local opinion. Control of diseasein wild animals often involves culling of infected individuals and this could be considered,especially as recovery of red squirrels with SQPx is rare in captivity. The evidence of this studywould suggest those squirrels that do survive SQPx pose no further risk to naive red squirrels, withno lesions being noted on the surviving individuals and no further pox cases were identified fromthe areas where the survivors originated.Any practice that has the effect of increasing squirrel densities and increasing the interactionbetween conspecific red and gray squirrels  should ideally avoided atanytime, but especially during periods of disease outbreak. Evidence suggests that the disease has adirect transmission route and environmental contamination is the main route of transmission.Modelling studies have tended to neglect the potential for red squirrels to survive SQPVinfection, with the exception of one  were overtlySQPx positive. Therefore, of 251, 206 deaths (82%) can be attributed to SQPx. However, of theremaining 45 that died of other causes, some will previously have survived exposure to SQPV. Theproportion doing so should be the same as the proportion of subsequent survivors that previouslysurvived exposure to SQPx (5 of 16), giving 14 individuals (of 45) that survived exposure, but thendied of other causes. Thus, we have a total number exposed of 225 , an exposure rate of 84% (225 of 267), and asurvival rate following exposure of 8.4% (19 of 225)  support. The success of such programswill hinge on sound disease control or prevention measures, for which there is still a largerequirement for further knowledge on the epidemiology of SQPV and other squirrel diseases. There isnow a need to confirm the general conclusions herein with similar studies in other areas of theUnited Kingdom to differentiate, if present, any local variation in the effect of disease of redsquirrel populations in the United Kingdom and potentially Europe.


Determining the shapes of a rotating liquid droplet bound by surface tension is an archetypal problem in the study of the equilibrium shapes of a spinning and charged droplet, a problem that unites models of the stability of the atomic nucleus with the shapes of astronomical-scale, gravitationally-bound masses. The shapes of highly deformed droplets and their stability must be calculated numerically. Although the accuracy of such models has increased with the use of progressively more sophisticated computational techniques and increases in computing power, direct experimental verification is still lacking. Here we present an experimental technique for making wax models of these shapes using diamagnetic levitation. The wax models resemble splash-form tektites, glassy stones formed from molten rock ejected from asteroid impacts. Many tektites have elongated or ‘dumb-bell' shapes due to their rotation mid-flight before solidification, just as we observe here. Measurements of the dimensions of our wax ‘artificial tektites' show good agreement with equilibrium shapes calculated by our numerical model, and with previous models. These wax models provide the first direct experimental validation for numerical models of the equilibrium shapes of spinning droplets, of importance to fundamental physics and also to studies of tektite formation. Developments in computing have enabled increasingly sophisticated numerical calculations of the shapes of equilibrium1417181920212223242526273023The question of how a spinning liquid drop deforms under the action of rotation can be traced back as far as the debate between Newton and Cassini concerning the shape of the Eartht = 0 s with an image showing the withdrawal of the pipette after formation of the droplet. The air flow was activated at t = 20 s. As the angular velocity of the droplet increased, its equatorial radius expanded, initially retaining an axisymmetric shape, as can be seen in the fourth (t = 30 s) and fifth (t = 40 s) images. This shape eventually became unstable to a tri-axial shape (t = 50 s), which with increasing angular momentum quickly evolved into a two-lobed shape (t = 60 s). At this point in this experimental run, the air flow was reduced to a level sufficient to maintain constant angular velocity, allowing the droplet to approach rigid body rotation. The droplet continued to rotate as a rigid body with no discernible change in the longest symmetry axis for ~20 s. At t = 80 s solidification began at the periphery of the two lobes, with complete surface solidification after a further 20 s. The total time from pipetting to complete surface solidification was of order 100 seconds, comparable to the estimated cooling time for some natural tektites33The still images in V = 0.5 ml. Each of these wax models were formed from liquid droplets that were spinning with different angular momenta when they solidified. From top to bottom, as pictured in Numerous ‘artificial tektites' were produced by this method, with a range of angular velocities and droplet volumes between 0.1 and 0.5 ml. b3/V versus c/a, where 2a is the length of the longest axis, 2c is the length of the axis that coincides with the axis of rotation, and 2b is the length of the axis perpendicular to a and c, as illustrated in c/a = 10 represents the axial ratios of a sphere. Data points at progressively smaller c/a values represent droplets with progressively larger angular momenta. Also plotted are ratios reported by Cohen et al.c/a, as shown in the figure. Our results are also in reasonable agreement with earlier calculations compiled by Cohen et al.Our initial calculations of the stable equilibrium shapes showed a poor fit with experiment for the most strongly deformed prolate shapes. Subsequently, the numerical calculation was stopped part way through and the mesh was recalculated on the deformed geometry. When this was done, the results of our numerical calculations show good agreement for all values of c/a ≈ 0.67 marks the switch in stability from the axisymmetric equilibrium shapes to tri-axial equilibrium shapes with increasing angular momentum7c/a < 0.65 because, during spin-up with constant airflow, the torque on the droplet changes rapidly immediately after axisymmetry has been broken, as the arms of the droplet catch the airflow like the sails of a windmill. This effect made it difficult to maintain a constant angular velocity in this region of c/a, especially since simulations show that this range of c/a corresponds to a very narrow window of angular momentum.The change in the gradient of the graph at c/a ≤ 0.45 correspond to tektites that have sufficient angular momentum to form a two-lobed shape with an identifiable neck. For these shapes, the dimensions b and c are the dimensions of the neck, not the lobes, as illustrated in c/a ≤ 0.22, or with simulation for c/a < 0.2 owing to the loss of equilibrium at the corresponding angular momentum; experimentally, we observed an accelerating narrowing and rapid pinch-off of the central neck between the two lobes at this point, leading to fission of the two lobes, often accompanied by the production of one or more small satellite droplets between the lobes  forces are taken into account. The surface tension on the outer surface of the drop is assumed constant. Surface tension imparts a normal stress on the outer boundary of the drop that is proportional to the curvature. The equations were solved numerically using the finite-element modeling package Comsol Multiphysics which includes an arbitrary Langrange-Eulerian formulation for deforming meshes. The outer boundary of the mesh was required to move at the fluid velocity. The surface tension induced normal stress was included using the formulation described in Walkley et al. levitated non-rotating wax cubes to obtain precise measurements of magnetic susceptibility2728We used an 18.5 T superconducting solenoid magnet with a vertical, 58 mm diameter bore  to levitate the droplets of wax. The temperature of the air in the bore was approximately 18°C. Prior to levitation, the wax was heated above its melting point (which is in the range 54–58°C) using a hot plate, to a temperature of 180 ± 4°C. The liquid wax was pipetted into the magnet bore at the point in the magnetic field where the wax levitated in stable mechanical equilibrium. Previously, Takahashi


The role of microRNAs (miRNAs) as small non-coding RNAs in regulation of gene expression has been recognized. They appear to be involved in regulation of a wide range of cellular pathways that affect several biological processes such as development, the immune system, survival, metabolism and host-pathogen interactions. Arthropod-borne viruses impose great economic and health risks around the world. Recent advances in miRNA biology have shed some light on the role of these small RNAs in vector-virus interactions. In this review, I will reflect on our current knowledge on the role of miRNAs in arbovirus-vector interactions and the potential avenues for their utilization in limiting virus replication and/or transmission. Flaviviridae (+ssRNA), Togaviridae (+ssRNA), Bunyaviridae (-ssRNA), Rhabdoviridae (-ssRNA) and Reoviridae (dsRNA) . In. InAe. aed cells .Ago1 gene resulted in no change in virus replication suggesting that the miRNA pathway may not have any effect on virus replication. For example, silencing of Ago1 gene in An. gambiae mosquitoes did not change O'nyong-nyong virus replication [Ago1 in Ae. aegypti Aag2 cells had no effect on Semliki Forest virus [Ago2 or Ago3, known to be involved in the RNAi and piRNA pathways, respectively, had significant impacts on virus replication. Given that recent evidence suggests that miRNAs could also be loaded into Ago2 [Drosha or Dicer1 gene, which are not yet known to be involved in the RNAi or piRNA pathways, has any effect on alphavirus replication.In a number of alphaviruses transmitted by mosquitoes, knockdown of lication . Similarst virus  or CHIKVst virus . Howevernto Ago2 ,8,9,10, nto Ago2 . It willAedes taeniorhynchus. However, it is not known how these binding site sequences facilitate virus infection in mosquitoes. Given that miR-142-3p is not produced in insects, it is possible that a mosquito-specific miRNA may interact with the sequences resulting in strong selection for the sequences; although there could be other explanations such as the presence of RNA secondary/tertiary structures. Of relevance, a recent study that analyzed the secondary structures in the 3’UTR region of a number of flaviviruses showed the presence of duplicated RNA structures and numerous repeated sequences in the region that could impede the insect immune response by serving as molecular sponges to sequester host proteins or miRNAs [Viruses can also utilize host miRNAs for their own benefit. For example, in American eastern equine encephalitis virus transmitted by mosquitoes, binding of the mammalian host miR-142-3p to the 3’ UTR of the virus genome is important for tissue tropism and suppression of the host innate immune system . The miRr miRNAs . TherefoAedes mosquitoes when infected with DENV-2 and CHIKV. They may be part of a common response to viral infection or alternatively both manipulated by the viruses. Future research is needed to elucidate the role cellular miRNAs play in arbovirus-vector interactions.So far, we only have a very small number of examples of the impact of arbovirus infections on the miRNA of arthropod vectors and even these mainly provide preliminary information in regards to differential expression of miRNAs which await experimental validations to demonstrate their role in arbovirus-vector interactions. Furthermore, it remains to be determined if the differentially abundant miRNAs following infection are the result of host exploitation by the virus or part of the host response to infection. Another area of interest is to find out how common or different are miRNA changes in a particular vector to different viruses that it transmits and in turn whether the same virus elicits similar changes in different vectors. From the little we know, it appears that miR-2b and miR-1000 are both repressed in The skepticism in regards to the production of miRNAs by RNA viruses, as detailed above, has meant that investigations in this area have been very limited. However, since several studies have shown that the production of mature miRNAs (endogenous or exogenous) from RNA viruses is possible, interest in looking for miRNAs or miRNA-like viral small RNAs from RNA viruses has increased. Arboviruses being mainly RNA viruses fall into this group. So far, there have only been two reports demonstrating production of functional miRNA-like small RNAs from arboviruses, WNV and DENV , althougFlaviviridae family and are exclusively transmitted by mosquitoes. Their genomes consist of a single positive strand RNA of around 11 kb with one long open reading frame encoding three structural proteins involved in the formation of virions and seven non-structural (NS) proteins mainly involved in virus replication but also in virus assembly and suppression of the host antiviral response . Theiewed in , for somiewed in ,57). Froiewed in ,59. In aiewed in ; althougiewed in . FurtherBioinformatics analysis of the WNV sfRNA followed by Northern blot detection and molecular cloning led to the identification of a 21 nt miRNA, KUN-miR-1, from the terminal stem-loop structure (3’SL) in the Kunjin strain of WNV . KUN-miRAe. albopictus) cells prior to virus exposure led to significant increases in DENV-2 replication only in the case of vsRNA-5. This 23-nucleotide vsRNA mapped to the very first stem-loop in the 3’UTR. These small RNAs, including vsRNA-5, appeared in low copy numbers in deep sequencing; hence, vsRNA-5’s significance was questioned suggesting that it is an RNAi degradation product [ in vitro synthesized single stranded precursor stem-loop RNA transfected into mosquito cells, or when the precursor was overexpressed from a plasmid in the cells independent of the virus. In contrast to the effect of the vsRNA inhibitor in improving virus replication, application of the vsRNA-5 mimic led to reductions in DENV-2 replication. Using bioinformatics and deep sequencing analysis, six miRNA-like viral small RNAs (vsRNAs) were identified in DENV-2 infected mosquitoes that mapped to the stem-loops in the 5’UTR (one vsRNA) and 3’UTR (five vsRNAs) of the virus genome . Transfe product . However product . For exa product . FurtherBioinformatics analysis followed by experimental validation showed the presence of target sequences of vsRNA-5 in the non-structural protein 1 (NS1) region. Considering that one long transcript is produced during DENV replication, which consists of structural as well as non-structural proteins, targeting NS1 by vsRNA-5 basically means that the virus genome is targeted. A follow up bioinformatics analysis revealed that vsRNA-5 and its NS1 target sequences might be mainly conserved in DENV serotype 2 and not in the other serotypes .Heliothis virescens ascovirus [–H2-30 and miR-H6 from Heliothis zea nudivirus 1 [In all instances found so far in which virus genes are the targets of virus-encoded miRNAs, the miRNA-virus interaction leads to regulation of virus replication. For example, miR-BART2 from Epstein Barr virus , HvAV-miscovirus , and miRivirus 1  all targDrosophila melanogaster flies and Aag2 cells. Taking a blood meal by an arthropod vector results in massive changes in the expression of a large number of genes, including miRNAs, that are involved in reproduction and blood-meal processes  and 3-fold reduced REL1 levels (negative regulation) as compared with the control mimic-injected mosquitoes. Interestingly, DENV-2 replicated more efficiently in the presence of aae-miR-375 mimic as compared to controls, which suggested that alterations in miRNA levels following blood feeding might have an indirect impact on replication of arboviruses.As demonstrated in gulation . Overallgulation . These c aegypti . Similar aegypti . These wo midgut . The dowlication . In fact-miR-375   that contribute towards this anti-viral property; reviewed recently by Rainey et al. [Wolbachia on virus replication may vary from inhibition [Wolbachia strain, the host , and the virus. Strikingly, a recent study showed that the wAlbB Wolbachia strain enhanced WNV replication in transiently transinfected Culex tarsalis mosquitoes [Ae. aegypti Aag2 cells infected with wMelPop strain of Wolbachia, in which replication of WNV genomic RNA was enhanced as compared to non-infected cells, however, virus secretion and/or assembly in this host (cell line and whole mosquitoes) was inhibited leading to less virion secretion [Considering the lack of available vaccines and drugs against most arboviruses, management of the diseases caused by arboviruses has heavily concentrated on controlling vector populations or inhibiting virus-transmission by the vectors. Issues with chemical insecticides such as the development of resistance in vector populations, affecting non-target insects and environmental contamination have made non-chemical control alternatives more attractive. These include application of microbial control agents such as entomopathogenic bacteria and fungi, or utilization of symbionts.  species ,88. Due pulation . In two nogaster ,91. Thisexplored . Notablynhibited . Due to iewed in ). Althouy et al. . It is whibition ,91,93, nhibition  to even hibition  dependinsquitoes . This isecretion . Ae. aegypti mosquitoes with and without wMelPop Wolbachia infection revealed that about 13 miRNAs were differentially expressed in Wolbachia-infected mosquitoes. The mosquito-specific miRNA, aae-miR-2940, was highly induced and exclusively detected  in Wolbachia-infected mosquitoes. Initially, MetP was determined as one of the targets of the miRNA and was shown to be important for replication/maintenance of Wolbachia [Wolbachia density. Interestingly, in a subsequent study it was found that MetP enhances WNV replication in Aag2 cells [Wolbachia-infected mosquitoes and mosquito cell lines [Wolbachia induces MetP expression via induction of aae-miR-2940  undergo rapid, positive and diversifying selection, and that refractoriness of mosquitoes to DENV infection positively correlates with nucleotide diversity indices in the Dicer-2 gene [Based on evolutionary studies carried out in ath rate . New miRath rate . Althougr-2 gene . This maArboviruses of medical and veterinary importance continue to impose large numbers of deaths, health risks and financial losses around the world. In many instances, there are no effective drugs or vaccines available for diseases caused by arboviruses. Despite many groups around the world working on arboviruses, there are still many gaps in our knowledge in regards to the interaction of arboviruses with their hosts, in particular, the arthropod vector. Deeper understanding of molecular mechanisms regulating interplaying events such as blood-feeding, reproduction and anti-viral responses may lead to development of new approaches in arbovirus control through blocking/limiting transmission of arboviruses by arthropod vectors. MiRNAs have appeared as key molecules in the regulation of gene expression at both transcriptional and post-transcriptional levels, playing important roles in development and response of animals to environmental stimuli. Current knowledge, although limited, points to the fact that miRNAs are involved in the regulation of various aspects of arbovirus-vector interactions. However, we have only scratched the surface. Many differentially expressed miRNAs upon blood feeding, exposure to arbovirus infections, and at different developmental stages, have been identified, but their significance in those events or interactions have not been experimentally tested. Considering the short length of miRNAs and the absence of host immune responses to miRNA, they are ideal for incorporation into innovative strategies to control arboviruses; for example, generating genetically modified vectors that are refractory to virus infection by overexpressing miRNAs that boost the host anti-viral response or directly target arboviruses. Wolbachia in arthropod vectors has provided a unique opportunity to explore the endosymbiont in control of arboviruses and in fact other arthropod-borne pathogens [The discovery of the virus blocking property of athogens ,104; altathogens . Explora


In Ohio, United States, in early 2014, a deltacoronavirus was detected in feces and intestine samples from pigs with diarrheal disease. The complete genome sequence and phylogenetic analysis of the virus confirmed that the virus is closely related to a porcine deltacoronavirus (porcine coronavirus HKU15) reported in Hong Kong in 2012. Nidovirales, family Coronaviridae, subfamily Coronavirinae) are single-stranded, positive-sense, enveloped RNA viruses with a genome size ranging from 25.4 to 31.7 kb , as described by the International Committee for Taxonomy of Viruses (http://www.ictvonline.org/virusTaxonomy.asp?msl_id = 27). Virus from each coronavirus genus has been found in diverse host species, including mammals and birds  was lower than that typically observed with PEDV infection. Test results for samples from Farm 1 were negative for PEDV, transmissible gastroenteritis virus, rotavirus, and http://blast.ncbi.nlm.nih.gov/Blast.cgi) search of the sequences of both M and N fragments showed 99% nt identity with the porcine coronavirus HKU15 (PorCoV HKU15)-155. Therefore, the virus detected in this study was PorCoV HKU15, which belongs to the Deltacoronavirus genus.Because deltacoronavirus has been reported in pigs . NucleotRT-PCR was then run on samples from Farms 2–5. The results from the 5 farms are summarized in On the basis of 2 complete genome sequences from GenBank, of PorCoV HKU15-44 and HKU15-155, we designed 16 pairs of primers to determine the whole genome of extracted RNA samples (OH1987) from Farm 1 . The genPhylogenetic analysis of the complete genome of PorCoV HKU15 OH1987 showed that the OH1987 strain clustered with the other 2 PorCoVs, HKU15-155 and HKU15-44, and was distinct from the bird deltacoronaviruses . In addiDeltacoronavirus, when and how this virus was introduced into United States remain unknown. Further investigation is needed to determine whether infection with PorCoV HKU15 results in disease in pigs and if the virus was responsible for the clinical disease observed in outbreaks on the 5 Ohio farms in this study. In addition, surveillance should be conducted in other US states to define the distribution of the virus among the US pig population. Moreover, whole-genome sequence analysis should be performed for other strains from different locations to determine whether the virus was introduced into the United States by a single entry or by multiple entries.We detected a porcine deltacoronavirus in pigs in the United States. Although the genetic and phylogenetic analyses showed that the newly emergent strain, PorCoV HKU15 OH1987, was closely related to 2 strains from China, HKU15-155 and HKU15-44, in the genus


Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus.We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid,  Diaphorina citri, tentatively named Diaphorina citri-associated C virus (DcACV) (D. citri transcriptome and confirmed by reverse transcription-PCR (RT-PCR) followed by Sanger sequencing. The nucleotide sequences of both ends of the genomic RNAs were determined by rapid amplification of cDNA ends (RACE) using the SMARTer 5′/3′ RACE system according to the manufacturer’s instructions . The complete sequences of the DcACV genomic RNAs are 2,376 and 1,817 nucleotides (nt) for RNA1 and RNA2, respectively, and both RNAs are nonpolyadenylated. The 5′ and 3′ untranslated regions (UTRs) of RNA1 are 180 and 184 nt, respectively, flanking two predicted overlapping open reading frames (ORFs). ORF1 (nt 181 to 1083) and ORF2 (nt 654 to 2192) encode putative proteins of 300 and 512 amino acids, respectively. RNA2, which contains the 5′ and 3′ UTRs of 127 and 140 nt, respectively, encodes two putative proteins of 161 and 398 amino acids from predicted overlapping ORF1 (nt 128 to 613) and ORF2 (nt 481 to 1677), respectively. The first 17 nucleotides of both 5′ UTRs are identical.In our previous study, we reported partial sequences of a novel positive-sense, bipartite RNA virus in the Asian citrus psyllid,  (DcACV) . Here, wScaptodrosophilla deflexa named Tartou virus (accession no. AMO03231)  (accession no. YP001911140), the causal agent of the chronic bee paralysis disease in honeybees around the world  and contains a conserved GDD motif. However, no significant similarities were found between the other DcACV deduced amino acid sequences and the known protein sequences in databases. BLASTp analysis of the putative RdRp encoded by ORF2 of RNA1 showed the highest significant amino acid sequence identity  to a recently discovered virus of KX235518 and KX235519 for RNA1 and RNA2, respectively.The complete genome sequences of DcACV genomic RNAs have been deposited in GenBank under the accession numbers


Influenza A and B viruses possess a neuraminidase protein that shows sialidase activity. Influenza virus-specific neuraminidase inhibitors (NAIs) are commonly used for clinical treatment of influenza. However, some influenza A and B viruses that are resistant to NAIs have emerged in nature. NAI-resistant viruses have been monitored in public hygiene surveys and the mechanism underlying the resistance has been studied. Here, we describe a new assay for selective detection and isolation of an NAI-resistant virus in a speedy and easy manner by live fluorescence imaging of viral sialidase activity, which we previously developed, in order to achieve high-efficiency capture of an NAI-resistant virus. An NAI-resistant virus maintains sialidase activity even at a concentration of NAI that leads to complete deactivation of the virus. Infected cells and focuses (infected cell populations) of an oseltamivir-resistant virus were selectively visualized by live fluorescence sialidase imaging in the presence of oseltamivir, resulting in high-efficiency isolation of the resistant viruses. The use of a combination of other NAIs  in the imaging showed that the oseltamivir-resistant virus isolated in 2008 was sensitive to zanamivir and laninamivir but resistant to peramivir. Fluorescence imaging in the presence of zanamivir also succeeded in selective live-cell visualization of cells that expressed zanamivir-resistant NA. Fluorescence imaging of NAI-resistant sialidase activity will be a powerful method for study of the NAI resistance mechanism, for public monitoring of NAI-resistant viruses, and for development of a new NAI that shows an effect on various NAI-resistant mutations. Influenza viruses circulate worldwide and can affect every year up to 10% of the world’s population .Neuraminidase (NA) is a major surface glycoprotein of the influenza A and B viruses and expresses on the surface of the virus-infected cell at a high amount. NA shows sialidase activity that hydrolyzes the bond between galactose and terminal sialic acid in the glycochain. The sialidase activity of NA is critical for the virus replication cycle and therefore has been considered a suitable target for designing agents against influenza viruses. NA inhibitors (NAIs), which are sialic acid analogues and specific competitive enzymatic inhibitors against influenza virus NA, are clinically used for prevention and treatment of influenza –5. Sever50) value of each NAI can be determined and susceptibilities to NAIs can be compared. However, an enzymatic assay involves troublesome procedures for hygiene workers who must examine numerous samples, such as procedures for measurement of sialidase activity and for growth and isolation of a virus. A genotypic assay is used to detect a mutation of the NA gene that confers NAI resistance. Most genotypic assays are used for detection of H275Y conferring oseltamivir resistance. Genotypic assays are generally used in laboratories of environmental hygiene including regional laboratories because they easily enable highly sensitive detection of an NAI-resistance mutation directly from clinical samples or from virus cultures by genetic analysis and/or real-time PCR of the NA gene. However, the application of a genotypic assay is limited to already known mutations and cannot be used to evaluate NAI resistance attributed to unknown mutations. In addition, a genotypic method cannot be used to compare NAI susceptibilities such as IC50 values. In the case of a mixture of NAI-sensitive and NAI-resistant viruses, each virus strain must be isolated by the conventional plaque-forming assay before the enzymatic assay or genotypic assay [Methods for detection of NAI-resistant viruses can be divided into enzymatic assays and genotypic assays of viral NA , 20, 22.ic assay –28. Thusd-glycero-d-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac) [d-N-acetylneuraminic acid (Neu5Ac) by sialidases from viruses including influenza A and B viruses, the product BTP3 is a benzothiazolylphenol-based water-insoluble fluorescent compound [We previously developed a sialidase fluorescence imaging reagent, 2-(benzothiazol-2-yl)-4-bromophenyl-5-acetamido-3,5-dideoxy-α--Neu5Ac) , 30. BTPcompound . BTP3 locompound , 32–34. Cells were obtained from ATCC. Madin-Darby canine kidney (MDCK) cells (ATCC-CCL-34) were grown in a minimum essential medium (MEM) supplemented with 5%fetal bovine serum (FBS). African green monkey kidney COS7 cells  were grown in a Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS. Influenza A virus strains, oseltamivir-sensitive A/Shizuoka/838/2009 (H1N1pdm) (838) and oseltamivir-resistant A/Shizuoka/738/2008 (H1N1) (738), were grown in MDCK cells. Hemagglutination units (HAU) of the viruses were determined as described previously . HAU werVirus titers were determined by a plaque-forming assay . Virusesd-N-acetylneuraminic acid (4MU-Neu5Ac) or 1 mM BTP3-Neu5Ac was added on ice and then incubated at 37°C for 1 hr. Enzyme reaction for 4MU-Neu5Ac was stopped by 50 μl of 100 mM sodium carbonate buffer (pH 10.7). Fluorescence from sialidase reaction was quantified by using a microplate reader  with wavelengths of excitation and emission at 355/460 nm for 4-metylumbelliferone (4MU) and at 372/526 nm for BTP3. The concentration of NAI that reduced NA enzyme activity by 50% relative to a control mixture with no NAI was determined as IC50 by plotting the percent inhibition of the activity at respective NAI concentrations using GraphPad Prism 5 software .Viruses were standardized to equivalent NA enzyme activity in the linear range of the curve. Forty microliters of virus suspension in PBS was mixed with 5 μl of ten-fold dilutions of oseltamivir or distilled water only on a 96-well black plate . The mixture was incubated at 37°C for 15 min. Five microliters of 1 mM 2'-(4-methylumbelliferyl)-α-4 cells/well) were cultured for 24 hr. The cells were infected with oseltamivir-resistant 738 or -sensitive 838 at a multiplicity of infection (MOI) of 0.01 in 250 μl of an SFM and incubated at 37°C for 12 hr. After removing the supernatant, the infected cells were incubated with 150 μl of 20 μM BTP3-Neu5Ac with or without oseltamivir or zanamivir  in an SFM at 37°C for 10 min. Fluorescent images of the cells were observed under UV irradiation by using an IX71 fluorescence microscope  equipped with fluorescent filters .MDCK cells on a 48-well plate  according to the manufacturer’s instructions and incubated for 24 hr at 37°C. After removing the supernatant, the infected cells were incubated with or without 100 nM oseltamivir or 100 nM zanamivir in an SFM at 37°C for 5 min. Then, BTP3-Neu5Ac  was added to cell culture medium at 37°C for 5 min. Fluorescent images of the cells were observed under UV irradiation by using an IX71 fluorescence microscope equipped with fluorescent filters .Site-specific mutations of NA genes were introduced as described previously . The mutA monolayer of MDCK cells on a 12-well plate was infected with oseltamivir-resistant 738 or -sensitive 838 at 20 pfu/well in 500 μl of an SFM at 37°C for 30 min and cultured in 2 ml of an SFM containing 0.5% agarose and 2 μg/ml acetylated trypsin. After 2 days at 37°C, 50 μl of 2 mM BTP3-Neu5Ac with each NAI , oseltamivir, zanamivir, peramivir, and laninamivir, was added onto the overlaid agarose-containing SFM. After 1 hr at 37°C, fluorescent images of the plate were observed on a UV Transilluminator  at 365 nm.Mixtures of oseltamivir-resistant 738 and -sensitive 838 were prepared at three ratios of 100:0, 20:80, and 4:96 in virus infectious titers, respectively. A monolayer of MDCK cells in a 6-well plate was infected with each mixture (200 pfu/well) at two wells, respectively. Cells were infected at 37°C for 30 min and cultured in 4 ml of an SFM containing 0.5% agarose and 2 μg/ml acetylated trypsin. After 2 days at 37°C, 100 μl of 2 mM BTP3-Neu5Ac with or without oseltamivir  was added onto the overlaid agarose-containing SFM. After 1 hr at 37°C, fluorescent images of the plate were observed on a UV Transilluminator at 365 nm.A mixture of oseltamivir-resistant 738 and -sensitive 838 was prepared at a ratio of 1:1 in virus infectious titers. A monolayer of MDCK cells in a 6-well plate was infected with the mixture (40 pfu/well). Cells were infected at 37°C for 30 min and cultured in 4 ml of an SFM containing 0.8% agarose and 2 μg/ml acetylated trypsin. After 2 days at 37°C, 100 μl of 2 mM BTP3-Neu5Ac with or without oseltamivir  was added onto the overlaid agarose-containing SFM. After 1 hr at 37°C, fluorescent images of the plate were observed on a UV Transilluminator at 365 nm.Viruses were isolated from eight fluorescent focuses in live-focus fluorescence imaging in the presence of oseltamivir. The isolates were grown in MDCK cells within 3 passages until RNA extraction. The experiment was repeated three times .™ One Step RT-PCR Kit Ver. 2  with the following primer pairs: 08seasonNA-275Y-F (5’-GAGTTGAATGCACCCAATTTTTAT-3’) and UniNA-R (5’-TATTGGTCTCAGGGAGCAAAAGCAGGAGT-3’) for 275Y detection of 738, and 09pdmNA-275H-F (5’-AAATGAATGCCCCTAATTATCAC-3’) and UniNA-R for 275H detection of 838. RT-PCR were performed by using a TP600 PCR machine  with 1 cycle at 50°C for 30 min and 1 cycle at 94°C for 2 min (RT), 28 cycles at 94°C for 30 sec, 58°C for 30 sec, and 72°C for 45 sec (PCR), and 1 cycle at 72°C for 45 sec. The PCR products were analyzed by 1% agarose gel electrophoresis.Viral RNA was extracted from virus cultures by using RNeasy mini Kit  according to the manufacturer’s instructions. To distinguish genes of oseltamivir-resistant 738 and -sensitive 838, oseltamivir resistance mutation of H275Y (based on N1NA amino acid numbering) in NA was detected by one-step RT-PCR using PrimeScript50 values of oseltamivir using BTP3-Neu5Ac. IC50 values (means ± standard deviations) of oseltamivir for oseltamivir-resistant 738 and -sensitive A/Shizuoka/838/2009 (H1N1pdm09) (abbreviated as 838) were 175.7 nM  and 1.122 , respectively  and 0.8393 , respectively , was used in this study for detection for oseltamivir-resistant influenza A virus A/Shizuoka/738/2008 (H1N1) (abbreviated as 738), which was an epidemic virus in nature during the 2008–2009 season in Japan. We have reported that zanamivir, one of influenza A virus- and B virus-specific NAIs, inhibited live-cell fluorescence imaging of influenza A virus-infected cells for BTP3-Neu5Ac usage . Howeverectively . We alsoectively . IC50 vaWe examined selective fluorescent visualization of oseltamivir-resistant virus-infected cells using BTP3-Neu5Ac with oseltamivir. MDCK cells were infected with oseltamivir-resistant 738 or -sensitive 838. The infected cells at 12 hr postinfection were incubated with BTP3-Neu5Ac with or without oseltamivir or zanamivir  at 37°C for 10 min . OseltamTo examine whether the live-cell fluorescence imaging can be applicable to zanamivir resistance, we gave NA of zanamivir-sensitive virus A/USSR/92/1977 (H1N1) the property of zanamivir resistance by amino acid substitution of Q136K , 12. BTPWe demonstrated that BTP3-Neu5Ac could detect NAI-resistant virus-infected cells. Some virus cultures and clinical samples are thought to contain a variety of NAI-resistant and -sensitive mutants, because of rich variations and liable mutations as a property of influenza virus. A mixture of NA-resistant and -sensitive viruses should show sialidase activity to some extent even in the presence of an NAI. In this case, it is necessary to determine whether a virus sample contains only an NAI-resistant virus or whether it contains various mutants and to efficiently isolate the NAI-resistant virus if NAI-resistant viruses were confirmed in the sample. We have developed live-focus fluorescence imaging that can fluorescent-visualize influenza A virus-forming focuses in a plaque-forming assay using BTP3-Neu5Ac, which is applicable to virus titration and virus isolation , 29. TheIn addition, we tried selective detection and isolation of an NAI-resistant virus from a mixture of NAI-resistant and -sensitive viruses. MDCK cells were infected with mixtures of oseltamivir-resistant 738 and -sensitive 838 at different ratios of virus titers. The cells were cultured in an SFM containing 0.8% agarose and 2 μg/ml acetylated trypsin for 2 days. All focuses were fluorescent-visualized by adding BTP3-Neu5Ac onto the overlaid agarose-containing SFM . The numWe have developed a novel sialidase substrate, BTP3-Neu5Ac, for sensitive, rapid, and easy fluorescence imaging of cells infected with influenza A and B viruses. Influenza A virus has various antigenic subtypes (1–18 in HA and 1–11 in NA)  and ther50 values of oseltamivir in viral sialidase activity using 4MU-Neu5Ac were similar to those using BTP3-Neu5Ac. IC50 values reported in many previous studies using 4MU-Neu5Ac can be referred to for the determination of the appropriate concentration of NAI. Live-cell fluorescence imaging by BTP3-Neu5Ac with an NAI enables detection of an NAI-resistant virus at the cellular level, even very small titers of virus that cannot be detected by using 4MU-Neu5Ac. Moreover, after observation of fluorescent infected cells of an NAI-resistant virus, we can readily proceed to the virus culture.In surveillance and research of influenza virus sensitivity to NAIs, 4MU-Neu5Ac is most commonly used as a commercial sialidase substrate –43. SincPlaque-forming assay is a conventional virological method to measure infectious titers and to isolate a virus strain. Plaque-forming ability of a virus is generally dependent on the virus strain and cell type. Laboratory virus strains used in many previous studies have a tendency to form larger plaques. When such laboratory strains were used, plaques of an NAI-resistant virus are also selectively visualized by virus culture in an overlaid agarose-containing medium with an NAI, and the plaques have been usually used for isolation of an NAI-resistant virus , 45. HowIsolation of NAI-resistant viruses is important for analysis of NAI resistance mutations. An NAI-resistant virus can be high-efficiently isolated from the fluorescent focus by live-focus fluorescence imaging using BTP3-Neu5Ac with an NAI. In the present study, selective isolation of an oseltamivir-resistant virus from mixtures of oseltamivir-resistant 738 and -sensitive 838 was also successfully performed by live-focus fluorescence imaging with oseltamivir. A representative method on identification of various NAI resistance mutations is to generate escape virus mutants through successive virus cultures in the presence of an NAI. Isolation of NAI-resistant escape mutants is essential to analyze mutations in the NA gene of each virus strain. For such study, live-focus fluorescence imaging by BTP3-Neu5Ac with an NAI is the most useful method to efficiently isolate NAI-resistant escape mutants selectively from virus cultures containing NAI-sensitive mutants and parent viruses. Counting the number of fluorescent focuses with and without NAI will also enable calculation of ratios of NAI-resistant and -sensitive viruses in clinical samples and virus cultures.As shown in Oseltamivir-resistant 738 was detected in all of 24 fluorescent focuses by live-focus fluorescence imaging with oseltamivir. On the other hand, oseltamivir-sensitive 838 was detected in 2 of 24 fluorescent focuses by the imaging, indicating contamination of 838 to 738 isolates. A probable reason is that an invisible focus of oseltamivir-sensitive 838 overlapped with a fluorescent focus of oseltamivir-resistant 738. In such a case, use of lower titers of a virus is supposed to reduce the risk of overlapping among focuses. The use of an agarose-containing medium with an NAI will reduce the focus size of an NAI-sensitive virus through inhibition of the growth of an NAI-sensitive virus. These methods would improve the efficiency of selective isolation of an NAI-resistant virus by live-focus fluorescence imaging.We conclude that the use of BTP3-Neu5Ac with an NAI enables selective detection of an NAI-resistant virus at the levels of virus culture and infected cells (focus) in a sensitive, rapid, and easy manner. It is a powerful tool for public health workers who handle numerous samples and for researchers studying resistance mechanism of NAIs. It is also a useful tool for the development and screening of a novel NAI that has little risk of resistance acquisition and maintains effectiveness against NAs with various NAI-resistance mutations.S1 Fig(A) Viral RNA was extracted from virus cultures of oseltamivir-resistant 738 and -sensitive 838 strains. NA genes were amplified by RT-PCR from each viral RNA with UniNA-R primer and specific primers (08seasonNA-275Y-F and 09pdmNA-275H-F) for 275H or 275Y detection. (B) MDCK cells on a 6-well plate were infected with a mixture of oseltamivir-resistant 738 and -sensitive 838 strains at a ratio (%) of 1: 1 in terms of virus titers (pfu) . The cells were cultured in an SFM containing 0.8% agarose and 2 μg/ml acetylated trypsin. After 2 days at 37°C, 100 μl of 2 mM BTP3-Neu5Ac with or without oseltamivir  was added onto the overlaid agarose-containing SFM. After 1 hr at 37°C, fluorescence images of the plate were observed under UV irradiation at 365 nm. Selective live-focus fluorescence imaging of oseltamivir-resistant 738 from the virus mixture was repeated three times by the same experimental method. Fluorescent images from each experiment are shown as “1st experiment”, “2nd experiment” and “3rd experiment”. Eight isolates per an experiment were obtained from fluorescent focuses using live-focus fluorescence imaging with oseltamivir. To distinguish genes of oseltamivir-resistant 738 and -sensitive 838 strains, 275H for 838 and 275Y for 738 in an NA amino acid difference were detected by RT-PCR from viral RNA of each isolate.(DOC)Click here for additional data file.


Childhood overweight and obesity have health and economic impacts on individuals and the wider society. Families participating in weight management programmes may foresee or experience monetary and other costs which deter them from signing up to or completing programmes. This is recognised in the health economics literature, though within this sparse body of work, costs to families are often narrowly defined and not fully accounted for. A societal perspective incorporating a broader array of costs may provide a more accurate picture. This paper brings together a review of the health economics literature on the costs to families attending child weight management programmes with qualitative data from families participating in a programme to manage child overweight and obesity.A search identified economic evaluation studies of lifestyle interventions in childhood obesity. The qualitative work drew on interviews with families who attended a weight management intervention in three UK regions.We identified four cost-effectiveness analyses that include information on costs to families. These were categorised as direct (e.g. monetary) and indirect (e.g. time) costs. Our analysis of qualitative data demonstrated that, for families who attended the programme, costs were associated both with participation on the scheme and with maintaining a healthy lifestyle afterwards. Respondents reported three kinds of cost: time-related, social/emotional and monetary.Societal approaches to measuring cost-effectiveness provide a framework for assessing the monetary and non-monetary costs borne by participants attending treatment programmes. From this perspective, all costs should be considered in any analysis of cost-effectiveness. Our data suggest that family costs are important, and may act as a barrier to the uptake, completion and maintenance of behaviours to reduce child obesity. These findings have implications for the development and implementation of child weight initiatives in particular, in relation to reducing inequalities in health. Childhood overweight and obesity carry significant costs to individuals, families and wider society. Obesity in children is a major public health concern, with obese children and adolescents at increased risk of health and social problems in childhood and as adults .The globThe economic costs of obesity are significant. A systematic review of 32 articles published between 1990 and 2009 estimated that obesity accounted for between 0.7% and 2.8% of an average country’s total healthcare expenditure. Obese individuals were found to have medical costs approximately 30% greater than their normal weight peers . FiguresThere is growing interest in interventions to reduce the prevalence of childhood overweight and obesity and—given demands on health care services—increased interest in the cost-effectiveness of interventions to ascertain whether these interventions represent good value for money.When assessing the cost-effectiveness of public health interventions, a societal approach to measuring costs  may be mall costs should be identified and entered into the analysis of cost-effectiveness.When evaluating the cost and cost-effectiveness of health care programmes to reduce child overweight and obesity, health economics studies tend not to take account of the costs, financial and otherwise, carried by participating families. This may be because they are harder to measure  or because they are considered unimportant if a health service or public sector perspective is taken . Yet sucThe aim of this paper is to describe different kinds of costs—monetary and otherwise—incurred by families participating in a child weight management programme. In doing so, we contextualise qualitative data drawn from an empirical study , 12, 13 An online literature search was performed using PubMed and the NHS Economic Evaluation Database [NHS EED] to identify economic evaluation studies of lifestyle interventions in childhood obesity. To be included in the review studies had to (1) evaluate a family-based lifestyle intervention programme (2) in relation to overweight and/or obese children, and (3) investigate the impact of the programme on costs borne by parents/families. Search terms used in combination with ‘child/childhood obesity’ were “lifestyle intervention”, “parent”, “family”, “cost”, “cost-effectiveness”, “economic evaluation”.Searches were limited by date from 1990 until April 2014 and to articles available in the English language. Additional studies were identified using reference lists and PubMed related citations.The search of PubMed and the NHS EED provided a total of 256 citations . After aWe identified four papers reporting findings from three studies. Three fully met our inclusion criteria; the fourth , whilstWe provide both a PRISMA checklist  14], no, no14], Two Australian papers were identified. The first of these  assessedThe second report  estimateThe other two papers reported findings from two UK-based studies. One  evaluateThe second UK study  assessedThe heterogeneity of cost components, type of lifestyle interventions, age of the children participating and methodologies used to obtain final results did not allow an overall synthesis of the data reported in these studies. However, across the four studies, costs to families could be either direct (e.g. monetary) or indirect (e.g. time) and could be associated with either taking the child to the programme and/or helping the child attain and maintain a healthy weight.Below, we explore the scope and type of costs incurred by families drawing on qualitative data from a mixed methods evaluation of a family-based intervention to address overweight and obesity in children initially shown to be useful in a trial , 12, 13,Our sampling strategy for the qualitative data aimed to maximise variation across multiple deprivation at the small area level, family structure, parent-reported participant ethnicity, housing tenure and completion status of a weight management programme. In addition, we purposively selected two rural families. Full methodological details are available elsewhere , 12, 13.The data were collected in three locations in England . A sample of families who had participated in a weight management programme were interviewed and asked about their experience of attending the scheme (N = 23). The family groups varied in size and composition, but every group contained at least one adult and one child. Some groups were multi-generational, with children, parents and grandparents present. Respondents were asked about referral to the intervention, their experiences on the programme, weight loss during and after participation and any costs they had incurred. ‘Costs’ were not defined but the researchers indicated that these could be monetary or non-monetary. The qualitative dataset was analysed using Framework analytic techniques .UCL Ethics Committee granted approval for this study in February 2011 (REF: 2842/001). We conducted the project with reference to the framework for research ethics produced by the Economic and Social Research Council. Written informed consent was obtained from all participants. The interview material reported here is identified only by a family (F) number, and whether a child, mother or father is speaking.For the families who attended the programme, costs were associated with participation and maintaining a healthy lifestyle post-intervention. Respondents reported three types of cost which were frequently inter-related: time, social/emotional and monetary. Time-related costs of attendance were primarily about fitting a programme with twice weekly attendance into busy working and family lives and school. Families changed working patterns and shifts in order to attend and sometimes travelled long distances. For one North East England-based family, for example, the nearest programme would have been hard to get to in rush hour so they went to one 16 miles away which meant that the mother had to take time off work (F21). Undertaking an extra activity could be difficult for children with other after-school commitments including religious observance and sports activities. For parents, understanding employers or flexible working hours could help in taking children to the programme, but not all were able to do this.The emotional and social costs of participating were rarely identified as such by families. However, it was evident that families had had to make changes during and after the intervention, ones which were related to lack of time and exerted a social or emotional toll. Parents, usually mothers, spoke of the strain on their children. For some children, choosing the healthy option meant being ‘different’ or missing out on other opportunities. The changes needed for success on the programme could create friction between parents and children, exposing fragile relationships and threatening family dynamics. One mother spoke about the damage she felt had been done to her relationship with her daughter by spending her teenage years fighting about food. Her daughter acknowledged that her diet was her choice and responsibility while simultaneously blaming her mother for giving her bad habits and allowing her to make unhealthy choices. Both mother and daughter felt they were to blame. Tensions and contradictions were evident in some family discussions with participants wanting to ‘do the right thing’ in relation to weight management. Aside from differences between respondents in recollection of programme logistics, such as timing and frequency, most differences arose from assertions or defences of moral character, or challenges to someone else’s, in relation to behaviour or weight. For example, one child contested his mother’s claim that they visited the sports centre to check for activities at the beginning of last month, saying that it was more than six months ago . Several children disagreed with their parents that they had regained weight lost on the programme. More than one Bangladeshi or Pakistani family described moving to what they described as ‘non-Asian’ foods. A perception of ‘Asian’ food as unhealthy implies a change that—in part—rejects cultural norms and identity. This conflict between healthy eating and traditional practices has been noted by others  and evokRespondents were asked if they had incurred monetary costs while participating on the programme or after it had finished. Few explicitly did so. While many of the barriers to attending the programme described by parents would have resulted in costs, they were not usually identified as such by families. Time off work, childcare and, in particular, driving to the venue carried costs but were often described as ‘normal’ costs. Additional costs included trips to the cinema if targets were met, or joining children up to exercise classes. Those who did identify costs usually related them to switching to ‘healthier’ foods, with respondents describing the more expensive, ‘healthier’ food as beyond the reach of families on a low income. Some discussed the fact that low cost ranges and low cost supermarkets do not include the healthier alternatives they had been encouraged to buy while on the programme.Most analyses of the cost-effectiveness of childhood obesity interventions do not include the costs borne by families during or after participation in the programme. We identified four studies from the health economics literature that did include this detail, and analysis of these showed that, when costs borne by families have been included, these tend to be significant and are largely about time and money.These findings are supported by qualitative data collected during an evaluation of a family-based child weight management programme. While parents and carers, as well as children often reported enjoying the programme, families struggled to fit it into work and school schedules, or combine it with post-school activities. Respondents also reported emotional and social costs as attempts to maintain new diet or exercise behaviours impacted on family dynamics and relationships, or exposed tensions between family members. These costs could also be incurred or experienced beyond the family; some children and parents reported social embarrassment about attending the scheme, and some parents initially viewed walking through the door as an implicit admission of parental culpability. Whilst families rarely referred to direct monetary costs as a result of being on the scheme, respondents did speak of the higher costs of ‘healthier’ foods and, in some cases, this had deterred them from purchasing these foodstuffs.To enhance generalizability and quality, qualitative data were collected in several English locations by experienced qualitative researchers. We recruited a  broad array of respondents, and attempted to include those who had declined the intervention or had minimal participation. However, the latter proved difficult to achieve and our respondents were largely programme participants who had stayed the course and may, therefore, have incurred fewer costs and experienced more benefits than those who had dropped out or failed to attend even one session. There was, in some cases, a significant time lag between attendance on the programme and the collection of evaluation data, so that some respondents could not accurately recall the scope or type of costs incurred during or after the programme.In summary, our data suggests that family costs are an important cost component and may act as a barrier to the successful uptake and completion of childhood lifestyle interventions to reduce obesity. We make four observations here aimed at those commissioning and designing child weight management programmes.First, a societal perspective that utilises direct and measurable, as well as indirect time, social and emotional, costs to families should be the default analytic approach in the assessment of the cost-effectiveness of child weight management programmes. NICE recently produced a costing template to estimate the financial impact to the NHS of implementing guidance on tackling childhood overweight and obesity in England  designedSecond, as with all NICE costings approaches, the national assumptions used in the template described above can be altered to reflect local circumstances. Those working at the service frontline are well-placed to consider this. While it is unlikely that a metric could be produced, this is an area where local ‘know-how’ as a form of knowledge is crucial to help mitigate the type of time, emotional and monetary costs identified in our analysis. The Localism Bill  has granThird, although we refer throughout to families, the time, practical, emotional and culinary work was highly gendered, falling disproportionately to mothers. This is likely to be the case with a whole range of parenting and family-based interventions. Creative ways of mitigating these costs need to be considered, in particular in relation to reducing inequalities in child health.Finally, there is a socio-economic gap in childhood overweight and obesity. Trial and cost data with relatively short term follow-up may not capture the information needed to successfully implement programmes on a large scale and maintain gains. Data such as those in this paper can provide practical pointers to the kinds of implementation of programme design and delivery that may be more feasible for the most disadvantaged families.S1 Fig(ZIP)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Training the bench press exercise on a traditional flat bench does not induce a level of instability as seen in sport movements and activities of daily living. Twenty participants were recruited to test two forms of instability: using one dumbbell rather than two and lifting on the COR bench compared to a flat bench. Electromyography (EMG) amplitudes of the pectoralis major, middle trapezius, external oblique, and internal oblique were recorded and compared. Differences in range of motion (ROM) were evaluated by measuring an angular representation of the shoulder complex. Four separate conditions of unilateral bench press were tested while lifting on a: flat bench with one dumbbell, flat bench with two dumbbells, COR Bench with one dumbbell, and COR Bench with two dumbbells. The results imply that there are no differences in EMG amplitude or ROM between the COR bench and traditional bench. However, greater ROM was found to be utilized in the single dumbbell condition, both in the COR bench and the flat bench. Both sport and various activities of daily living (ADLs) require individuals to create pushing forces while standing. Most noticeably, this is seen in highly physical sports, like football or wrestling, but the same relative movement is seen when someone pushes a car or moves furniture around their home . TraditiFirst, in a supine bench press, either a bar or two free weights being used simultaneously create the resistance in the exercise, making it a bilateral movement. Unfortunately, in most sports and ADLs, movements are predominantly, or entirely, unilateral . Thus, tSecondly, there are many factors that limit one’s ability to develop pressing strength while standing, including stability and neuromuscular control of the core musculature, the individual’s weight, the base of support the individual utilizes, and the direction in which the pressing motion is performed . Since aAdditionally, when performing a bilateral, seated pressing motion, the EMG amplitude of core stabilizers, such as the rectus abdominis and external obliques, is reduced significantly when compared to a standing, unilateral pressing motion . Since mOne final drawback to the bench press is the limitation of a full range of motion (ROM). It is possible that the flat surface of the bench, on which a bench press is completed, may limit an individual’s ROM depending on their shoulder width. If individuals are only completing the bench press movement to parallel, they are likely not training through their full ROM. Previous research indicates that training should occur through a person’s entire functional ROM .Because traditional exercises like the bench press do not translate well into standing pressing strength, it is the goal of many researchers today to find ways to make the bench press more similar to an actual movement that would be performed outside of the gym. Researchers have found that exercising on unstable surfaces can increase electromyographic (EMG) amplitude of the core musculature, such as the rectus abdominis , externaTo create an unstable environment, some researchers simply use equipment designed to introduce instability to an exercise. One such piece of equipment is the CÖR™ Bench (COR Bench). The COR Bench is an inflatable 400-denier nylon urethane tube that is supported with a stand much like a normal bench. It is the combination of the rounded surface of the COR Bench and the fact that it is filled with air  that create an unstable environment for the individual, which may, in turn, increase both ROM and activation; however, this has yet to be substantiated. Being able to work through a larger ROM and targeting more muscles for stabilization can provide greater neurological and neuromuscular stimulation .Therefore, the purpose of this study was to examine the following questions: does performing a chest press on the COR bench elicit greater EMG amplitudes of the core muscles, and possibly the prime movers, when compared to a traditional bench? Will a greater ROM be used on the COR bench than the traditional flat bench? And finally, will a greater ROM be used with one arm when compared with two?To test whether or not the COR Bench provides a greater stimulus for resistance training, participants performed a one dumbbell and two dumbbell unilateral bench press on both the COR Bench and a traditional bench. Participants were asked to come to the lab to complete two different sessions, the first was to identify the participant’s bench press one repetition maximum (1RM) on both the COR Bench and flat bench, and the second was to test muscle activation and the kinematics of their movement during unilateral bench presses with one and two dumbbells on the COR and flat benches.M = 23.1, range = 20–28 years; mass, 88.7 ± 11.9 kg; height, 183.8 ± 5.6 cm) and 7 females  were recruited to participate over the course of a fall semester; however, the experiment was conducted in a laboratory (controlled environment) with a temperature of ∼23 °C. Participants were required to be 18–30 years old, have previous experience in participating in resistance training, be free from current or previous injury that may be aggravated by participating in upper extremity resistance training, and also be familiar with the bench press. All participants were recreationally active. Participants were asked to abstain from training for 24 h prior to testing. Before beginning, all participants completed an Informed Consent and Physical Activity Readiness-Questionnaire. The Institutional Review Board at Arizona State University approved this study and its relevant documents (IRB ID: 1211008510).Fifteen males , with a diameter of 1 cm and an inter-electrode distance of 2 cm, were placed on one prime mover muscle , one shoulder girdle stabilizer (right middle trapezius), and two muscles associated with core stabilization  . The lefBefore electrode placement, the skin over each muscle was prepared for EMG analysis by removing any hair and abrading and cleaning the skin with alcohol wipes. The EMG signals, collected at 1500 Hz, were analyzed using MyoResearch 3.0 . The collected data was rectified, filtered using a 10–500 Hz bandpass filter, and smoothed using root mean square with a window of 50 ms. Average amplitude was then determined for the duration of each trial.To assess the kinematics of the participant’s movements, reflective markers were placed on the skin over the acromion process of the scapula and the lateral epicondyle of the humerus, allowing for the analysis of ROM during the movement. As the electrodes were reaching a stable impedance condition, the participants engaged in a brief warm-up consisting of 10 arm circles forward, 10 arm circles backward, and 10 wall push-ups. A camera was placed behind the bench to capture the motion of the participant’s shoulder complex and their ROM. The shoulder complex ROM (SC ROM) was measured by subtracting the minimum from the maximum 3- point angle and averaging the value for the five trials. Each 3- point angle was determined from points at a line held vertical, the acromioclavicular joint, and the lateral epicondyle. This was thought to be the best angular representation of the shoulder complex as a whole .In a randomized fashion, each participant completed five repetitions at 70% of their 1RM of unilateral bench presses holding two dumbbells on both the COR Bench and the flat bench as well as five repetitions at 70% of their 1RM of unilateral bench presses holding one dumbbell on both the COR Bench and flat bench, for a total of 4 separate trials. When they were holding two dumbbells, they completed five repetitions for each arm in an alternating style, starting with both hands raised. In other words, starting with both hands in the air, their right arm came down first, then pushed up, followed by the left arm coming down, then being pushed back up, equaling one repetition per arm. All movements were controlled for speed with a 40 beats per minute tempo metronome , and participants were allowed two minutes rest between sets.There existed a difference in height between the two benches, so to control for any confounding factors, an elevated footrest was put at the end of the COR Bench to make the heights equal. Additionally, participants were required to keep their feet and legs in similar positions between both 1RM tests and all four experimental trials. This was accomplished by measuring the knee angle (via goniometer) and distance between the participants’ feet (via tape measure) while lying on the flat bench, and both of these were hold constant throughout the entire experiment for each participant individually.t-tests were performed. Non-parametric data that required comparisons were compared using Wilcoxon paired-samples signed-rank tests. A Benjamini–Hochberg procedure was carried out to control for false discovery rate for all pairwise comparisons . If data were parametric but did not meet sphericity assumptions, Greenhouse-Geisser corrections to degrees of freedom were applied to the ANOVA. Friedman’s test was used to compare within-subject effects across groups for nonparametric data. For parametric data that required pairwise comparisons, two-tailed paired-samples parisons . Alpha wferences . This meectively . Non-parectively . In ordeectively ; althougOne participant’s flat bilateral trial was corrupted (both EMG and ROM) due to a disk writing error, and one participant’s middle trapezius EMG signal during the bilateral COR Bench trial appeared to be invalid, as there were impossibly high amplitudes, potentially due to pressure on the electrode, so these values were not included in the statistical analyses.Using the calculated 1RM values, there was a −11.59% change in 1RM value when going from the flat bench to the COR Bench.χ2(3) = 16.200; p = 0.001], external oblique , and internal oblique  were not due to chance alone. However, it cannot be said that the pectoralis major EMG amplitudes between all four testing conditions were statistically different . The results of the pairwise comparisons were used to address the following questions.The mean EMG (±SD) amplitudes for each muscle under each testing condition are displayed in z = 0.859; p = 0.5207; Pearson’s r = 0.19), middle trapezius (t(19) = 2.7428; p = 0.0516; Cohen’s d = 0.61), external oblique , or internal oblique (t(19) = 0.6912; p = 0.5431; Cohen’s d = 0.15)] during the one dumbbell trials on the COR bench versus the one dumbbell on the flat bench were not due to chance alone.It cannot be concluded that any differences in EMG amplitude in the muscles analyzed  during the two dumbbell trials on the COR Bench versus the two dumbbells on the flat bench were not due to chance alone.It cannot be concluded that any differences in EMG amplitude in the muscles analyzed . Pairwise comparisons revealed that a statistically greater ROM was utilized in one dumbbell trials when compared to two dumbbells for both the COR bench (t(19) = 3.826; p = 0.0066; Cohen’s d = 0.86) and flat bench (t(18) = 4.115; p = 0.0066; Cohen’s d = 0.94). However, no statistical differences were observed between the COR bench and flat bench with two dumbbells (t(18) = 2.509; p = 0.0657; Cohen’s d = 0.58) or one dumbbell .The mean ± SD ROM for each testing condition is displayed in It was hypothesized that the introduction of instability to a unilateral bench press movement would increase the EMG amplitude of the muscles being analyzed. In other words, an increase in co-contraction should be observed in the conditions with a less stable environment (COR bench) when compared to the conditions with a more stable environment . Additionally, it was hypothesized that an increase in ROM should be observed in the conditions performed on the COR bench due to its curved, narrow, and inflated surface. And lastly, it was hypothesized that participants would utilize a larger ROM with one arm than with two.When performing the 1RM estimation test  on the fThe COR Bench was designed to provide instability to an exercise by not fully supporting the trunk like a traditionally used flat bench. Additionally, its design may allow for a greater ROM. Theoretically, these factors should elicit greater EMG amplitude of the core and shoulder girdle stabilizers and when performing lifts on the COR Bench when compared to a flat bench. However, in this present study, no statistical differences in EMG amplitude were observed for any muscle  when comparing the COR bench to its flat bench counterpart, for both one and two-dumbbell variations. These findings indicate that the COR Bench alone does not add enough instability to truly increase EMG amplitude in either core stabilizer muscles or prime movers analyzed in this study. This is similar to the results of With regards to ROM, it does not appear that the narrower, air compressed surface of the COR bench transfers to greater ROM when compared to that of the traditional bench, for both one- and two-dumbbell variations. However, with both the COR bench and traditional bench, single dumbbell variations utilized a statistically greater ROM than did the two dumbbell variations. Such results suggest that the single dumbbell variation can be utilized to train with greater ROM, but whether these small (∼7°), but statistically different, differences in ROM are sufficient to elicit superior training adaptations cannot be said for certain.a priori alpha are indicative that this study may have been underpowered and type-II errors may be present, especially following the correction for multiple comparisons. Lastly, the 1RM estimation protocol utilized in this study has not been validated for the dumbbell bench press, and it is therefore possible that the estimated 1RM differed from participants’ actual 1RM. Notwithstanding these limitations, our data are congruent with previous studies (There are a few limitations that should be noted when interpreting the results of this present study. Firstly, no specific inclusion or exclusion criteria existed with regards to training age and frequency. It is possible that different responses may have been elicited from those with different training experiences, but this cannot be said for certain. Second, although participants abstained from training for 24 h prior to the second day of this study, some did complete the testing protocol on the preceding day. Therefore, it is possible that some participants may have been sore or fatigued on the EMG testing day, but because a within-subject design was utilized, participants were familiar with the movement, and the testing protocol was relatively light, it is unlikely that this would have had a substantial impact on the observed outcomes. In addition, the medium to large effect sizes observed for comparisons that did not reach the  studies .In conclusion, the present study showed that the COR Bench did not prove to be an important of a source of instability, as it failed to elicit both greater EMG amplitude and ROM. Furthermore, it appears that using a single dumbbell may be a better option if larger ROM is desired. More comparative research—including training studies—involving athletes and rehabilitation patients with and without a history of upper extremity injury is warranted. Such further assessments will promote a better understanding of the other potential implications and advantages of the COR bench.10.7717/peerj.1365/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.1365/supp-2Supplemental Information 2Click here for additional data file.


Small RNA sequencing (sRNA-seq) can be used to detect viruses in infected hosts without the necessity to have any prior knowledge or specialized sample preparation. The sRNA-seq method was initially used for viral detection and identification in plants and then in invertebrates and fungi. However, it is still controversial to use sRNA-seq in the detection of mammalian or human viruses. In this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify viruses in human cells or tissues, particularly from some clinical samples. Six viruses including HPV-18, HBV, HCV, HIV-1, SMRV, and EBV were detected from 36 runs of data. Four viruses were consistent with the annotations from the previous studies. HIV-1 was found in clinical samples without the HIV-positive reports, and SMRV was found in Diffuse Large B-Cell Lymphoma cells for the first time. In conclusion, these results suggest the sRNA-seq can be used to detect viruses in mammals and humans. Infection by pathogens is one of the main risk factors for many diseases –4, partiTo overcome these limitations, next generation sequencing (NGS) technologies have been applied for virus and viroid discovery in plants and animals , 8. CompIn this study, we used 931 sRNA-seq runs of data from the NCBI SRA database  to detechttp://www.ncbi.nlm.nih.gov/sra/advanced), we retrieved 2,820 runs of data by the combined keywords including Illumina, small RNA, and Homo sapiens . We subsequently filtered these data based on the following criteria: (1) to remove non-small RNA-seq data by reading the annotations; (2) to remove data containing keyword “cell line”; (3) to remove data from cDNA library selection during library construction. Ultimately, we used 931 runs of data from 42 previous studies in this study (Using NCBI SRA advanced searching tools (is study .The software Fastq_clean  was usedhttp://dx.doi.org/10.1155/2016/2596782). We used the reference genome coverage and the average depth to quantify the detected viruses. The genome coverage represents the proportion of read-covered positions against the genome length. The average depth is equal to the total base pairs of the aligned reads divided by the read-covered positions on the reference genome  is one of the first viruses to be causally linked to a human tumor and is a major global cause of hepatocellular carcinoma (HCC). HBV, hepatitis C virus (HCV), and cirrhosis between them contribute to the genesis of almost all global HCCs . ConventIn the previous study SRP002272 from the NCBI SRA database , 15 clinThe assembled HBV in the data SRR039620 covered 54.6% of the reference genome JQ688404 with an average depth 6 (Supplementary File 1). In the data SRR039620, seven long viral contigs (≥ 40 bp) covered the HBV x (HBx), HBV core (HBc), and HBV polymerase (HBp) gene regions but did not cover the HBV surface (HBs) gene region. The long viral contigs (≥ 40 bp) in the data SRR039614 and SRR039621 only covered the HBx gene region. The assembled HCV in the data SRR039622 covered 10.8% of the reference genome D85516 with an average depth 1 (Supplementary File 1). HCV was also detected in the data SRR039623 with genome coverage 8.3% and average depth 1. Lentivirus causes acquired immunodeficiency syndrome (AIDS). As technology evolves, HIV testing assays are being improved on sensitivity and specificity [HIV as a member of the genuscificity . Howevercificity . In the cificity . These pSMRV, an endogenous virus of squirrel monkeys, had been isolated by cocultivation of squirrel monkey lung cells with canine cells . In prevIn this study, SMRV was detected in the data SRR1563015 and SRR1563017 . The assEpstein-Barr virus (EBV) has been firmly linked to some cancers and proliferative diseases, including Burkitt's lymphoma (BL), nasopharyngeal carcinoma, immunoblastic lymphoma, a subset of gastric carcinomas, rare T- and NK-cell lymphomas or leiomyosarcoma, acute infectious mononucleosis, and Hodgkin's disease. Almost 100% of BL cases in Equatorial Africa carry EBV. Children infected early in life with the highest antibody titres to the virus are at the highest risk of developing the tumor . EBV-posIn the previous study SRP046046 from the NCBI SRA database, 12 samples had been sequenced to distinguish the small RNA composition in six B cells from their exosomes. Six B cells included three EBV-positive lymphoblastoid B cells (LCLs), one EBV-positive Burkitt's lymphoma (BL) cell, one EBV-negative BL cell, and one Diffuse Large B-Cell Lymphoma (DLBCL) cell. As a result, EBV had been detected from two EBV-positive BL samples and six EBV-positive LCL samples . In thisThe plant sRNA-seq data had been shown to contain adequate information for studying nucleotide polymorphism of the actual virus . Among tConsistent with our previous results in plant virus detection, the distribution of vsRNA coverage over the human virus genomes was not even, with some read-enriched regions (hotspots) in the vsRNA-covered regions on both of the positive and negative strands (Supplementary File 3). In HPV-18, HBV, HCV, and SMRV, the vsRNA-covered region on the positive strand was more than nine times larger than the vsRNA-covered region on the negative strand, while HIV-1 and EBV had little difference between vsRNA-covered regions over the positive and negative strands. Using the data SRR941591 as an example , the numTo investigate the RNAi responses using 36 virus-containing runs of data, we analyzed the length distribution of the reads aligned to the virus reference sequences. Viral small RNA read lengths of HIV-1 in the data SRR941591 had the distribution pattern expected from a RNAi response, similar to what had been found in previous studies . This pahttp://www.mirbase.org/) and identified three known mature virus miRNAs. They were rlcv-mir-rL1-1-3p, ebv-mir-BART1-5p, and ebv-mir-BART1-3p located at positions 53,801, 151,640, and 151,676 on the EBV reference M80517. Three mature miRNAs came from two miRNA precursors (pre-miRNAs) rlcv-mir-rL1-1 and ebv-mir-BART1. Compared to two other miRNAs, the ebv-mir-BART1-5p was expressed at a very high level of 17,987 read counts. As for the remaining four duplexes, we confirmed they could not be matched to the human genome. Then, we used RNAfold online server (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) to predict the second structures of their pre-miRNAs (Supplementary File 4). As a result, we identified a new EBV pre-miRNA, with a length of 90 bp and a very high minimal folding free energy index (MEFI) over 1.0 , piwi-interacting RNAs (piRNAs), or degraded mRNA fragments. DNA viruses produce their own miRNAs facilitating the detection of DNA viruses, for example, EBV. In the data SRR1563063, we found seven miRNA-like duplexes from EBV vsRNAs (Supplementary File 3). Then, we blasted these duplexes to the miRBase database (over 1.0 . Furtherover 1.0 . Each unIn this study, we used 931 sRNA-seq runs of data from the NCBI SRA database to detect and identify human viruses. Six viruses were detected and two of them were not found in previous studies. These results suggest the sRNA-seq can be used to detect viruses in mammals and humans. The sRNA-seq data contains the heterozygosity information that can be used to investigate the pathogen evolution in one person and design therapies to deal with a specific virus population. The sRNA-seq data can also be used to find new virus miRNA or to investigate the RNAi responses in mammals and humans. However, sRNA-seq data used in this study were not from virus-infection experiments with knowledge of the exact virus sequences. In place of the exact virus sequences, the putative virus sequences were used to investigate the RNAi responses. Although using the putative virus sequences brought some uncertainties, the results of this study still shed light on the studies of virus induced RNAi in mammals.Supplementary Material includes four files, which are Detection reports of six viruses, Read distribution on six virus genomes, The sequences and loci of seven duplexes, and The predicted second structures of seven duplexes.