Land managers of natural areas are under pressure to balance demands for increased recreation access with protection of the natural resource. Unintended dispersal of seeds by visitors to natural areas has high potential for weedy plant invasions, with initial seed attachment an important step in the dispersal process. Although walking and mountain biking are popular nature-based recreation activities, there are few studies quantifying propensity for seed attachment and transport rate on boot soles and none for bike tires. Attachment and transport rate can potentially be affected by a wide range of factors for which field testing can be time-consuming and expensive. We pilot tested a sampling methodology for measuring seed attachment and transport rate in a soil matrix carried on boot soles and bike tires traversing a known quantity and density of a seed analog (beads) over different distances and soil conditions. We found % attachment rate on boot soles was much lower overall than previously reported, but that boot soles had a higher propensity for seed attachment than bike tires in almost all conditions. We believe our methodology offers a cost-effective option for researchers seeking to manipulate and test effects of different influencing factors on these two dispersal vectors. Invasive alien species of plants (weeds), together with animals, fungi and microbes are widely recognised as posing a major threat to global biodiversity, second only to habitat destruction in their impact Randall . Weeds hOne human activity with high potential for unintentional dispersal of weed seeds is tourism (including recreation). People today, especially in economically developed countries, have increasing time for leisure Molitor  and inteA small but growing number of studies have shown capacity for unintentional human-mediated dispersal of seeds by tourists, either attaching directly to hikers’ clothing or equipment, embedded in soil picked up by vehicles, or animal dung/feed   available surface area of soles vs. tires  or Brassica nigra [black mustard], again taking 20 steps on the spot. Assuming Tray 1 was filled to a soil depth of 20 mm and Tray 1 and Tray 2 were of equivalent dimensions, this would suggest a soil area of 100,000 mm2 and density of seeds 100/100,000 mm2 = 0.001 seeds/mm2, although the actual density of seeds exposed to the boot soles was probably much higher than this: ‘probably artificially high’ . We used a commercially-obtained loam-based soil , i.e. 50 ‘seeds’/l of soil –24.7 [SE = 3.25]) and tires (0.00 [SE = 0.00]–34.6 [SE = 4.42]) under both moist and wet soil conditions (Table Our “long” test distance ≈150 m) was broadly comparable to that employed by Mount and Pickering (50 m was ns Table  study (i2] trays containing (i) wetted soil (moisture % level not reported) and (ii) 100 seeds. They recorded high attachment rates, ranging from  4–93 % attachment, mean 52% and 42 %, variability unreported and  26–52 % attachment, mean % and variability unreported]. The authors noted that their sampling protocol did not match the “real situation” and that their recorded attachment rates were ‘probably artificially high’ : as previously noted our beads were specifically selected to be a comparable size and shape to seeds used in that study.Firstly, we were using plastic beads as an analog for seeds, not real seeds. However, seeds of different species exist in a wide range of morphologies and adhesive qualities, masses and sizes and we therefore argue that our beads can be considered as a representative analog of real seeds on all three parameters except for the small hole centring the beads. The only two previous controlled studies of direct seed attachment on boots that we found This study is a pilot test of a potentially very flexible and cost-effective sampling methodology; the possible occurrence and scale of the potential attach/detach/reattach issue would benefit from further testing.(ii)The % of beads attaching from the available bead reservoir on a ‘short’/single circuit was very low overall ; this suggests that the probability of the same individual beads re-attaching during multiple circuits is likely to be very low.(iii)The ‘short’/single circuit distance test is unaffected by this potential issue and estimates of seed attachment over longer distances can therefore be arrived at via simple multiplication.Secondly, although our use of the circular test track allowed us to simulate a realistic walking and riding pattern and beads were available for attachment from on top of/within shallow surface soil, similar to conditions likely to be the case in a natural environment, the methodology employed in the “long” (≈150 m) test distance, necessitating repeatedly walking/riding the same track, meant that some beads might have become attached, detached and subsequently reattached on boot soles and bike tires. Although we were unable to quantify this, we regularly observed soil dropping back onto the track from both boots and bike tires during circuits. This was especially marked for the bike under “wet” conditions, with soil (possibly containing beads) picked up on the tires often unable to pass through the caliper brake pads and subsequently ejected back onto the track. This issue was probably less likely to occur for boot soles because, as previously noted, boots predominantly tended to pick up soil and beads in the heel treads, with soil tightly compacted and requiring beads to be physically extracted by the researchers, with very few beads (estimated <5 %) attaching to the remainder of the soles. In defence of the sampling methodology, however, we argue:Thirdly, time and funding limits meant that our small-scale experiment used the same, single walker/rider for all tests and only one pair of boots and one bike. Boots and bike tires obviously come in a very wide variety of materials, sizes and tread patterns and these may affect seed attachment rate. Different walking/riding behaviour of individuals may also have an effect. Wichmann et al.  found seFor the above reasons, our results presented here are necessarily case-specific and cannot be generalised more widely to define the absolute relative propensity for seed attachment and transport rate in a soil matrix on boot soles and bike tires. We nevertheless suggest that the methodology as trialled here shows significant promise for researchers to use it more comprehensively to test the attachment rate of different seed types under a range of densities and soil conditions across a variety of different compounds and sizes of boot soles and bike tires, in a way that is cost-effective and that reflects real-world walker and biker behaviour.


This paper investigates the friction coefficient of a moving liquid marble, a small liquid droplet coated with hydrophobic powder and floating on another liquid surface. A floating marble can easily move across water surface due to the low friction, allowing for the transport of aqueous solutions with minimal energy input. However, the motion of a floating marble has yet to be systematically characterised due to the lack of insight into key parameters such as the coefficient of friction between the floating marble and the carrier liquid. We measured the coefficient of friction of a small floating marble using a novel experimental setup that exploits the non-wetting properties of a liquid marble. A floating liquid marble pair containing a minute amount magnetite particles were immobilised and then released in a controlled manner using permanent magnets. The capillarity-driven motion was analysed to determine the coefficient of friction of the liquid marbles. The “capillary charge” model was used to fit the experimental results. We varied the marble content and carrier liquid to establish a relationship between the friction correction factor and the meniscus angle. Although we have previously estimated the correction factor of the ideal Stokes friction coefficient based on indirect measurements and calculationsx) direction, the component of the resultant force FR(x) acting on a particle is:Two floating spherical particles, placed close to each other, experience a resultant force, m is the mass of the particle, FC(x) and Ff are the capillary and friction force, respectively. The friction force can be calculated using Stokes’ law30where r is the particle radius, μ is the dynamic viscosity of the carrier liquid, β is the correction factor for the coefficient of friction.where et al.The seminal work by Kralchevsky L is the distance between the particles and the first order of the modified Bessel function of the second kind K1 that can be approximated as:where z is a dimensionless quantity (z = qL in our case), γ the surface tension between the fluids, q the inverse capillary length of the carrier fluid:where ρw is the density of the carrier liquid. Q1 and Q2 are the capillary charges of individual particles that can be calculated as:where r0 is the contact radius of the TPCL, which is typically much smaller than the distance between the two marbles L ref. r0 is on the order of 5 × 10−4 m, whilst L is set to be 1.5 × 10−2 m. In where Substituting (7) into (6) results in:Q = Q1 = Q2; L = 2x given that x is measured from the midpoint between the two marbles. Substituting m = (4πr3/3)ρm, where ρm is the effective density of the liquid marbles, and making the appropriate arrangement in (1) gives:For two liquid marbles of the same composition and radius, μ, r, ρm and ρw) are known. From the experiments, x is measured as a function of time, and the resultant data can be differentiated with respect to time to obtain both velocity, β for a known meniscus angle ψ.In (9), the physical properties :The meniscus angle Fg is the weight of the marble, Fb is the buoyancy force, and FC(y) is the vertical component of the capillary force. Making the appropriate substitutions for Fg, Fb and FC(y) gives:where h is the height of the meniscus, measured vertically from the TPCL to the free liquid surface sufficiently far away from the marble; h is a function of r and ψ, and can be solved using methods described in our previous workwhere ψ for a given set of values for r, γ, ρm and ρw. With the value of meniscus angle ψ then, β can subsequently be obtained from (9). Note that the analysis given above is only valid for small particles, i.e. (qr)2 ≪ 1, and small meniscus slopes, i.e. qr)2 and −1.β is known to be dependent on the amount of perturbation of the liquid interface, which in turn depends on the meniscus angle ψ and the submerged depth of the liquid marble. Since this depth is a function of ψ for a known volume, β should be a function of ψ as well. For a small floating liquid marble, the buoyancy force Fb is small compared to the surface tension force FC(y)Fb in (10), The correction factor, β should be a function of ψ for a given volume, it should also be a function of the liquid surface tension, γ and ρm. Note that the liquid density and marble surface tension terms are missing, because buoyancy force and marble deformation are neglected. This assumption gives us a convenient way to estimate β based on reasonably measurable quantities. In this paper, we vary the effective marble density ρm and the carrier liquid surface tension γ to determine the relationship between the correction factor β and sin ψ. The marble radius r is kept constant, whereas ρm is measured for marbles containing different NaCl concentrations. The surface tension γ and the density ρm were changed in our experiments, independently.As 12. We also assumed that the marbles maintain the spherical shape during the motion. Although the marble is spherical in shape, its physical properties differ from that of a solid spherical particle. For a solid spherical particle, the contact angle is constant regardless of floating position due to its rigidity. The meniscus angle can hence be calculated if the contact angle and surface inclination at the TPCL are known. For a floating marble, the apparent contact angle changes due to the deformation of the marble itselfWith an aspect ratio of ε ≈ 0.9 for a 5-μL marble, the liquid marbles were assumed to be spherical in our experimentsCapillary force arising from the edge of the container is neglected, as the meniscus created by the container is miniscule and far away from the floating marbles. The marble is assumed to maintain its initial volume throughout the experiment as the time scale of the experiments (~30 seconds from sample preparation until the end of collision) is much smaller than that of the evaporation process of the marble of approximately one hour.® iron oxide  5 μm nominal diameter) was mixed with the NaCl solutions at 0.25 wt%. Weight measurements were conducted using an electronic balance . The mixture was shaken thoroughly to increase particle suspension before being dispensed onto a bed of polytetrafluoroethylene (PTFE) powder , as reported previouslyA 5 m (mole/kg) NaCl stock solution was prepared and diluted to desired concentrations . NaCl solutions of 0 to 5 m with 1 m interval were used as carrier liquids and liquids of the marble. Magnetite ; five runs were conducted with NaCl solution as carrier liquid (1–5 m) and marbles containing DI water; one run was conducted with DI water as both carrier liquid and liquid marble content. Each run was repeated three times. All experimental works were carried out at a temperature of 293.5 ± 0.5 K and atmospheric pressure.β.A 140-mm-diameter Petri dish with an opaque white base was centred on a stationary platform with two square-shaped through holes spaced 15 mm apart. The Petri dish diameter is 140 mm and made of polystyrene, which has a contact angle of about 90° with waterWhen the marbles were floating above the magnets, they experienced vertical forces from the magnets. Hence the floating position would be lower with a larger contact radius. Once the magnets were dropped, the liquid marbles rapidly recover their stable positions without any influence of the magnetic field. Therefore neither the magnets nor the magnetite play any role in the subsequent marble motion generated by the capillary force. Video recordings show that marble pairs collided and bounced off each other elastically. Some runs with 5 m NaCl marbles collided with enough impact to rupture one of the marbles. A sample video recording is provided in the ρm of the marble needed to solve for β. Although NaCl can be dissolved up to 6 m, solid precipitation was observed after extended period of time. Therefore, 5 m was the maximum concentration attempted. NaCl solution was used because: (i) NaCl salt is cheap and readily available; (ii) Properties of NaCl solution are well understood and widely reported; (iii) NaCl has high solubility in water; (iv) NaCl is one of the few additives which increases the viscosity and surface tension of water, hence reducing errors generated by ambient air currents. At 5 m, the surface tension, the density and the dynamic viscosity of NaCl solution are 80.0 mN/m3 Based on the measured initial values, a curve (solid line) is fitted for each experimental run using different values of x. A smoothing spline was fitted to the data points to reduce data spread caused by centroid calculations in the image analysis process. The smoothed curve was differentiated with respect to time to yield the velocity data; and differentiated twice to yield the acceleration data. As the smoothing magnitude was relatively small, minute spreads in the position data were amplified in the acceleration data.β values from each data point, plotted against the marble position. β values are relatively constant for a large distance (x > 3 mm) but increases rapidly as the distance shortens. Thus, a two-term exponential fitting function was fitted onto the data points to obtain an averaged β:a and b are fitting parameters. Ideally, the correction factor should be independent of the position, assuming that the floating body is a rigid sphere. However, this assumption might not be true as the correction factor is a function of the position, β values. The data spread increases with increasing distance. This uncertainty is likely caused by the fact that the marbles dwelled at the vicinity of the starting position (≈7.5 mm) with low velocities (<1 mm/s). Thus, they were very susceptible to micro currents and minute vibration sources. Since β is relatively linear in the region of (3 mm < x < 7.5 mm), we took the average value of β at these two position as the correction factor for the coefficient of friction β versus the sine function of the meniscus angle where ψ is zero. Consequently, the value of β should be 0.5. In our case, the liquid marble floats relatively high on the free surface with a small meniscus angle ψ. Therefore, we expect a smaller correction factor of β < 0.5. Our results disagrees with this hypothesis to a certain extent, as experiments of water marble floating on water have an average β of 0.52, higher than the expected value. This discrepancy is likely contributed by the perturbation of the water surface due to the weight of the marble and rough contact area caused by the particle coating. Nevertheless this value is very close to our estimated value reported earlierIf the marble floats with its equator intersecting the interface, its meniscus angle ψ. Hence, the correction factor β is reduced . β for small floating marbles without the need to run an experiment for every parameter change.where Cβ to the meniscus angle ψ is limited for small, floating and non-wetting spherical objects because the buoyancy forces are relatively small. Since this model assumes spherical marbles, it is valid for small Bond numbers γm is the marble effective surface tension. For future works, using compatible liquids of larger differences in surface tension and density can further expand the range of the meniscus angle ψ.This model relating the correction factor This paper reports the experimental determination of the Stokes friction correction factor of a small floating liquid marble for a small range of meniscus angles. The experimental setup was designed to simplify the calculations of the various force terms involved in the marble motion generated by capillary force. We exploited the non-wetting properties of a liquid marble and its magnetic actuation to design the experiments. The correction factor of a 5-μL water marble was found to be about 0.52. We found that the correction factor could be estimated to vary linearly with the sine function of the meniscus angle, which itself could be expressed in terms of marble size, effective density, and carrier liquid surface tension. The method reported here provides a convenient way to estimate the correction factor of a small floating liquid marble without the need of characterising its motion. The known friction factor enables proper designing and modelling liquid marbles as a digital microfluidics platform for various applications.How to cite this article: Ooi, C. H. et al. Measuring the Coefficient of Friction of a Small Floating Liquid Marble. Sci. Rep.6, 38346; doi: 10.1038/srep38346 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Pseudocowpox virus (PCPV) infects cattle worldwide with zoonotic potential but has not been isolated in Japan. Thus, the epidemiological status of PCPV infection in cattle is undetermined.B2L gene encoding viral envelope. Phylogenetic analysis showed that the isolated virus was classified into PCPV.In May 2016, a cattle in a farm in Yamaguchi Prefecture showed white vesicles and hyperemia in the mucosa under the tongue surface, but not on the teats and coronary cushions. A parapoxvirus was isolated from the oral lesion swab and was genetically characterized based on the full-length sequence of This case indicates its potential spread in Japan. This is the first report of isolation of PCPV in Japan.The online version of this article (10.1186/s12985-017-0840-3) contains supplementary material, which is available to authorized users. Parapoxvirus in the family Poxviridae, which includes bovine papular stomatitis virus (BPSV) and orf virus (ORFV) [Pseudocowpox virus (PCPV) is a member of the genus s (ORFV) . Parapoxs (ORFV) .In Japan, although serological surveys have revealed that seroprevalence of parapoxvirus is very high in cattle and sheep , 4 and mB2L gene encoding viral envelope of this isolate, and evaluated its phylogenetic relation to known members of this virus group.We here report the first case of the isolation of PCPV in Japan. We determined the full-length sequence of the In May 2016, a breeding cow  in a farm in Yamaguchi Prefecture, in the western part of Japan showed anorexia, mild fever, frothy salivation, and hyperemia in the mucosa under the tongue surface. No lesions were observed on the teats or coronary cushions. A few days after the onset of clinical signs, the cattle showed white vesicles on the tongue surface  and used for virus isolation and DNA extraction.An oral swab sample was collected from the mucosal lesions of the affected cattle and was homogenized with Eagle’s minimum essential medium (MEM). The sample was centrifuged at 800×l-glutamine, and 5–10% fetal bovine serum at 37 °C. The cells were cultured in rolling tubes and 24-well plates, washed with maintenance medium , and inoculated with 0.1 ml of the processed samples. After adsorption for 60 min at 37 °C, the inocula were replaced with 0.5 ml of maintenance medium. The rolling tube-cultured and 24-well plates-cultured cells were incubated by rotary cultures and stationary cultures, respectively, and observed daily for cytopathic effects (CPE) for at least 7 days. Cultures without CPE were passaged twice in a blinded manner.Virus isolation was performed through inoculation in primary bovine testis (BT) cells; two cell lines, hamster lung (HmLu-1) and Madin-Darby bovine kidney (MDBK) cells were also tested for comparison. The cells were grown in Eagle’s MEM with kanamycin  supplemented with 0.295% tryptose phosphate broth (TPB), 0.015% sodium bicarbonate, 0.03% B2L gene encoding envelope of parapoxvirus with the primer sets OVB2LF1/OVB2LR1 [DNA was extracted both from the oral swab sample and from the BT cells showing a CPE using magLEAD 12gC . Polymerase chain reaction (PCR) amplifications were carried out with TaKaRa Ex Taq Hot Start Version  using a SimpliAmp Thermal Cycler  for detection of the full-length 1137 bp) and partial-length 554 bp) /OVB2LR1  and PPV154 bp /OV bp and p/OVB2LR1 . Phyloge/OVB2LR1 , epizoot/OVB2LR1 , blueton/OVB2LR1 , ovine hAt the third passage, a distinct CPE was observed from 2 to 3 days after inoculation in the rolling tube-cultured BT cells, characterized by a rounded morphology and cell detachment  less herpes simplex virus was produced in stationary than in rotary cultures. Although the procedures of rotary cultures are more burdensome than those of static cultures, we suggest that rolling of inoculated cultures should be conventionally applied in clinical virology laboratories to aid in the isolation of PCPV. In the affected cattle in this study, there was no evidence of infection on the teats and udder, which are the more common lesion sites of pseudocowpox infection. The classification of parapoxviruses was formerly based on the natural host range, clinical signs, and serology [As noted above, seroprevalence of parapoxvirus is very high in cattle in Japan and multiple BPSVs have been isolated , but no serology , howeverserology  as evideB2L gene determined from the PCR product (accession no. AB921003) was identical to that of the present strain YG2828 (data not shown). Thus, our results confirmed that PCPV can be isolated from atypical sites besides the teats and udder, and suggest that YG2828-like PCPV may cause oral lesions in cattle. Moreover, since parapoxviruses cross-react antigenically and two similar strains infected cattle in different locations in separate years, YG2828-like PCPV might be spreading among the cattle population in Japan. It is known that cattle are frequently infected with parapoxvirus subclinically [Previously, there has only been one report describing the PCR detection of PCPV DNA in Japan, in which PCPV DNA was detected from oral lesions in a calf in Iwate Prefecture, in the northern part of Japan, but virus isolation was unsuccessful . Notablyinically  and PCPVB2L gene revealed that the isolated virus was genetically close to strains isolated from cattle in the USA and Finland. PCPV is responsible for significant economic losses in the cattle production. Further virological and epidemiological studies to characterize this strain and the possibility of its spread in Japan are highly required.A PCPV was firstly isolated in Japan from the oral lesion swab of cattle showing white vesicles and hyperemia in the mucosa under the tongue surface, but not on the teats and coronary cushions, by rotary cultures. Genetic characterization based on the full-length sequence of


Infection with Zika virus is an emerging public health crisis. We observed prolonged detection of virus RNA in vaginal mucosal swab specimens and whole blood for a US traveler with acute Zika virus infection who had visited Honduras. These findings advance understanding of Zika virus infection and provide data for additional testing strategies. Zika virus is a rapidly emerging mosquitoborne virus . A previously healthy, nonpregnant, 26 year-old non-Hispanic white woman returned to the United States from Tegucigalpa, Honduras, during mid-May 2016. Five days after her return (day 0), signs and symptoms consistent with Zika virus infection developed, beginning with rash and subsequent fever, headache, and conjunctivitis . Fever aSerial specimens were longitudinally collected for >11 weeks. The first specimens were collected on day 0, two hours after onset of rash and 2 h before development of fever. All remaining specimens were collected at 3, 8, 14, 21, 28, 35, 42, 53, 64, and 81 days after onset of illness. Specimens included serum, whole blood (EDTA anticoagulated), urine, saliva, and vaginal mucosa swabs. The patient was not menstruating when vaginal swab specimens were collected.RNA was extracted from serum, whole blood, and urine samples by using the QIAamp MinElute Virus Spin Kit  according to the manufacturer’s instructions. Oral and vaginal mucosal swab specimens were collected by using the BBL CultureSwab Collection and Transport System . Specimens were incubated in 250 μL of AL/carrier RNA lysis buffer for 10 min at room temperature; 200 μL of phosphate-buffered saline was added before RNA extraction. ,Eluted RNA from all samples was tested in a quantitative reverse transcription quantitative PCR (qRT-PCR) that included a TaqMan Fast Virus 1-Step Master Mix  and a TaqMan ZIKV 1107 assay . Results5 PFU/mL by plaque assay. Attempts to isolate virus from the day 64 erythrocyte fraction showed no evidence of cytopathic effects, and first and second passages were negative by qRT-PCR. Because the day 81 whole blood specimen was still positive by qRT-PCR, we used ficoll to separate peripheral blood mononuclear cells and erythrocytes and found that erythrocytes were the only fraction positive for Zika virus RNA. The partial sequence of the virus we isolated was submitted to GenBank under accession no. KX928077.Virus isolations were performed for Vero cells in complete Dulbecco’s modiﬁed Eagle’s medium containing 10% heat-inactivated fetal bovine serum. Cells were infected with day 0 serum samples (or mock-infected with cell culture medium) and observed for cytopathic effects. Cell culture supernatants were sampled 13 days after cell culture infection, and RNA was extracted and tested for Zika virus RNA. Supernatant was collected on day 14, and viral titer was 8.5 × 10On day 8, plasma was evaluated by using an ELISA (Given recent concerns regarding the ongoing epidemic of Zika virus disease, there is an urgent need to document the natural history of infection and assess transmission risk through nonvector routes. We had the unique opportunity to prospectively monitor the clinical and virologic course of Zika virus infection in a patient starting on day 0.We detected viral shedding in vaginal secretions up to day 14. Only 1 human study reported Zika virus RNA in cervical mucous up to day 11 after onset of signs and symptoms (We could not determine whether positive results by qRT-PCR indicated replicating virus. With the recent finding of possible female-to-male virus transmission (We detected viral RNA in serum up to 8 days and in whole blood up to 81 days after onset of illness. Diagnosis of infection currently relies mostly on PCR detection of Zika virus in serum. With concerns for Zika virus infection during pregnancy, screening of whole blood might be more sensitive in identifying infected patients, particularly if an asymptomatic patient has traveled from an area where exposure is a concern, had high-risk sexual contact, or is convalescing and PCR for a serum sample would probably yield a negative result.,Our observation is further supported by another recent study that found whole blood samples positive for Zika virus by PCR up to 2 months postinfection (In conclusion, this case study advances understanding of the natural history of Zika virus. It provides new findings, including detection of Zika virus RNA in vaginal secretions up to day 14 and in erythrocytes up to day 81, the longest reported duration of detection in this sample type. A desquamating rash developed on the hands and feet of the patient, which we presume was related to her infection. To our knowledge, this finding has not been previously described.Additional studies involving larger cohorts of acutely ill Zika virus–infected patients tested over a longer period would solidify our understanding of the natural history of infection, duration of viral detection, and clinical outcomes. These studies will enable further development of evidence-based policies regarding diagnosis and clinical management of Zika virus–infected patients.


In order to define strategies to curb the continuing increase in healthcare costs, we describe the cost breakdown of open tibial fractures. Twenty-seven clinical and process variables were recorded retrospectively, and five main hospital related cost categories were defined. Three multivariate linear models were fitted to the data. Total healthcare costs of open tibial fractures were almost twice as high compared to closed fractures and mainly existed of hospitalization costs. Length-of-stay (LOS) was found to be the most important variable driving the healthcare costs of open tibial fractures. Deep infection lead to a 6-fold increase of LOS and 5-fold increase in total healthcare costs of open tibial fractures. Therefore, appropriate international consensus guidelines are required to improve not only the patient outcome (infection prevention) but also reduce overall healthcare cost by focusing on reducing the LOS. Healthcare costs have increased significantly over the past decades. In Belgium, health expenditure currently accounts for approximately 10% of its GDP (gross domestic product), up from 8% in 2000 . Length-In parallel, we want to understand the cost breakdown of open tibial fractures, since these fractures are associated with an increased complication rate  and treatment costs –6. The dHere we report a retrospective study of a large consecutive cohort of adult patients with either an AO/OTA type 41, 42 or 43 tibial fracture. We describe open tibial fractures in relation to patient demographics and operative characteristics. We define all hospital-related healthcare costs within the Belgium's healthcare system. Next, in order to define clear-cut clinical pathways and keep the healthcare system vital, we identify a subset of clinical variables that have a significant influence on costs and LOS, and determine whether these variables are actually related to the total healthcare costs. We hypothesized that Gustilo type 3 open tibial fracture patients will incur higher costs.n=29), other musculoskeletal trauma surgery during the follow-up (n=46), primary treatment elsewhere (n=23), , pathological fractures (n=1), and amputation (n=1) within 5 days after the accident. This study was conducted in compliance with national legislation and the guidelines of the ethics committee of University Hospitals Leuven. All tibial fractures related hospital stays and ambulatory consults of the selected patients were included in the analysis.A total of 358 patients with an acute AO/OTA type 41, 42 and 43 tibial fracture were included in this study. All patients were treated at the Traumatology Department of University Hospitals Leuven between January 2009 and January 2014. Follow-up was until January 2016, allowing a maximum follow-up time of two years for all patients. Exclusion criteria were non-acute fractures (diagnosed more than 4 weeks after the accident), age < 18 years (Twenty-seven variables were retrospectively recorded from an electronic database (KWS – UZ Leuven); 20 clinical variables , hardware removal, re-osteosynthesis, other operations, and mortality) and 7 process variables .Comorbidity was recorded using the ASA  -score. The cardiovascular risk factors (CVRF) include: age, current cardiovascular diseases , diabetes, rheumatoid arthritis, smoking, use of blood vessel narrowing drugs , dyslipidemia, hypercholesterolemia, and hypertension. All fractures were classified according to the AO/OTA classification system ; fracturFive main hospital related cost categories were defined: honoraria, materials, hospitalization (cost of daily patient care), daycare stay, and pharmaceuticals . In summThe costs described in this paper relate to the Belgium’s healthcare financing context and are exclusively hospital care-related. Furthermore, these costs comprise the majority of reimbursements paid to the hospital (by any party involved) in financing the care for a specific patient either directly or indirectly.U test.Continuous variables are presented as medians with interquartile distribution, whereas categorical variables are presented as numbers and percentages. Categorical variables were compared with the Pearson Chi-Square test. Comparison of continuous variables was performed using the Mann-Whitney y = a + b1x1 +  ⋯  + bnxn). Leave-one-out (LOO) cross validation was used to analyze generalizability In order to determine the variables that drive healthcare costs and LOS of open tibial fractures, three multivariate linear models were fitted to the data  and total costs are summarized in Table A univariate comparison of clinical and process -related variables for patients with and without an open fracture is shown in Table AV-CM) had an R2 of 0.98 and an adjusted R2 of 0.97. Only the variable total LOS was deemed significant , again with total costs as the dependent variable. The variables denoted with excluded in Table 2 of 0.67 and an adjusted R2 of 0.58  .Univariate analysis for open tibial fractures showed a 5-fold increase in the total healthcare costs  and 18 [9 – 34] vs. 11 [7 – 18], p = 0.138, respectively).The treatment costs and total LOS of open tibial fractures in patients with or without CRFV did not differ significantly   compared to open AO/OTA type 41 tibial fractures.Despite open AO/OTA type 42 tibial fractures were the most important bone segment fractures driving the total healthcare costs and total LOS, open AO/OTA type 43 tibial fractures turned out to be significantly more expensive  , as well as prolonged use of antibiotics  for type 3 compared to type 1 open tibial fractures. Subsequently, most deep infections were observed in patients with Gustilo type 3 open tibial fractures.Univariate subanalysis for Gustilo type open tibial fractures showed significantly higher total healthcare costs . As a consequence, total healthcare costs of open tibial fractures were almost double as high compared to closed fractures. They mainly consisted of hospitalization costs. Total LOS was the most important variable driving the healthcare costs of open tibial fractures (t = 30.56). After exclusion of all process variables related to hospital stay, deep infection was identified as the most important clinical parameter driving the LOS (model LS-M) and total healthcare costs (model NSRV-CM) of open tibial fractures. In addition, CVRF were identified as significant driver of the total healthcare costs. Patients with open tibial fractures that were complicated by deep infection, showed a 6-fold increase in LOS and 5-fold increase in total healthcare costs. Despite the fact that open AO/OTA type 43 fractures were on average the most expensive tibial fractures, the multivariate analysis showed that the presence of an AO/OTA type 42 tibial fracture (in combination with other factors) was found to be a more important factor in driving the LOS and total healthcare costs. Gustilo type 3 open tibial fractures had a significantly higher total LOS and amount of healthcare costs, due to a higher rate of deep infection (30%).Our results are in line with Page et al. , who demvs. €2,758,172 for closed fractures. Since the total number of open tibial fractures was limited , the impact of strategies to curb the total costs of open tibia fractures  was also limited. However, Belgium’s hospitals are financed through a mixture of patient co-payments the health insurance system and the Ministry of Health [Although the median total cost per patient for the treatment of open tibial fractures was approximately twice as a high compared closed tibial fractures, total cost for the treatment of open tibial fractures was €955,026 Compared to the literature, our deep infection rate (3.4%) was rather low . PatientIt is also important to notice that, especially in patients with open tibial fractures, we observed a strong variance in terms of costs. We assume that most likely the total LOS is not only a result of surgical strategy, patient characteristics and surgeon’s decision, but is also a function of local guidelines, historically developed processes, habits and the individual preferences of the surgeon. Therefore, appropriate international consensus guidelines are required to improve not only the patient outcome but also reduce overall healthcare cost by focusing on reducing the LOS.Deep infection was found to be the leading cause of the high total LOS and healthcare costs of open tibial fractures. Strict antibiotic guidelines and surgical protocols are adopted to minimize infection. Provided that the guidelines and protocols should be in the first place clinically beneficial, they may also serve as a tool to curb the total LOS and healthcare costs. Following the results of this study, we updated our treatment strategies for open fractures in order to improve patient outcome and reduce healthcare costs. First, current evidence states that long term systemic prophylactic antibiotic administration is not necessary . ShortenPlease note, that the analysis presented here is based on an arguably limited sample of open tibial fractures, due to this a cautionary approach has to be taken when inferring properties about this sample and, in turn, about the population. This has been taken into consideration by limiting the number of variables in the multivariate model (see Methods) and by verifying the conclusion drawn below though bivariate analysis.In conclusion, total healthcare costs for open tibial fractures were approximately twice as high compared to closed fractures and mainly consisted of hospitalization costs, i.e. the cost of daily patient care. This study shows that total LOS was the most important variable driving total healthcare costs of open tibial fractures. Guidelines on appropriate LOS can help to improve efficiency, decrease variability, and curb the total healthcare costs. Open tibial fractures complicated by deep infection presented the most significant increase in LOS and in total healthcare costs. Besides that infection prevention is essential in high-quality care, it can also be an important contribution to make trauma care more cost efficient. To that end, patients with open tibial fractures must be treated according to strict antibiotic and surgical guidelines. In cases of deep infection, updated antibiotic protocols make it possible to administer intravenous antibiotics in outpatients (OPAT) with the same level of outcome and a shorter LOS. Multiple possible preventive measures like local antibiotics and implant coatings can be addressed. Finally, as has been evident, trauma research faces the problem of studies publishing heterogeneous small sized patient cohorts, therefore (multicentre) randomized clinical trials on infection prevention are mandatory to ultimately ensure the quality as well as standardization of trauma protocols and hospitalization processes (i.e. LOS).


Prune dwarf virus (PDV) is one of the members of Bromoviridae family, genus Ilarvirus. Host components that participate in the regulation of viral replication or cell-to-cell movement via plasmodesmata are still unknown. In contrast, viral infections caused by some other Bromoviridae members are well characterized. Bromoviridae can be distinguished based on localization of their replication process in infected cells, cell-to-cell movement mechanisms, and plant-specific response reactions. Depending upon the genus, “genome activation” and viral replication are linked to various membranous structures ranging from endoplasmic reticulum, to tonoplast. In the case of PDV, there is still no evidence of natural resistance sources in the host plants susceptible to virus infection. Apparently, PDV has a great ability to overcome the natural defense responses in a wide spectrum of plant hosts. The first manifestations of PDV infection are specific cell membrane alterations, and the formation of replicase complexes that support PDV RNA replication inside the spherules. During each stage of its life cycle, the virus uses cell components to replicate and to spread in whole plants, within the largely suppressed cellular immunity environment. This work presents the above stages of the PDV life cycle in the context of current knowledge about other Bromoviridae members. Prune dwarf virus is a member of one of the six genera in the family Bromoviridae. Genus Ilarvirus includes 19 species. Historically, this genus was divided into four subgroups according to serological characteristics [Ilarvirus, new systematics were presented by International Committee on Taxonomy of Viruses (ICTV) , and now this group of viruses includes American plum line pattern virus (APLPV), Apple mosaic virus (ApMV), Asparagus virus 2 (AV-2), Blackberry chlorotic ringspot virus (BCRV), Blueberry shock virus (BlShV), Citrus leaf rugose virus (CiLRV), Citrus variegation virus (CCV), Elm mottle virus (EMoV), Fragaria chiloensis latent virus (FCILV), Humulus japonicus latent virus (HJLV), Lilac leaf chlorosis virus (LLCV), Lilac ring mottle virus (LiRMoV), Parietaria mottle virus (PMoV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Spinach latent virus (SpLV), Strawberry necrotic shock virus (SNSV), Tobacco streak virus (TSV), Tulare apple mosaic virus (TaMV) [Ilavirus class T = 3 usually consist of 180 molecules of coat protein and encapsidate (+)ssRNA [Anulavirus, Bromovirus, and Cucumovirus. Certain virus species in the genus Ilavirus, including PDV, form two types of particles at the same time: icosahedral and bacilliform [Alfamovirus, Ilarvirus, and Oleavirus.eristics ,3,4. In s (TaMV) . Similars (TaMV) . The gen(+)ssRNA ,8. Icosailliform . The icoilliform , whereasBromoviridae members, including PDV, consist of three single-stranded (+)RNA (ssRNA) components, identified as RNA1, RNA2, and RNA3. Each of the (+)ssRNA particles possesses a cap structure at the 5′-end and a 3′-untranslated region (3′UTR), that forms a tRNA-like structure (TLS) [Cucumovirus, and in the majority of Ilarviruses, an additional open reading frame (ORF2b) has been identified within RNA2, that is expressed from an additional subgenomic sgRNA4A; sgRNA4A encodes a suppressor of RNA interference (RNAi) [Bromoviridae is the occurrence of high concentrations of CP in the infected cells, but rather low amounts of non-structural proteins. The knowledge about replication cycle of PDV is still poorly understood, and limited mostly to the aspects of the structure and RNA genome organization.The genomes in all re (TLS) . Furthere (RNAi) . Another−6 Da), RNA2 (0.95 × 10−6 Da) [−6 Da) [The genome of PDV is divided into three single-stranded positive RNA segments RNA1 1.3 × 1010−6 Da) ,14, and  [−6 Da) ,16,17. E10−6 Da , [−6 Da) .Prunus avium trees of the cultivar Salomo. RNA1 had a length of 3374 nucleotides, and contained single open reading frame (ORF) identified as ORF1, comprising 3168 nucleotides and coding for a non-structural protein P1 (putative replicase component) (The monocistronic PDV-RNA1 was first sequenced by Ramptish and Estwell  from themponent) .Ilarviruses, as most of them carry the additional ORF 2b [Cucumber mosaic virus (CMV). Contrary to RNA1 and RNA2, PDV-RNA3 is a dicistronic molecule  and one German isolate (PDV-15/28) demonstrated that the number of nucleotides in RNA3 was 2129, similar to those in PDV-137. The homology of the RNA3 nucleotide sequences and the MP and CP amino acid sequences among PDV-D1, PDV-D2, and PDV-15/28 isolates are 96.9%, 93.8%, and 98.6%, respectively [Similarly to RNA1, PDV-RNA2 is a monocistronic molecule carrying ORF 2 . Determil ORF 2b   and Cucumber mosaic virus (CMV), it targets the tonoplast [PDV-RNA1 , and cones to ER , whereasonoplast ,31. Kozionoplast  observedonoplast .Moreover, PDV P1 localized strongly inside tonoplast A,B and iP2 protein—this protein contains 788 amino acids with molecular weight 89 kDa [Bromoviridae family [t 89 kDa . P2 is lt 89 kDa . Translae family .MP (movement protein)—a transport protein with molecular weight of 32 kDa, contains 293 amino acids, and is subjected to direct translation from RNA3 CP  RNA4 [Ilarvirus is rich in arginine (R) and/or lysine (K) residues that are responsible for binding to the 3′UTR hairpin structure of viral RNA [2+ [Prunus necrotic ring spot virus (PNRSV), a virus closely related to PDV, a specialized arginine-rich domain of CP displays the affinity to 3′UTR in both RNA3 and sgRNA4A. The domain is located between 25 and 50 N-terminal amino acids, probably in all Ilaviruses [Apple mosaic virus (ApMV) and PNRSV, a zinc finger motif was identified within CP, which probably increases the affinity for RNA binding.sg) RNA4 ,8. The Niral RNA ,38. Thisiral RNA . The pse RNA [2+ , which raviruses ,41. In tIlaviruses.The C-terminal region plays key role in CP dimerization. Bol  suggestsBromoviridae family like PDV) that collecting and individual packaging of RNA particles to the capsids are influenced by the spatial conformation of CP. Coat protein has always been considered to be responsible for supporting the replication process and later encapsidation, via in trans effects on RNA 1 and RNA2 but in cis—on RNA3. Rao [Moreover, Neelman and Bol  postulatNA3. Rao  also devNA3. Rao . At thisNA3. Rao . In the NA3. Rao  by usingNA3. Rao  have demNA3. Rao .Moreover, these authors observed PDV CP inside endoplasmic reticulum (ER) A, near pPhylogenetic analyses of sequences of PDV CP showed a high level of diversity among the diverse isolates from remote parts of the world. Kalinowska et al.  showed aBromoviridae could be absorbed by cellular membrane via pinocytosis [Frankliniella occidentalis), with the transmission level nearly 20%.The initial stage of viral infection is mechanical or biological discontinuity of the plant cell wall, to enable virus penetration through cellular membrane. So far, no specific receptors have been identified for penetration of plant viruses ,46. Howeocytosis ,47. In tocytosis . Greber ocytosis  showed aBromoviruses induce proliferation of endoplasmic reticulum (ER) in the form of spherules and/or the formation of small vacuoles from the external nuclear membrane, which constitute the site of initial viral infection. In the case of PDV, the presence of CP epitopes inside the tobacco spherules has been observed near vacuole rather than ER  requires the presence of CP molecule at both the 3′ and 5′ sides to maintain a correct RNA conformation. Moreover, the translated replicase proteins are stored in different cellular compartments, depending upon a particular virus. BMV stores them on the ER membrane, AMV, and CMV in the tonoplast or in the vicinity of the vacuole [Each viral particle encapsidates a single, translation-competent (+)ssRNA, and proteins 1a and 2a are first to be translated. The translation process is stimulated by the 3′TLS region that interacts with the cap at the 5′ side, and secures the circularization of the RNA template ,49. Pall vacuole ,50,51. K vacuole  suggeste vacuole . The resThe immunogold labeling of CP and P1 PDV demonstrates that, as for AMV, its replication is primarily associated with the tonoplast . The repBromoviridae, the two RNA strands are synthesized asymmetrically; one (−) RNA molecule accounts for approximately 100 (+) RNA molecules [Ilarviruses like PDV. It seems that CP acts as the factor responsible for asymmetrical synthesis of RNA strands [With olecules . Bol [28 strands  proposed strands . On the  strands ,56. The Plant viruses are specialized pathogens able to move in plants by using cellular structures of the host ,57,58. IBromoviridae.The long-distance transport of viruses is closely related to the transport that occurs from the phloem parenchyma cells or accompanying cells to the interior parts of the sieve elements in the phloem, where the virus moves rapidly . Then, the virus is actively transported together with assimilates inside the sieve elements ,64,65,66Tobacco mosaic virus (TMV)  does not require CP for transport (1), that is bound to the plus end of microtubules, which then implies the possibility of MP elongation during transport. Application of herbicides that block microtubule polymerization also blocked the transport of viral complexes [Cowpea chlorotic mottle virus (CCMV), the Bromoviridae representative. In this case the transport protein locates itself in plasmodesmata and causes considerable increase of the lower level of size exclusion limit, SEL. As a result, the plasmodesmata widened to the extent that the complex with the viral genome was able to penetrate through to the next cell [The first group includes two subtypes . One subransport . The virransport . Studiesomplexes . Howeveromplexes . The queomplexes ,84. Thusomplexes  for Cowpext cell ,86.Cucumber mosaic virus (CMV) [The second subtype depends on both CP and MP . It is cus (CMV) . Canto aus (CMV)  demonstrus (CMV)  suggesteus (CMV)  demonstrus (CMV) .Cowpea mosaic virus  or in two Bromoviridae: Alfalfa mosaic virus (AMV) and Brome mosaic virus (BMV) [NbNACa1. This protein has a similar sequence to MP–BMV, and is probably involved in the translocation of newly formed virions.The second group of movement mechanisms operates e.g., in us (BMV) ,92,93,94us (BMV) ,95. Kaidus (BMV)  establisNbNACa1 limited both the location of MP in plasmodesmata and the BMV movement. Both N- and C-MP termini are responsible for the induction of cytoskeleton protein synthesis, as demonstrated with corresponding MP mutants that blocked the formation of tubules in protoplasts [Mutations in the gene coding for toplasts . MP not toplasts . Deletiotoplasts . For thiBromoviridae members. The authors showed that the sequences of MP RBD (movement protein RNA binding domain) among PDV isolates were most similar to AMV, suggesting a similar mechanism of cell-to-cell transport, likely in the virion form. Van der Vossen et al. [Kozieł et al.  used bion et al.  demonstrn et al.  showed tn et al. .Bromoviridae members of known transport mechanisms revealed similarities between AMV and PDV [Apart from the local transport via plasmodesmata, an equally important issue is the fast long-distance movement to secure efficient viral infection within distant plant organs. Phylogenetic comparison of the amino acid sequences of the RBD region (residues 56–85) among PDV strains and with those in the  and PDV , suggest and PDV .Thus, PDV is most likely transported in a form of viral particles, not only via plasmodesmata but also over long distances.Long-distance transport, also referred to as systemic, often requires the virus to move from epidermis or parenchyma cells to penetrate the phloem parenchyma cells, followed by movement to the accompanying cells and/or sieve tubes. Until recently, it was believed that phloem is the only tissue involved in systemic transport. Indeed, Garbaczewska et al.  demonstrThere are two key points in the pathway to enter and to leave the sieve elements. Plasmodesmata connecting the sieve elements with the accompanying cells display a unique morphology, namely the occurrence of extensive branching at the side of the accompanying cells . At the Bromoviridae require unaltered CP to move systemically [Bromoviridae (including PDV), regardless of the differences in the mechanisms of cell-to-cell transport. Virion formation/encapsidation is the last stage in the virus life cycle. CMV is transported between cells as a ribonucleoprotein, and CMV encapsidation takes place within the wall elements of the tube after penetrating to smaller sieve tubes [Bromoviridae is most likely the phloem. Until now, there was no reports about the type of vascular tissue responsible for PDV transport. However, Kozieł et al. [Not all viruses can penetrate to the sieve elements through phloem parenchyma; the plasmodesmata that connect directly the phloem parenchyma to the sieve elements can constitute an alternative potential route to phloem ,101. Sieemically ,84,103. emically  showed temically ,105. It ve tubes . As demove tubes , CMV is ve tubes . Pallas ve tubes , Tenlladve tubes , and Bolve tubes  indicateł et al.  showed tResponse reactions to PDV infection are mainly undescribed. One example of available information is related to various symptoms induced by different strains in plant hosts . Among PSchattenmorelle fruits reached 94–96%. Reduction of the number of cherry fruits was accompanied by 9–15% height reduction in infected trees in comparison to healthy plants. In the case of sweet cherry, the range of fruit reduction was 30–90%. Brunt et al. [Regardless of type of disease, PDV infection manifests as a significant decrease in fruit yield. Nemeth  noted tht et al.  and Kajat et al.  observedt et al.  showed tBromoviridae have been only described for CMV. The effects strongly depend upon CMV gene expression at the beginning of infection. Arabidopsis thaliana plants (ecotype C24) were resistant to CMV strain Y (yellow) [RCY1 104 kDa protein that belongs to the family of nucleotide-binding leucine-rich repeat proteins (CC-NBS-LRR). These proteins are responsible for signal transduction related to ethylene and salicylic acid pathways, inducing necrotic changes in the infected tissue. Necrosis occurred at the inoculation sites, localizing the virus and limiting its spread to other plant organs [Arabidopsis thaliana was caused by the interaction of a CMV suppressor (coded by ORF2b) with a plant catalase. Alike for numerous plant viruses, CMV infection induces the RNA interference (RNAi)-based response, which is suppressed by CMV protein 2b. The interaction of RNAi response machinery with sgRNA2b triggers the silencing of CHLI, the host gene that is involved in chlorophyll biosynthesis, and thus, resulting in the yellowing of infected leaves [Until now, the mechanisms of plant response to (yellow) ,109. Thet organs  demonstrd leaves .Bromoviridae. The available data mostly concern local transport, missing however, the information about molecular or ultrastructural effects in the infected tissue. Further studies are required for identification of cell components that contribute to PDV infection, and for characterization of pathological changes in the infected plant tissue. It is likely that the accumulating knowledge will reveal new means of resistance against PDV, one of the most dangerous plant viruses debilitating the stone fruit trees.Prune dwarf virus remains an enigmatic pathogen, even in the context of accumulated knowledge about other


Bicoid gradient formation and function in the Drosophila pre-syncytial blastoderm. Published 17, February 2016Fixation and de-vitellinization section of the Materials and methods, for Embryo in situ hybridization the wash buffer was listed as containing 1% Triton X-100. The wash buffer should have been described as containing 0.1% Triton X-100, and the relevant sentence should be: Embryos were de-chorionated in 50% bleach, washed with 0.1% Triton X-100 and transferred to a 20 ml vial containing fixative , 50 mM EGTA (pH 8.0), 10.24% formaldehyde (Ultrapure), and shaken for 1.5 hour.In the published article the Pre-hybridization section of the Materials and methods, for Embryo in situ hybridization the pre-hybridization solution (HybS) was listed as containing 50% formaldehyde. HybS should have been described as containing 50% formamide. The correct sentence is: Embryos were incubated with rocking for: (1) 10 min at room temperature in 1 ml 1:1 hybridization solution (HybS):PBT ; (2) 10 min in 1 ml HybS at 55°C; (3) 45 min in 1 ml fresh HybS at 55°C; and (4) overnight in fresh HybS at 55°C.In the The article has been corrected accordingly.


During recent West Nile virus (WNV) outbreaks in the US, half of the reported cases were classified as neuroinvasive disease. WNV neuroinvasion is proposed to follow two major routes: hematogenous and/or axonal transport along the peripheral nerves. How virus spreads once within the central nervous system (CNS) remains unknown.Using immunohistochemistry, we examined the expression of viral antigens in the CNS of rhesus monkeys that were intrathalamically inoculated with a wild-type WNV. The localization of WNV within the CNS was mapped to specific neuronal groups and anatomical structures. The neurological functions related to structures containing WNV-labeled neurons were reviewed and summarized. Intraneuronal localization of WNV was investigated by electron microscopy. The known anatomical connectivity of WNV-labeled neurons was used to reconstruct the directionality of WNV spread within the CNS using a connectogram design. Anatomical mapping revealed that all structures identified as containing WNV-labeled neurons belonged to the pathways of motor control. Ultrastructurally, virions were found predominantly within vesicular structures (including autophagosomes) in close vicinity to the axodendritic synapses, either at pre- or post-synaptic positions , strongly indicating transsynaptic spread of the virus between connected neurons. Neuronal connectivity-based reconstruction of the directionality of transsynaptic virus spread suggests that, within the CNS, WNV can utilize both anterograde and retrograde axonal transport to infect connected neurons.This study offers a new insight into the neuropathogenesis of WNV infection in a primate model that closely mimics WNV encephalomyelitis in humans. We show that within the primate CNS, WNV primarily infects the anatomical structures and pathways responsible for the control of movement. Our findings also suggest that WNV most likely propagates within the CNS transsynaptically, by both, anterograde and retrograde axonal transport. West Nile virus (WNV) is a mosquito-borne neurotropic flavivirus that has emerged as a human pathogen of global scale. During recent WNV outbreaks in the US, half of the reported human cases were classified as neuroinvasive disease. Although much research has been done, there are still gaps in our understanding of WNV neuropathogenesis. While WNV neuroinvasion is proposed to occur by the hematogenous route and/or by axonal transport along the peripheral nerves, how virus spreads once within the central nervous system (CNS) remains unknown. In this study, we examined the expression of viral antigens in the CNS of monkeys that were intrathalamically inoculated with WNV. Next, we mapped the localization of WNV-infected neurons to specific anatomical structures, identified the intraneuronal localizations of WNV particles and investigated the role of neuronal connectivity in the spread of WNV within the CNS. Our results revealed that all structures containing WNV-labeled neurons belonged to the pathways of motor control. Virions were found in close vicinity to the axodendritic synapses, strongly indicating transsynaptic spread of the virus. Neuronal connectivity-based reconstruction of the directionality of transsynaptic virus spread suggests that, within the CNS, WNV can utilize both anterograde and retrograde axonal transport to infect connected neurons. West Nile virus (WNV) is a mosquito-borne neurotropic flavivirus that has emerged as a human pathogen of global scale . During in vitro system of compartmentalized neuronal cultures showed that WNV can spread between neurons in both anterograde and retrograde directions via axonal transport  and 10 dpi [n = 1]). WNV-infected animals developed a fulminant encephalomyelitis by 9/10 dpi . BrainsImmunohistochemical detection of WNV antigens in the CNS of rhesus monkeys was performed using WNV-specific primary antibodies in hyperimmune mouse ascitic fluid  and subsequent steps were according to previously described procedures ; howeveiewed in . To thisiewed in  to preveiewed in ,62, WNV Intraneuronal movement of WNV most likely involves microtubules –65 and tSince the directionality of virus spread cannot be determined based on the “snapshots” revealed by immunohistochemistry and EM, we next attempted to reconstruct the directionality of transsynaptic spread of WNV based on the neuroanatomical connectivity between identified infected structures. For this purpose, we compiled known connectivity information and designed connectograms to capture and visualize the complex neuroanatomical connections between WNV-infected structures in an intuitive and concise way. We created two connectograms  showing Each connectogram contains two concentric rings and a black core. The names for each major CNS region are given on the outside periphery of outermost ring  in no particular order. For the outermost ring, each CNS region was assigned a spectrum domain color counterclockwise as follows: subcortical regions (red), motor cortex (orange), midbrain (yellow), spinal cord (green), cerebellum (light blue), medulla oblongata (blue), and pons (purple). The next ring toward the center of the connectogram is divided in sixteen segments. Each segment representing a specific structure/type of neurons includes an abbreviation and is assigned a unique color within the spectrum domain color of the corresponding larger anatomical CNS region. Within the black core, the directions of proposed spread of WNV between the structures/types of neurons are depicted by arrows . Each arrow has the same color as the segment representing a structure/type of neurons from which it originates. The direction of the arrow indicates a proposed direction of virus spread between one neuronal order to the next. The proposed orders of neurons are indicated by circled numbers.Assuming that the neurons of motor thalamus  represent the neuronal order “0” (starter cells), the next neuronal order “1” will be neurons of the deep cerebellar nuclei (DCN), basal ganglia (BG), and corticospinal motor neurons (CSMN) . The arrBy the terminal stage of neuroinfection  in the medulla oblongata and Clarke’s column (CC) in the spinal cord is intriguing. These structures relay proprioceptive information from the upper (ACu) and lower (CC) parts of the body to the cerebellum. The axons of these relay neurons terminate as mossy fibers on the granule neurons. It is conceivable that these structures may have been infected by retrograde spread of the virus along the dorsal spinocerebellar tract from granule neurons. However, we found very few WNV-labeled granule neurons in the cerebellum . SimilarIn summary, our reconstruction of the directionality of WNV spread within the CNS of intrathalamically inoculated NHPs suggests both anterograde and retrograde axonal transport. The connectograms  show eleCollectively, our results of the anatomical and ultrastructural mapping of WNV neuronal infection in the primate CNS, together with the connectivity-based reconstruction of the directionality of virus spread strongly suggest the following:WNV exclusively infects neurons residing within the specific anatomical structures, each of which plays a major role in the control of movement by the CNS;in vivo ultrastructural evidence of the pre- and post-synaptic localization of WNV particles;WNV can spread within the CNS transsynaptically, as demonstrated by our first WNV can spread in both, anterograde and retrograde directions through axonal tracts comprising motor control pathways, which is supported by the connectivity-based reconstruction of virus spread.Progression of WNV neuroinfection transsynaptically along specific pathways governing motor control in both, anterograde and retrograde directions suggested by this study may open a way for future therapeutic approaches. Although it seems unlikely that antiviral interventions would be justified for asymptomatic or self-limiting WNV cases in humans, we cannot neglect the necessity for development of rational treatments of flavivirus neurological disease. Our findings imply that the focus of such treatments should not only be on limiting virus replication but also on blocking neuron-to-neuron virus transmission, thus preventing further damage to the CNS.S1 FigA) 3 dpi ; (B) 7 dpi ; and (C) 9/10 dpi . The limit of virus detection was 1.7 log10 PFU/g of tissue (gray-shadowed in the center of each graph). The motor thalamus (site of virus inoculation) was the major site of virus replication at an earliest time point (3 dpi). Although in lower titers, WNV was also detected in the basal ganglia, motor cortex, and midbrain. Over the course of next 4 days, the virus continued to replicate in the above regions, but the most dramatic increase in virus loads occurred in the remote CNS regions, such as pons/medulla oblongata, cerebellum, and spinal cord. During the next 2–3 days (9/10 dpi), all remaining animals developed a fulminant encephalitis. The pons/medulla oblongata, cerebellum, and spinal cord  were the structures harboring the highest virus loads at the terminal stage of the CNS infection with WNV. Thalamus, cortex, basal ganglia, and midbrain supported WNV replication transiently. The virus loads in these structures returned to their initial levels by 9/10 dpi even though animals succumbed to encephalitis at that time. Thus, after transient replication at the site of inoculation and neighboring structures, WNV had spread to the pons/medulla oblongata, cerebellum, and spinal cord and continued to replicate there.Radar graphs were constructed using previously reported virus titer data  for clar(TIF)Click here for additional data file.S2 FigWNV antigens are present in the perikarion (yellow arrow), axon (red arrowhead), and dendrites (red arrows) of a Purkinje cell (7 dpi). The underlying granule cells are not labeled in this field. Scale bar: 10 μm.(TIF)Click here for additional data file.S3 FigA) Cerebellar cortex (likely a Purkinje cell) (9 dpi): newly assembled immature virus particles accumulate within the endoplasmic reticulum (ER) creating PCA. (B) Another PCA in the cerebellar cortex at higher magnification (9 dpi). One row of the array is outlined by yellow dashed lines and the height of the row (40 nm) is consistent with the approximate size of assembling virions. (C) This PCA is situated close to the nucleus in the ventral horn of lumbar spinal cord  (9 dpi). Note a continuity of all shown PCAs with the membranes of endoplasmic reticulum (yellow arrowheads). Nu, nucleus; Nuo, nucleolus. Scale bars: (A) 500 nm;  100 nm.Perikarial sites of virus replication/accumulation appear as paracrystalline-like arrays . ((TIF)Click here for additional data file.S4 FigA) and degenerating granule neurons of WNV-inoculated animals (B), both at 9/10 dpi. Note a disappearance of Purkinje cells in B. Purkinje cells in A appear normal and do not express NeuN. Scale bars (in μm) are shown at the bottom of each image as well as images provided by the magnifier tool of Image Scope (Aperio).Shown is the NeuN immunoreactivity highlighting a normal granule neurons of mock-inoculated animals ((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


H9N2 avian influenza viruses are endemic in poultry in Asia and the Middle East. These viruses sporadically cause dead-end infections in pigs and humans raising concerns about their potential to adapt to mammals or reassort with human or swine influenza viruses. We performed ten serial passages with an avian H9N2 virus (A/quail/Hong Kong/G1/1997) in influenza naïve pigs to assess the potential of this virus to adapt to swine. Virus replication in the entire respiratory tract and nasal virus excretion were examined after each passage and we deep sequenced viral genomic RNA of the parental and passage four H9N2 virus isolated from the nasal mucosa and lung. The parental H9N2 virus caused a productive infection in pigs with a predominant tropism for the nasal mucosa, whereas only 50% lung samples were virus-positive. In contrast, inoculation of pigs with passage four virus resulted in viral replication in the entire respiratory tract. Subsequent passages were associated with reduced virus replication in the lungs and infectious virus was no longer detectable in the upper and lower respiratory tract of inoculated pigs at passage ten. The broader tissue tropism after four passages was associated with an amino acid residue substitution at position 225, within the receptor-binding site of the hemagglutinin. We also compared the parental H9N2, passage four H9N2 and the 2009 pandemic H1N1 (pH1N1) virus in a direct contact transmission experiment. Whereas only one out of six contact pigs showed nasal virus excretion of the wild-type H9N2 for more than four days, all six contact animals shed the passage four H9N2 virus. Nevertheless, the amount of excreted virus was significantly lower when compared to that of the pH1N1, which readily transmitted and replicated in all six contact animals. Our data demonstrate that serial passaging of H9N2 virus in pigs enhances its replication and transmissibility. However, full adaptation of an avian H9N2 virus to pigs likely requires an extensive set of mutations. Avian influenza viruses (AIVs) of the H9N2 subtype emerged from the natural waterfowl reservoir and have become endemic in poultry in Asia and the Middle East since the early 1990s . In 1999in vitro [et al. reported that HA-Q226L increased virus replication and direct contact transmission of H9N2 in ferrets, which are considered the “gold standard” model for human influenza pathogenesis and transmissibility studies [Influenza A virus infection is mediated via the attachment of the viral hemagglutinin (HA) receptor-binding site (RBS) to sialyloligosaccharides present on the host cell surface. Avian influenza viruses preferentially bind sialic acids linked to galactose by an α2,3 linkage . Conversely, most human and swine influenza viruses more readily bind to receptors that contain terminal α2,6-linked sialic acid  . Becausein vitro , 21. In  studies . However studies . This raPigs are important natural hosts for influenza A viruses. These animals have a similar sialic acid receptor distribution as humans , and theThe experiments were authorized and supervised by the Ethical and Animal Welfare Committee of the Faculty of Veterinary Medicine of Ghent University, with the EC2013/63 number.A/quail/Hong Kong/G1/1997 (H9N2) was isolated from quail at a live bird market and underwent seven passages in eleven-day-old embryonated chicken eggs, three passages in Madin-Darby Canine Kidney cells (MDCK), and two passages in pigs. After each pig passage the virus was also passaged once in MDCK cells to grow a large stock to be used in the experiment. The virus stock used in the experiment shares at least 99% identity at the nucleotide and protein levels with the original A/quail/Hong Kong/G1/1997 virus . This strain was selected because it is representative for the G1-like lineage and contains the HA-Q226L substitution that is known to enhance human-like receptor specificity.A/California/04/2009 pandemic H1N1 (pH1N1) virus underwent three passages in MDCK cells, one in eggs and a last passage in MDCK cells before use. This virus is representative of the pH1N1 viruses that circulate worldwide in swine.Forty-seven three-week-old piglets were purchased from a commercial herd that is serologically negative for swine influenza and porcine reproductive and respiratory syndrome virus. Before the start of the experiments, all animals were free of influenza virus antibodies as demonstrated by a commercial blocking anti-influenza A nucleocapsid ELISA (ID-VET) and by virus neutralization (VN) tests using A/swine/Belgium/1/98 (H1N1), A/swine/Flanders/1/98 (H3N2), A/swine/Gent/7625/99 (H1N2) swine influenza viruses (SIVs), as well as A/California/04/09 (pH1N1). Upon arrival, the animals were housed in a biosafety level-2 (BSL-2) HEPA filtered isolation unit for at least two days to allow their acclimatization.6.2 TCID50 of A/quail/Hong Kong/G1/1997 (H9N2). For inoculation unanesthesized pigs were held in a vertical position with the neck stretched. The inoculum (1.5 ml per nostril) was gradually instilled into the nasal cavity by insertion of a fifteen mm canula attached to a syringe. To determine virus excretion in the nose, individual pigs were swabbed daily from 0 to four days post-inoculation (dpi). At four dpi both animals were humanely euthanized by slow injection of an overdose (≥ 100 mg/kg) of pentobarbital in the jugular vein. At the time of necropsy, the following tissue samples were collected for virus titrations: nasal mucosa respiratory part , nasal mucosa olfactory part (i.e. ethmoid labyrinth), trachea  and five different samples representative of the entire lung. The nasal mucosae (respiratory part) were pooled and a 20% (w/v) tissue homogenate was prepared and used to inoculate two pigs with this passage one virus inoculum. Ten blind  serial passages were performed in this way. Nasal swabs from all pigs, tissue samples and the inocula used for the subsequent passages were titrated in MDCK cells.For the first passage, two pigs were housed in a biosafety level-3 (BSL-3) HEPA filtered isolator and inoculated intranasally with three ml of phosphate-buffered saline (PBS) containing 106.5 TCID50 of the respective virus. Forty-eight hours pi, two pigs were introduced in each isolator allowing direct contact with the inoculated pig. All animals were clinically monitored and nasal swabs for virus isolation were collected daily from all pigs during eleven days after cohousing. Sixteen dpi, the animals were relocated to a BSL-2 HEPA filtered isolation unit. Serum samples were collected at 0, 16, 23 and 30 dpi and 0, 14, 21 and 28 days post-contact (dpc).The inoculum giving the highest virus isolation rates and virus titers throughout the respiratory tract and in nasal swabs was examined for its transmission between pigs. We compared the transmissibility of mentioned virus to that of the parental H9N2 and that of the pH1N1. Nine three-week-old pigs were used. At 0 dpi (= 2 days before the start of contact transmission), three pigs were housed in three separate BSL-3 HEPA filtered isolators (one index pig per isolator) and intranasally inoculated with 10To compare the single-step growth curves of the parental, passaged virus and pH1N1 confluent monolayers of MDCK cells were inoculated with an identical multiplicity of infection of 5 MOI per cell at 37°C. After one hour of incubation the cells were washed with phosphate-buffered saline (PBS) containing 10 IU/ml penicillin and 10 μg/ml streptomycin to remove unbound virus particles and overlaid with Minimal Essential Medium (MEM) containing supplements . The three viruses were tested in triplicate, supernatants were collected at 0, 4, 6, 12 and 24 hours post inoculation and titrated in MDCK cells as described below.2, virus positive MDCK cells were visualized by immunoperoxidase staining. The cells were fixed with 4% paraformaldehyde for ten minutes at room temperature and subsequently incubated with mouse anti-NP monoclonal HB-65 antibody  for two hours. Incubation with horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody  for one hour was followed by a development step with H2O2 as substrate and 3-amino-9-ethyl-carbazole (AEC) as precipitating agent. Virus titers were calculated by the method of Reed and Muench (1938) and expressed as log10 50% tissue culture infective doses (TCID50) per 100 milligram  or per gram (tissues).Cotton swabs were weighed before and after collection to determine virus titers per 100 milligram nasal secretions. Nasal swab samples from both nostrils were suspended in 1 ml PBS supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin and mixed vigorously at 4°C for 1 hour. Tissue samples were weighed and grounded in PBS with 10 IU/ml penicillin and 10 μg/ml streptomycin to obtain 20% (w/v) tissue homogenates. Nasal swab samples and tissue homogenates were clarified by centrifugation and stored at -70°C until titration on MDCK cells. Briefly, confluent monolayers of cells were inoculated with 10-fold serial dilutions of the sample. After five days of incubation at 37°C with 5% CO50 of virus per well as previously described [50 of MDCK cell-grown virus. MDCK cells (800.000 cells per ml) were incubated with the virus-serum mixture for 24h, after which virus positive cells were visualized by immunoperoxidase staining.Virus neutralization tests were performed on MDCK cells in 96-well plates, using 100 TCIDescribed . Briefly10 TCID50 per 100 milligram nasal secrete or gram tissues, samples that tested negative for virus were given a numeric value of 1.6 log10 TCID50 per 100 milligram nasal secrete or gram tissues. Samples that tested negative in the virus neutralization assay were assigned a value corresponding to half of the minimum detectable titer. For each transmission experiment, the basic reproduction ratio (R0) was estimated on the basis of the outcome of the experiment  with use of the maximum likelihood estimator. Thus, the most likely reproduction ratio was estimated using the total number of animals in the experiment, the total number of susceptible and infectious animals at the beginning of the experiment, and the number of animals that became infected during the experiment. A pig was considered to be infected when it experienced seroconversion. The 95% confidence intervals (CIs) were constructed symmetrically around the estimated value of R0 [Because the detection limit was 1.7 logue of R0 .In addition, nasal virus shedding of individual pigs was quantified by calculation of the area under the curve (AUC). The means of the AUCs were compared between different viruses using standard two–sample Mann-Whitney U tests. Differences were considered significant when p<0.05. GraphPad Prism Software, version 5, was used for statistical analyses.Amino acid sequences of HA proteins from H9N2 viruses isolated from swine and humans in the field were obtained from Genbank. Different human and swine amino acid sequences were compared with the HA of the A/quail/Hong Kong/G1/1997 (H9N2) strain, with special attention to amino acids at positions 190, 225, 226 and 228 because these contribute to transmission between mammals , 16. AmiGCCGGAGCTCTGCAGATATCAGCGAAAGCAGG) and CommonUni12A (GCCGGAGCTCTGCAGATATCAGCAAAAGCAGG). Subsequently, all eight genomic segments were amplified in one PCR reaction using a mix containing an 1:1 mix of CommonUni12G and CommonUni12A cDNA, primer CommonUni13 (GCCGGAGCTCTGCAGATATCAGTAGAAACAAGG) (200 nM) and the Phusion High Fidelity polymerase (Thermo Scientific) [We compared the genetic diversity of the parental wild-type H9N2 virus and two samples from the passage four virus : one from the nasal mucosa  and another from the lungs (lower respiratory tract). The RNA from these samples was isolated using the QIAamp Viral RNA Mini Kit (Qiagen). cDNA was synthesized with the Transcriptor First Strand Synthesis kit (Roche), as described previously . Two sepentific) –37. RT-Pentific) , with th150 ng of each purified RT-PCR sample was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 400 bp. The fragment ends of 50 ng of these DNA fragments were repaired using the NEBNext Ultra DNA Library Preparation kit (New England Biolabs), followed by addition of the adaptors by using the NEBNext Multiplex Oligos for Illumina kit . The resulting fragments were size-selected at 300 to 400 bp using Agencourt AMPure XP bead sizing (Beckman Coulter). Afterwards, indexes were added in a limited-cycle PCR (10 cycles), followed by purification on Agencourt AMpure XP beads. Fragments were analyzed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies) before loading on the sequencing chip. After the 2×250 bp MiSeq paired-end sequencing run, the data were base called and converted to Illumina FASTQ files (Phred +64 encoding).de novo assembly of the sequencing reads for the A/quail/Hong Kong/G1/1997 (H9N2) virus stock sample, since this virus stock was used to start the virus adaptation. This consensus sequence is available through GenBank accession number (KY785896-KY785903). The raw sequencing data were submitted to the NCBI Sequence Read Archive where they can be found under project number SRP078326.Sequence data analyses were performed on the resulting Illumina FASTQ files (Phred 64+ encoding) using CLC Genomics Workbench (Version 7.0.3) following the analysis pipeline as described . The proTo evaluate if the mutations described in passage four were maintained throughout the subsequent passages, the partial coding sequences of HA, M and PB1 genes were determined for all inocula from passage five onwards by direct Sanger sequencing of overlapping RT-PCR products. RNA was extracted from virus with the QIAamp Viral RNA Mini Kit . Reverse transcription and amplification of the genes was done by one-step RT-PCR  using custom specific primers (primer sequences available upon request). After amplification, RT-PCR products were purified using the Nucleo Spin Gel and PCR clean up  DNA purification kit. Samples were sequenced by GATC Biotech AG  using the Sanger ABI 3730 xl platform. Sequencing was done with custom designed primers (primer sequences available upon request). HA, M and PB1 gene segments were compared at the nucleotide and amino acid (aa) level using MEGALIGN program within the DNASTAR 5.01 software package . The complete genome sequences of A/quail/Hong Kong/G1/1997 (H9N2) were already available from GenBank (GenBank accession numbers: AF156378.1 (HA), AF156463.1 (M), AF156421.1 (PB1). The sequences generated in this study are available through GenBank accession numbers (KY785881-KY785895)The prime goal of this study was to assess the capacity of an avian H9N2 virus to adapt to swine. For this, ten blind serial passages were performed in influenza naïve pigs. Animals were clinically scored and samples from the upper and lower respiratory tract were isolated to monitor viral loads. None of the pigs showed clinical signs. An overview of the virus titers in nasal swabs of individual pigs through the different passages is shown in On day four after inoculation, pigs were euthanized and samples from the upper and lower respiratory tract were collected to determine the viral load. Infectious H9N2 virus was found in the respiratory tract of all pigs except for those of passage ten . The numTo assess the level of adaptation of the virus after serial blind passages in pigs, three different viruses were tested in three independent direct contact transmission experiments. As a positive control, we used the A/California/04/2009  pH1N1 virus, which is representative of swine-adapted influenza viruses –40. The 10 TCID50/100 mg of secrete. All inoculated animals developed antibodies against the homologous virus  for A/Qa/HK/P0 and 2.27 (95% CI: 0.54–9.29) for A/Qa/HK/P4. Because all A/Cal/04/09 contact pigs became infected, the estimated R0 value was ∞ (95% CI: 0.83-∞) for this experimental setting. In summary, A/Qa/HK/P4 showed enhanced transmission compared to A/Qa/HK/P0, but both viruses transmitted less efficiency than A/Cal/04/09.Only one out of six direct contact animals in the A/Qa/HK/P0 group shed virus during more than five days resulting in a mean AUC of 2.8. In contrast, all six animals that were co-housed with an A/Qa/HK/P4 virus inoculated pig, excreted virus for five days or more. The virus excretion was not homogeneous over time (mean AUC of 5.9) and peak viral loads in contact animals were approximately 100-fold lower compared to the viral titers in the nasal excretions of the A/Qa/HK/P4 index pigs . In contin vivo transmission of the A/Qa/HK/P4 virus did not have an impact on the in vitro replication kinetics of the virus in a continuous cell line.In addition, we evaluated the replication kinetics of the viruses tested in the transmission experiments by determining the single-step growth curve in MDCK cells for each virus. As demonstrated in de novo. Illumina MiSeq deep sequencing was performed on full genome RT-PCR products of the samples. Nucleotide variants that differ from the consensus sequence of the A/quail/Hong Kong/G1/1997 (H9N2) virus stock sample that was used to start the serial passages and that occur at a frequency of 5% or more are shown in We compared the viral diversity present in the H9N2 virus stock that was used to start the serial passages with the viral diversity present in the virus sampled after passage four from the nasal mucosa (after a single round of amplification on MDCK cells) and the lung. The viral diversity was analysed in virus (pooled from two pigs) sampled after passage four, since higher virus shedding and higher viral replication in the upper and lower respiratory tract was obtained upon inoculation with this virus. It was anticipated that a comparison of the viral diversity present in the virus from the upper and lower respiratory tract would enable us to determine if these two airway compartments favour the acquirement of a different set of mutations for optimal adaptation. In addition, sequencing of the virus stock is needed to determine if the detected pig mutations were already present in the virus inoculum at the start of the serial passages or if they arose To evaluate if the substitutions present at the highest frequency in the passage four virus  were maintained during the subsequent passages we sequenced the HA, M and PB1 coding-genes of the virus present in the inocula used to infect pigs from passages six to ten. Interestingly, the substitutions observed in the A/Qa/HK/P4 nasal mucosa were maintained during all passages. However, the substitutions detected in the lung sample were no longer found in further passages.Avian H9N2 viruses play a pivotal role in the ecology of influenza in poultry in Eurasian countries. Since the late 1990’s, recurrent dead-end infections with H9N2 viruses have been reported in pigs and humans , 41, 42.et al. (2009) inoculated pigs intranasally with A/chicken/Belgium/818/1979 (H9N2) and found that this strain was not excreted at all by the inoculated pigs [et al. (2012) used the same A/quail/Hong Kong/G1/1997 (H9N2) strain to inoculate pigs intratracheally and they detected nasal virus excretion in only one out of twelve inoculated animals, albeit at low virus titers [0. This virus showed also higher transmission efficiency when compared with previous studies with H9N2 as well as other avian influenza virus subtypes in pigs. In all of these studies the direct contact pigs excreted no or minimal amount of virus [in vivo phenotype and in vitro replication rates on continuous cell lines. Furthermore, from passage seven onwards the virus replication progressively decreased until it was lost. This result may be explained by the generation of a narrow bottleneck after passage four due to the strong genetic pressure posed by the experimental set up, which may decrease the diversity of the viral population which can hamper the adaptation process [Our results confirm the susceptibility of pigs to intranasal inoculation with avian H9N2 influenza viruses reported in previous experimental studies and in nature , 29, 30.ted pigs . Though s titers . The vers titers . Moreoves titers , 25, 46.s titers . For H9,s titers , 46, 48.s titers . Our datof virus , 29, 30. process , 50.de novo in both the upper and lower respiratory tract after four passages and it was associated with a higher replication efficiency during passage five. Interestingly, this mutation was also present in all forty-seven swine and twelve human H9N2 field isolates available in Genbank  and Obadan et al. showed that reassortant H9N2 viruses containing pH1N1 internal genes improved replication and transmissibility in pigs, although they were still less efficient than pH1N1 virus. In previous H9N2 adaptation studies in mammals, efficient transmission was only reached with ferret-passaged reassortant H9N2 viruses containing either human seasonal H3N2 or pH1N1 internal genes [In summary, our results do not reject the theory of the pig as an intermediate host for H9N2 AIVs, but demonstrate that adaptation of these viruses to pigs is a complex process and that the mutations selected after four passages in pigs were not sufficient to confer efficient pig-to-pig transmission. It also shows that additional molecular features may be required for efficient transmission of avian H9N2 viruses in pigs. Qiao al genes , 57. The