Rabbit haemorrhagic disease virus (RHDV) type 2 (GI.2/RHDV2/b) is an emerging pathogen in wild rabbits and in domestic rabbits vaccinated against RHDV (GI.1). Here we report the genome sequence of a contemporary RHDV2 isolate from the Netherlands and investigate the immunogenicity of virus-like particles (VLPs) produced in insect cells. RHDV2 RNA was isolated from the liver of a naturally infected wild rabbit and the complete viral genome sequence was assembled from sequenced RT-PCR products. Phylogenetic analysis based on the VP60 capsid gene demonstrated that the RHDV2 NL2016 isolate clustered with other contemporary RHDV2 strains. The VP60 gene was cloned in a baculovirus expression vector to produce VLPs in Sf9 insect cells. Density-gradient purified RHDV2 VLPs were visualized by transmission electron microscopy as spherical particles of around 30 nm in diameter with a morphology resembling authentic RHDV. Immunization of rabbits with RHDV2 VLPs resulted in high production of serum antibodies against VP60, and the production of cytokines (IFN-γ and IL-4) was significantly elevated in the immunized rabbits compared to the control group. The results demonstrate that the recombinant RHDV2 VLPs are highly immunogenic and may find applications in serological detection assays and might be further developed as a vaccine candidate to protect domestic rabbits against RHDV2 infection. Lagovirus) was first reported in 1984 in China as the causal agent of Rabbit Haemorrhagic Disease (RHD), an acute and highly lethal disease in wild and domestic rabbits (Oryctolagus cuniculus). Two years after the initial outbreak, RHDV reached the European continent. RHDV is highly contagious and can have a mortality rate of around 80–100% in adult rabbits [Rabbit haemorrhagic disease virus  in the Netherlands examined numerous dead rabbits that were found to be RHDV2-positive. In January 2017, the first case of RHDV2 in the Netherlands was confirmed as the cause of death in a wild hare. The dead hare presented with severe acute inflammation in the liver and spleen, and RHDV2 infection was confirmed by PCR analysis.At present, the emergence of RHDV2 raises concerns regarding the impact of the disease among domestic and wild rabbit populations. RHDV2 is replacing existing strains of RHDV and is expected to cause severe problems to the rabbit industry. Thus, there is a clear need for a safe and effective RHDV2 vaccine. The traditional approach is an inactivated vaccine, typically produced from the liver collected from artificially infected rabbits. Because inactivated RHDV2 vaccines may not always confer full protection against RHD, alternative vaccines including those based on a myxoma virus vector are in development. Another promising approach to develop safe and effective vaccines is the application of virus-like particles (VLPs). VLPs have been shown to induce a potent B-cell mediated immune response, and can induce highly effective CD4+ T cell proliferative responses and a cytotoxic T lymphocyte (CTL) response ,24,25,26In the present study, we identified the complete genome sequence of a contemporary RHDV2 strain from the Netherlands (NL2016) and expressed the VP60 capsid protein of RHDV2-NL2016 to generate VLPs. Subsequently, we have evaluated the immunogenicity of the RHDV2 VLPs in rabbits.All animal experiments involved in these studies were conducted following the guide for care and use of laboratory animals of the Ministry of Science and Technology of the People’s Republic of China. All experiments were performed in a secondary biosecurity laboratory. All animal procedures were approved by the Institutional Animal Care and Use committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (permit number: 20180511). Every effort was made to minimize suffering.O. cuniculus, no. 3151221028) was ground with liquid nitrogen in a mortar and total RNA was isolated with Trizol . Primers . The phylogenetic tree was constructed by Mega5.1 using a selection of VP60 sequences . Next, the VP60 gene was transferred to the Gateway destination vector pDEST10 , which drives expression of N-terminal his-tagged transgenes from the strong baculovirus polyhedrin promoter. The resulting pDEST10-VP60 plasmid was sequenced and used for the construction of a recombinant baculovirus.Primers with attB1/2 recombination sites and Spodoptera frugiperda Sf9 insect cells were maintained in Sf-900ⅡSFM medium  at 28 °C. The plasmid pDEST10-VP60 was transformed into DH10Bac Escherichia coli competent cells, to generate recombinant bacmid DNA . The recombinant bacmid DNA was used to transfect Sf9 insect cells to generate the corresponding recombinant baculovirus, Bac-RHDV2-VP60, using Cellfectin II transfection reagent . Viral titres were determined by an end-point dilution assay (EPDA) using Sf9 cells. Subsequent infections were performed by adding virus to cells at a multiplicity of infection (MOI) of 10 TCID50 units per cell. Cells were incubated for 2 h and then the medium was changed by fresh medium. Plaque purification was performed to isolate a pure viral stock. Briefly, the Sf9 cell monolayers were incubated with 10-fold serial dilutions of baculovirus Bac-RHDV2-VP60. After infection for 2 h, the medium containing virus was replaced by fresh medium, and the cells were overlaid with 1% low melting-point agarose  in an Sf-900™ Medium (1.3×) medium  and incubated at 28 °C. After 7 days, several plaques were randomly picked from serial dilutions. Later the recombinant baculoviruses from individual plaques were amplified, titrated and then used to inoculate Sf9 cells.Sf9 cells were infected with Bac-RHDV2-VP60 at a multiplicity of infection (MOI) of 10 followed by incubation at 28 °C for three days. Indirect immunofluorescence assay (IFA) was used to check the expression of VP60 in Sf9 cells. Sf9 cells were fixed with 4% paraformaldehyde, then an anti-His mouse monoclonal antibody was used for the first antibody and a FITC-conjugated rabbit-anti-mouse antibody was used for detection. To check the expression by western blot analysis, supernatants and cell lysates were collected. Cells were lysed in RIPA buffer  and anti His-tag antibodies were used for VP60 detection. Furthermore, Sf9 cells were infected with Bac-RHDV2-VP60 at an MOI of 10 to test the VP60 expression level at different timepoints post infection. Culture fluids and cells were collected from six-well plates, in volumes of 2 mL, and lysed by sonication with a 2 mm Ultrasonic Amplitude Transformer giving 5 s pulses at 300W with intervals of 9 s for a total of 10 min. Next, the lysates were clarified by centrifugation at 8000 rpm for 15 min to remove the cell debris. Then, the lysates were subjected to ultracentrifugation at 40,000 rpm for 3 h to pellet the VLPs. Finally, the pellets were resuspended in 80 μL PBS and analysed by SDS-PAGE and western blot analysis.To test the ability of RHDV2 VP60 to self-assemble into VLPs, the supernatants of lysed Bac-RHDV2-VP60 infected Sf9 cells were clarified and VLPs were pelleted as above and then subjected to caesium chloride density gradient centrifugation. The VP60 band was isolated with a needle, diluted in PBS and centrifuged again to remove the CsCl. Finally, the pellet was resuspended in PBS. The caesium chloride purified material was checked by TEM via negative staining with 0.5% aqueous uranyl acetate. The grids were observed with a transmission electron microscope  operating at 80 kV.To prepare RHDV2 VLPs for immunization trials, Sf9 cells were infected at an MOI of 1 for 72 h with plaque-purified Bac-RHDV2-VP60. Cells were lysed ultrasonically in the growth medium, and then clarified by centrifugation at 8000 rpm for 15 min. The supernatants were collected and the total protein concentration was measured by a BCA assay and then used for immunization experiments. To formulate the experimental vaccines, MONTANIDE ISA 201VG adjuvant  was mixed at a ratio of 1:1 with VLPs by Emulsifying Equipment  for injection. Sf9 cell lysates from uninfected cells were subjected to the same treatments to generate the negative control antigen.E. coli using a PGEX-4T-1 expression vector, followed by blocking the plates with 5% skimmed milk diluted in PBS-Tween (PBST) at 37 °C for 1 h. After washing with PBST, the plates were incubated for 1 h with 100 μL of serum samples (1:500 diluted in PBS), and then washed with PBST. Next, 100 μL of HRP-conjugated goat anti-rabbit IgG  was added at a dilution of 1:10,000 and again incubated for 1 h. The plates were washed three times and then 100 μL TMB substrate  was added and the plate was incubated in the dark for 15 min. Finally, 50 μL 2 M sulfuric acid was added to stop the colour development, after which the absorbance was measured at 450 nm by using a microplate reader . All samples obtained were measured in quadruplicate.Fifteen 8-week-old New Zealand White Rabbits which lacked anti-RHDV antibodies were purchased from the Laboratory Animal Centre of the Shanghai Veterinary Research Institute and raised in pathogen-free, isolated cages. All animals used in the immunization trials were approved in compliance with the guidelines of the Animal Research Ethics Board of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS). Rabbits were immunized with 2 mg VLPs, 5 mg VLPs or with control antigen via subcutaneous injection. Serum samples were collected from each immunized rabbit at 0, 7, 14 and 21 days after immunization. RHDV2-specific antibodies in immunized rabbits were detected by indirect ELISA assays as described . BrieflyThe level of cytokines at weeks 0, 1, 2 and 3 post-immunization was examined to investigate the efficiency of the cellular immune response induced by VLPs. Therefore, the serum collected above was used to determine the levels of the Th1-Type I related cytokine IFN-γ, while IL-4 levels were measured to check for Th2-type 2 cytokines. Commercial Elisa Kits  were used for these assays.p < 0.05 was considered significantly different.All statistical analyses were performed using GraphPad Prism Version 5. The data including antibody response and cytokine production were compared by one-way ANOVA among different groups. A O. cuniculus) was found dead on 22 December 2015 in a garden in Nederweert, in the province of Limburg in the Netherlands. Post-mortem examination showed it was a young adult male, in good condition (based on muscle development and degree of fat storage). The trachea mucosa was hyperaemic, and the anterior lung lobes were dark red and consolidated. The liver was light brown and soft. The gastrointestinal tract was filled with feed remains, and a few coccidia and cestode and nematode parasites were observed . Main histopathologic findings were diffuse swollen and hyper-eosinophilic hepatocytes, diffuse hyperaemia and necrosis of lymphoid cells in the spleen, and pulmonary hyperaemia with moderate to severe intra-alveolar oedema and focal haemorrhage. The observed per acute hepatic necrosis, splenic necrosis and acute pneumonia are fatal lesions consistent with RHDV2 infection, which was confirmed by PCR analysis.A specimen of the European rabbit  A, of whiIn order to produce RHDV2 VLPs in insect cells, the VP60 ORF was PCR amplified and cloned downstream of a His-tag into the expression plasmid pDEST10 under control of the very strong polyhedrin promoter. The pDEST10-VP60 plasmid was used to construct a recombinant baculovirus bacmid, which was then transfected into Sf9 insect cells to generate the recombinant baculovirus Bac-RHDV2-VP60.An indirect immunofluorescence assay (IFA) using anti-his antibodies showed that VP60 accumulated to high levels upon transfection of bacmid DNA A. SupernTo determine whether the overexpressed RHDV2 VP60 protein self-assembled into VLPs, CsCl density gradient purified material was visualized by electron microscopy analysis. TEM showed a high concentration of spherical particles A with diThe RHDV2 VLPs were then used to immunize rabbits. Groups of five rabbits were immunized subcutaneously with different dosages of VLPs or with control antigen. Serum was collected every week and RHDV2-specific antibodies in immunized rabbits were detected by indirect ELISA assays. The antibody titers of rabbits immunized with VLPs were shown to increase with time and are statistically different from the control group. As expected, the antibody titers in rabbits immunized with 5 mg VLPs are a little higher than those immunized with 2 mg VLPs .To investigate the cellular immune response to RHDV2 VLP immunization, IFN-γ and IL-4 production levels were measured in sera from immunized rabbits. The levels of IFN-γ A and IL-Lepus granatensis [O. cuniculus had been infected with RHDV. With the emerge of GI.2, more species have been observed to become infected, e.g., Lepus corsicanus [Lepus europaeus [Lepus capensis mediterraneus [Lepus timidus [RHD is a highly contagious and lethal viral disease of both wild and domestic rabbits and associated with large economic losses. RHD may be caused by related but different lagoviruses. Since the first discovery of RHDV2 in 2010 in RHDV-vaccinated rabbits, RHDV2 has spread through wild rabbit populations and has replaced RHDV in several countries. RHDV2 is genetically and antigenically distinct from RHDV and thus can overcome immunity to classical RHDV. The case-fatality in mature rabbits infected with RHDV2 is lower compared to classic RHDV infection, however, young rabbits are more susceptible to RHDV2 . Before natensis  and O. crsicanus , Lepus europaeus ,44, Lepu timidus . Thus, iTraditional RHD inactivated vaccines are effective, but production of the viral antigens can be cumbersome given the lack of cell lines supporting RHDV and RHDV2 infection. VLPs are a product of modern biotechnology and can be produced at large scale, at low cost, and generally at the lowest biosafety level. VLPs structurally resemble the authentic virus as they are the result of self-assembly of solely the viral structural protein(s). Due to the lack of viral genetic material inside the capsid, VLPs are non-infectious and have a similar safety profile as subunit vaccines. However, in comparison to subunits, VLPs display their antigenic epitopes in a structured, repetitive fashion that may serve as pathogen-associated molecular patterns (PAMPs). In this way, co-stimulatory signals are available for the activation of T-lymphocytes by antigen presenting cells. This is one of the reasons that VLPs in general tend to generate a more potent humoral and cellular immune response .RHDV VLPs have been produced in multiple expression systems, including the BEVS. The advantage of baculovirus expression in insect cells is the ease of use and robustness of the system, leading to high expression of almost any pro- or eukaryotic protein. Another advantage of the BEVS is the efficiency of post-translation modifications, such as phosphorylation or glycosylation, which is important for the functionality of complex viral (glyco)proteins . Indeed,In our study, we obtained the complete RHDV2 genome sequence from a diseased rabbit, which was found in the Netherlands in 2016. The contemporary RHDV2-NL2016 isolate appeared to be closely related to other European RHDV2 genotypes for which the VP60 sequence is available, suggesting that the virus quickly spread over the continent after its initial introduction. It is known that RHDV2 strains have evolved by recombination between the non-structural proteins and minor structural proteins, VP60 and VP10. Recombinants may, e.g., include the structural proteins of GI.2, with non-structural proteins derived from non-pathogenic lagoviruses (GI.3 and GI.4) or from pathogenic GI.1 strains ,52. HoweWe cloned the RHDV2-NL2016 VP60 gene in a recombinant baculovirus expression vector using Gateway and Bac-to-bac technology and demonstrated the successful expression of RHDV2 VLPs in Sf9 insect cells . The icoTogether, our results suggest that RHDV2 VLPs produced in insect cells with recombinant baculovirus technology are highly immunogenic in rabbits. The VLPs may be further developed as a promising, novel vaccine to protect rabbits against RHDV2. An attractive possibility would be to generate a bivalent RHD vaccine by combining the RHDV and RHDV2 VLPs in a single formulation. Finally, the VLPs may find application as a potent antigen in diagnostic serological assays to discriminate RHDV from RHDV2 infections.


Though TNF reverse signaling enhanced SCG axon growth, it did not affect PVG axon growth. Whereas activation of TNF forward signaling in PVG axons inhibited growth, TNF forward signaling could not be activated in SCG axons. These latter differences in the response of SCG and PVG axons to TNF forward signaling were related to TNFR1 expression, whereas PVG axons expressed TNFR1, SCG axons did not. These results show that both TNF reverse and forward signaling are physiological regulators of sympathetic innervation in different tissues.Members of the TNF and TNF receptor superfamilies acting by both forward and reverse signaling are increasingly recognized as major physiological regulators of axon growth and tissue innervation in development. Studies of the experimentally tractable superior cervical ganglion (SCG) neurons and their targets have shown that only TNF reverse signaling, not forward signaling, is a physiological regulator of sympathetic innervation. Here, we compared SCG neurons and their targets with prevertebral ganglion (PVG) neurons and their targets. Whereas all SCG targets were markedly hypoinnervated in both TNF‐deficient and TNFR1‐deficient mice, PVG targets were not hypoinnervated in these mice and one PVG target, the spleen, was significantly hyperinnervated. These  Studies supporting this idea were carried out on the experimentally tractable sympathetic neurons of the superior cervical ganglion (SCG) and their cranial innervation targets. TNFR1 is expressed by SCG targets and acts as a ligand for membrane‐integrated TNF expressed along SCG axons. TNFR1‐activated TNF‐mediated reverse signaling enhances SCG axon growth and branching in vivo analysis of TNF‐deficient and TNFR1‐deficient mice to additional SCG targets and to prevertebral ganglion (PVG) targets, and have also studied the innervating populations of sympathetic neurons in vitro. We find that PVG targets do not exhibit the pronounced hypoinnervation phenotype of SCG targets observed in TNF‐deficient and TNFR1‐deficient mice and show that this in vivo difference can be accounted by differences in the response of SCG and PVG neurons to TNF reverse and forward signaling in vitro. Our results demonstrate that distinctive signaling mechanisms in different populations of developing sympathetic neurons regulate and refine innervation in different tissues.Here, we have extended the Mus musculus) was approved by the Cardiff University Ethical Review Board and was performed within the guidelines of the Home Office Animals (Scientific Procedures) Act, 1986. Mice with null mutations in the Tnf gene  were dissected and processed for visualization of sympathetic fibers by tyrosine hydroxylase (TH) staining in either whole mount tissue preparations or immunolabeling‐enabled three‐dimensional imaging of solvent‐cleared organs (iDISCO) or both. The whole mounts were carried out on tissue from P10 pups as described . The same orientation and region was imaged for each organ using the following standard anatomical landmarks: pineal gland, TH‐positive axon bundles entering the gland; trachea, TH‐positive lateral axon bundles entering this structure; and spleen, TH‐positive axon bundles accompanying the splenic arteries. Because it was difficult to maintain the structural integrity of the stomach after removal of milk, the most intensely innervated region closest to the lesser curvature was imaged. To compare the extent of sympathetic nerve branching, a modified line intercept method was used. Using ImageJ, a grid of 24 squares  was aligned in a standard orientation on images of the trachea, spleen, and stomach. For the smaller pineal gland, the grid consisted of 6 × 6 squares of side length 50 μm per square. Fiber density was estimated using the formula πDI/2, where D is the interline interval in microns (158 or 50) and I is the mean number of fiber bundles intersecting along one side of each grid square. The data are expressed as a percentage of the mean of the wild‐type data. All imaging and quantification was performed blind.For iDISCO preparations, TH‐positive fibers were fluorescently labeled using anti‐TH antibody  and Alexa Fluor 594 secondary antibody . Using a 3D printed compartment, the stomach and spleen were imaged in their entirety using inverted and confocal microscopy (Zeiss Axio Z2 Imager and Zeiss LSM 710) and z‐stacks were constructed. Fiji‐Image J was used for semi‐automated quantification of TH‐immunoreactivity as a measure of sympathetic target organ innervation density. Images were converted to grayscale and the Feature Extraction (FeatureJ Hessian) tool was employed using the smallest eigenvalue of Hessian tensor with the smoothing scale set to 0.5. To ensure consistent analysis across all conditions, multiple images from all mice were initially analyzed to generate a uniform threshold value, which was applied to every image of the same organ analyzed followed by a user‐defined macro to provide quantitative measurement of TH‐immunoreactivity within each target organ. The data are expressed as a percentage of the mean of the wild‐type data. All imaging and quantification was performed blind.et al., Dissected P0 and P5 SCG and prevertebral coeliac and superior mesenteric ganglia of CD1 mice were freed of adherent connective tissue using tungsten needles and were trypsinized and plated at very low density (~200 neurons per dish/well) in poly‐ornithine and laminin‐coated 35‐mm tissue culture dishes  or 4‐well dishes  in serum‐free Hams F14 medium  of a two‐compartment microfluidic device  , anti‐TNF alpha , anti‐TNFR1 , and anti‐TNFR2 . Secondary antibodies were Alexa Fluor‐conjugated anti‐immunoglobulin from ThermoFisherScientific used at 1:500 . Images were obtained using a Zeiss LSM 710 confocal microscope.The cultures were fixed in 4% paraformaldehyde (PFA) for 20 min and were washed with phosphate‐buffered saline (PBS) before blocking nonspecific binding and permeablizing the cells with 5% bovine serum albumin (BSA) and 5% donkey serum plus 0.1% Triton X‐100  in PBS for 1 h at room temperature. Neurons were incubated overnight with primary antibody in 1% blocking solution at 4Tnf and Tnfrsf1a mRNAs were quantified by RT‐qPCR relative to a geometric mean of mRNAs for the house keeping enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and succinate dehydrogenase (SDHA). Total RNA was extracted from whole SCG, celiac, and mesenteric ganglia at P0 with the RNeasy Mini extraction kit , and 5 μl was reverse transcribed for 1 h at 45°C using the AffinityScript kit  in a 25‐µl reaction according to the manufacturer's instructions. 2 µl of cDNA was amplified in a 20‐µl reaction volume using Brilliant III ultrafast qPCR master mix reagents . QPCR products were detected using dual‐labeled (FAM/BHQ1) hybridization probes specific to each of the cDNAs . The PCR primers were: Tnf forward, 5ʹ‐TAC TTA GAC TTT GCG GAG‐3ʹ and reverse, 5ʹ‐AGA GTA AAG GGG TCA GAG‐3ʹ; Tnfrsf1a forward, 5ʹ‐TTC CCA GAA TTA CCT CAG‐3ʹ and reverse, 5ʹ‐AAC TGG TTC TCC TTA CAG‐3ʹ; Gapdh forward, 5ʹ‐GAG AAA CCT GCC AAG TAT G‐3ʹ and reverse, 5ʹ‐GGA GTT GCT GTT GAA GTC‐3ʹ; Sdha forward, 5ʹ‐GGA ACA CTC CAA AAA CAG‐3ʹ and reverse, 5ʹ‐CCA CAG CAT CAA ATT CAT‐3ʹ. Dual‐labeled probes were: Tnf, FAM‐CAG GTC TAC TTT GGA GTC ATT GCT C‐BHQ1; Tnfrsf1a, FAM‐CAC CGT GTC CTT GTC AGC‐BHQ1; Gapdh, FAM‐AGA CAA CCT GGT CCT CAG TGT‐BHQ1; Sdha, FAM‐CCT GCG GCT TTC ACT TCT CT‐BHQ1. Forward and reverse primers were used at a concentration of 150 nM each and dual‐labeled probes were used at a concentration of 300 nM. PCR was performed using the Mx3000P platform  using the following conditions: 45 cycles of 95°C for 12 s and 60°C for 35 s. Standard curves were generated in every 96‐well plate, for each cDNA for every real‐time PCR run, using serial threefold dilutions of reverse transcribed spleen total RNA . Four separate dissections were performed.The levels of t‐test or one‐way ANOVA followed by Dunnet's post hoc test and for non parametric data the Mann–Whitney test or one‐way ANOVA Kruskal–Wallis followed by Dunn's post hoc test as indicated.Statistical comparisons for normally distributed data were performed by independent Student's et al., et al., Tnf gene  to activate TNF‐mediated reverse signaling .Like TNFR1 immunoreactivity, TNF immunoreactivity was evident on both SCG and PVG neurons. However, in contrast to TNFR1 immunoreactivity, TNF immunoreactivity was observed on the cell soma and the axons of both SCG and PVG neurons Fig. B. Thus, et al., in vivo and in vitro.We also studied and compared TNFR2 immunoreactivity in cultured SCG and PVG neurons using a specific anti‐TNFR2 antibody. As previously reported, TNFR2 immunoreactivity was low compared with TNF and TNFR1 immunoreactivity in SCG neurons  and the spleen. lia Fig. C and thelia Fig. D. The leSMG Fig. C and thein vivo and in vitro approaches to assess the contribution of TNF and TNFR1 to the establishment of sympathetic innervation of different tissues in developing mice and to clarify the underlying signaling mechanisms. Our in vivo studies of the phenotypes of TNF‐deficient and TNFR1‐deficient mice have revealed significant decreases in the sympathetic innervation density of SCG targets compared with wild‐type mice but either no change or hyperinnervation of PVG targets. Our in vitro studies suggest that these phenotypic differences in innervation are related in part to differences in the specific growth responses of SCG and PVG neurons to TNF and TNFR1 applied to their axons. This implies that anatomically distinct populations of sympathetic neurons differ in their capacity to respond to target‐activated TNF forward and reverse signaling.We have used −/−Tnf and −/−Tnfrsf1a postnatal mice revealed highly significant 20%–30% reductions compared with wild‐type littermates. Highly significant reductions in sympathetic innervation of similar magnitudes (20%–50% compared with wild‐type littermates) were also reported in three other SCG targets, the submandibular salivary gland, nasal turbinates, and iris, in −/−Tnf and −/−Tnfrsf1a postnatal mice (Kisiswa et al., in vivo studies together with the previously reported expression of TNF, but not TNFR1, along SCG axons and the expression of TNFR1 by SCG target field cells (Kisiswa et al., 2+ influx via T‐type Ca2+ channels, which leads to activation of PKC and ERK (Kisiswa et al., Quantification of sympathetic innervation in the pineal gland and trachea of −/−Tnf and −/−Tnfrsf1a mice suggest that TNF and TNFR1, probably by a reverse signaling mechanism, make a significant contribution to the establishment of the sympathetic innervation of this tissue. Tracheal sympathetic innervation is not, however, eliminated in −/−Tnf and −/−Tnfrsf1a mice, suggesting that additional factors are involved. The demonstration that tracheal sympathetic innervation is significantly reduced to a small extent in mice lacking GDF5 suggests that this factor also makes a contribution to establishing the sympathetic innervation of this tissue (O'Keeffe et al., Our current demonstration of the importance of TNF and TNFR1 in the establishment of tracheal sympathetic innervation is especially intriguing because the trachea is the only structure in which sympathetic fibers grow and branch independently of NGF. Mice lacking NGF and BAX, a key protein required for apoptosis, retain sympathetic neurons but have reduced or absent sympathetic innervation in all tissues except the trachea (Glebova and Ginty, −/−Tnf and −/−Tnfrsf1a mice, tissues that are predominantly innervated by PVG neurons, or small statistically significant increases in the innervation of the spleen in −/−Tnfrsf1a mice quantified in both whole mount and iDISCO preparations. An increase in sympathetic innervation was also observed in −/−Tnf mice in whole mount preparations, although this did not reach statistical significance. This implies that TNFR1 at least plays a role in suppressing the ramification of sympathetic axons in the spleen. We are confident of these results because large numbers of mutant and wild‐type mice were examined blind. Moreover, we visualized sympathetic fibers using two methods, whole mount and iDISCO, and studied innervation at two ages, P6 and P10. While there is some difference in the mean increase in innervation of the spleen in −/−Tnfrsf1a mice compared with +/+Tnfrsf1a littermates in iDISCO and whole mount preparations, the statistical significance of the increase is similar in both cases. Although it is possible that this difference could be related to the different ages at which analysis was undertaken, sympathetic fibers first reach their targets during early fetal development and have ramified extensively in their target tissues by P6 and P10.In marked contrast to all SCG targets examined, there was either no significant decrease in the sympathetic innervation of the spleen and stomach of −/−Tnf and −/−Tnfrsf1a mice appears to be related to intrinsic differences in the response of SCG and PVG neurons to TNFR1 and TNF. First, while TNFR1‐Fc significantly enhanced NGF‐promoted axon growth from cultured postnatal SCG neurons, TNFR1‐Fc failed to enhance NGF‐promoted axon growth from PVG neurons of the coeliac and superior mesenteric ganglia. These differences in the response of SCG and PVG neurons to TNFR1‐Fc are not simply due to differences in expression of the reverse signaling receptor. TNF is expressed along the axons of both SCG and PVG axons. Moreover, it is likely that PVG axons are exposed to TNFR1 in vivo, as this is expressed in PVG targets. It will be interesting in future work to ascertain which step or steps in the signaling cascade leading from TNF activation to axon growth is blocked in PVG neurons. Second, while TNF suppresses NGF‐promoted axon growth from both SCG and PVG neurons grown in dissociated culture in which both axons and cell soma are exposed to TNF, TNF only suppresses axon growth from PVG neurons, not SCG neurons, when axons are exposed to TNF in compartment cultures. This difference in the response of PVG and SCG neurons in compartment cultures to the growth inhibitory actions of TNF appears to be related to the distribution of TNFR1, the forward signaling receptor that mediates this response, on the neurons. Whereas TNFR1 expression is restricted to the cell soma of SCG neurons, TNFR1 is expressed both at the cell soma and along the axons of PVG neurons. Thus, while TNF is expressed in the target tissues of both SCG and PVG neurons, only PVG neurons are capable of responding to target‐derived TNF. These intriguing findings raise several interesting questions for future research. For example, how is TNFR1 expression spatially restricted on SCG neurons and is the expression of TNFR1 on the soma of SCG neurons physiologically relevant?The marked difference between the SCG and PVG targets observed in et al., Although previous work has shown that the reduction in NGF‐promoted axon growth promoted by TNF is mediated by TNFR1 (Kisiswa et al., et al., et al., et al., et al., et al., et al., et al., The modulation of axon growth by TNF superfamily members, generally without affecting neuronal survival, is becoming an emerging theme in the developing mammalian nervous system. In addition to the opposite effects of TNF forward and reverse signaling on sympathetic axon growth and target innervation discussed here, TNF forward and reverse signaling also exert opposite effects on the growth of dorsal root ganglion axons (Wheeler In summary, our work has revealed unsuspected complexities in the ways in which TNF and TNFR1 contribute to the establishment of sympathetic innervation of different tissues. Axon growth and ramification within different tissues is differentially modulated by TNFR1‐mediated TNF activated forward signaling and by TNF‐mediated TNFR1‐activated reverse signaling in the innervating neurons. These differences are related to differences in the capacity of anatomically different populations of innervating sympathetic neurons to respond to TNFR1 and TNF, which in turn are due, in part, to differences in the cellular distribution of TNF and TNFR1 on responsive neurons.The authors declare no competing financial interests.C.E. carried out the background work and performed the whole mount experiments. C.E. and O.Y.C performed the iDISCO and cell culture experiments. O.Y.C. undertook the immuncytochemistry analysis. S.W. carried out the qPCR. L.K. helped with initial preliminary data. A.M.D. and S.W. supervised the project, and A.M.D. wrote the manuscript with input from all authors.The data that support the findings of this study are available from the corresponding author upon reasonable request.


N-acetylglucosamine oligomers (GlcNAc) and GlcNAc/branched mannose in BSA, IgG, TNF-α, and THP, but not in IFN-γ. These carbohydrate moieties mediated binding with THP. Small amounts of SiaαGal/ GalNAc, SiaGal/GalNAc, and mannose residues were also present in THP and TNF-α. Binding affinity (Kd) between THP and TNF-α by Scatchard plot analysis was 1.4–1.7 × 10−6 M, lower than antigen-antibody or ligand-receptor binding affinities. To elucidate the structure-binding relationship of THP-TNF-α, THP was digested with neuraminidase, β-galactosidase, O-sialoglycoprotein endopeptidase, carboxypeptidase Y, or proteinase K. β-galactosidase increased binding capacity of THP for TNF-α. Monosaccharide inhibition suggested that α-methyl-D-mannoside, GlcNAc, and GalNAc, but not sialic acid, suppress THP-TNF-α binding as detected by ELISA. We conclude that sugar-lectin and sugar-protein interactions between cognate sites in THP and TNF-α mediate their binding.In a previous study we noted significant THP binding to TNF-α, but did not explore the molecular basis of the structure-binding relationship. In this study, we used lectin-binding ELISA to assess the carbohydrate compositions of THP, BSA, IgG, TNF-α, and IFN-γ. We identified β- Tamm-Horsfall glycoprotein (THP), a 80–90 kDa macromolecule, is produced by the mammalian renal thick ascending limb of Henle’s loop ,2. THP i2 and Man5GluNAc2 that mediate interactions with type-1 fimbriated Escherichia coli , SNA-1 [SiaαGal/ GalNAc], GNA [mannose], DSA [β-GlcNAc oligomers], and ConA [GlcNAc/branched mannose] were obtained from E-Y Labs . Human recombinant TNF-α, IFN-γ, IL-6, and IL-1β were obtained from R&D Systems . Mouse monoclonal anti-human TNF-α antibody and HRP-conjugated anti-TNF-α antibody were purchased from R&D Systems. α-Methyl-D-mannoside (α-MDM), N-acetyl-D-glycosamine (GlcNAc), N-acetyl-D-galactosamine  and N-acetylneuraminic acid  were purchased from Sigma.Purified human IgG, BSA, neuraminidase, β-galactosidase, proteinase K, and carboxypeptidase Y were purchased from Sigma-Aldrich . Twenty-four-hour urine was collected in a clean glass bottle from a normal individual. We followed the purification procedures described in our previous report . THP puret al.  × [B]/[AB]. Thus, small Kd indicates high-affinity interactions and large Kd values indicate low-affinity interactions.A Scatchard plot was used to analyze the binding affinities of THP and TNF-α. We followed the method described by Kananick et al.  with modSoftware  to calcu450.α-MDM, GalNAc, or GlcNAc (10 µg) was incubated in TNF-α-coated microwells overnight at 4 °C. THP (2 μg/well) was added and incubated overnight at 4 °C. HRP-anti-uromucoid antibody was added. After reaction at room temperature for 2 h, THP-TNF-α binding was measured as OD450 nm.THP-coated microwells were preincubated with 10 μg of α-MDM, GalNAc, or GlcNAc overnight at 4 °C. TNF-α (2 μg/mL) was added and incubated overnight at 4 °C. HRP-anti-TNF-α antibody was added and incubated at room temperature. The binding of TNF-α to THP was detected by ODp < 0.05.Results are reported as mean ± S.D. Continuous variables were assessed by non-parametric Wilcoxon rank-sum test using commercially available software (Stata/SE8.0 Windows). Statistical significance was indicated by d of THP-TNF-α binding was around 1.5 × 10−6 M, less than antigen-antibody or ligand-receptor binding. (2) THP and TNF-α contain high amounts of β-GlcNAc oligomers and GlcNAc/branched mannose, which are relevant to THP-TNF-α binding. (3) Galactoside in THP carbohydrate side-chains hindered THP-TNF-α binding via sugar (in the THP molecule)-lectin (in the TNF-α molecule)-sugar (in the TNF-α molecule)-ZP domain (in the THP molecule) interactions. (4) Many monosaccharides such as α-methyl-D-mannoside, N-acetylglucosamine, or N-acetylgalactosamine, but not sialic acid, mediate THP-TNF-α binding. TNF-α is a crucial proinflammatory cytokine in many inflammatory disorders including rheumatoid arthritis, ankylosing spondylitis, psoriasis/psoriatic arthropathy, or inflammatory bowel diseases. Identification of the THP domain structure(s) responsible for TNF-α binding may yield a novel therapeutic strategy for acute and chronic inflammatory diseases.We have explored the molecular basis of the structure-binding relationship between THP and TNF-α. At least four original findings are reported here: (1) The K


Understanding virus shedding patterns of avian influenza virus (AIV) in poultry is important for understanding host-pathogen interactions and developing effective control strategies. Many AIV strains were studied in challenge experiments in poultry, but no study has combined data from those studies to identify general AIV shedding patterns. These systematic review and meta-analysis were performed to summarize qualitative and quantitative information on virus shedding levels and duration for different AIV strains in experimentally infected poultry species. Methods were designed based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Four electronic databases were used to collect literature. A total of 1155 abstract were screened, with 117 studies selected for the qualitative analysis and 71 studies for the meta-analysis. A large heterogeneity in experimental methods was observed and the quantitative analysis showed that experimental variables such as species, virus origin, age, inoculation route and dose, affect virus shedding  for highly pathogenic AIV (HPAIV), low pathogenic AIV (LPAIV) or both. In conclusion, this study highlights the need to standardize experimental procedures, it provides a comprehensive summary of the shedding patterns of AIV strains by infected poultry and identifies the variables that influence the level and duration of AIV shedding. There are several subtypes of AIV, which can be distinguished by two surface glycoproteins: Hemagglutinin (HA) and neuraminidase (NA). Currently sixteen HA and nine NA subtypes have been identified in birds [Avian influenza viruses (AIV) belong to the family Orthomyxoviridae, genus in birds . AIV arein birds . During in birds ,2. From in birds ,4. In adin birds . Besidesin birds ,7.Since AIV, especially HPAIV, has a major impact on poultry, much research is focusing on obtaining more knowledge on the infectiousness and transmission potential of this pathogen. Indirect indicators of the infectiousness of an infected bird are the level and duration of virus shedding. These parameters are often used to obtain information of the infection characteristics and the transmission potential of the virus to susceptible recipient hosts during challenge and transmission experiments. The level and duration of virus shedding was studied in different experimental settings, designed to answer a variety of research questions. First of all, shedding can be used to determine which poultry species, age groups or breeds could be potentially more infectious following infection ,9,10,11.In general, a meta-analysis of small-scale animal experiments has the potential of enhanced statistical power, obtained by combining the observations of a set of studies. However, in the case of virus shedding, comparison of the published information is complicated by the fact that different experimental approaches were used. The methodology differs mainly in: (1) The use of different hosts: Species, ages and breeds, (2) the use of different virus strains and (3) the experimental design: Inoculation doses and routes, the types of samples and interval of sampling during the experiment.The objective of this systematic review (SR) is to get a better understanding of the characteristics of AIV shedding in poultry. Therefore, information on experimental methods was collected and summarized to obtain qualitative information of experimental approaches to study virus shedding in poultry. Subsequently, a quantitative meta-analysis was performed, in which variables that influence both virus shedding levels and duration of different AIV strains were quantitatively identified, whilst correcting for experimental biases.A SR uses pre-specified and standardized methods to identify and critically appraise the relevant research, and to collect, report and analyze data from the studies that are included ,22. ThisThe purpose of the literature search was to identify data on virus shedding levels and the duration of AIV in poultry at a high sensitivity, capturing all relevant citations. Literature was searched in the electronic database PubMed and the Ovid search engine. Using the Ovid search engine CAB Abstracts, Biological Abstracts and AGRICOLA databases were searched simultaneously. No restrictions were imposed on publication date. The last search was performed on 19th July 2017.To find all relevant search terms related to AIV shedding in the databases, the Mesh database of PubMed was visited. Furthermore, G. Koch and J. L. Gonzales, both AIV experts in respectively virology and veterinary epidemiology, working at Wageningen Bioveterinary Research (WBVR), were consulted. Boolean operators AND and OR were used to narrow down the search. 50/mL, TCID50/mL, viral RNA copies and PCR cycle thresholds from real time PCR) and virus shedding duration (days). The first screening aimed to identify relevant studies, the following criteria were used:The publication is written in English;The study described is primary research;Influenza A virus is the subject being studied;The study was performed in poultry;Virus shedding is quantified.After searching the literature, the titles and abstracts of the publications were screened. The first literature screening consisted of basic criteria, while the second screening assessed the quality of the studies that passed the first screening. The screening was performed by one reviewer, in case of doubt a second reviewer was consulted. As a result of the low specificity of the literature search, the aim of the first screening was to quickly remove non-relevant citations. Primary research studies written in English were assessed, no restrictions were imposed on the publication date. The study population evaluated consisted of healthy poultry of all ages, experimentally infected with AIV. Only experimentally infected poultry was considered, as infection conditions are unknown for poultry and wild birds naturally infected. Studies experimentally evaluating virus shedding of AIV were assessed. In addition, studies evaluating the effect of either experimental or commercial vaccination on virus shedding were considered, as the challenge control group could provide data for the meta-analysis. Types of outcomes include virus shedding level  is known;The experimental units are confirmed negative for AIV in advance of the study;The virus inoculation route and dose are described;Virus shedding is quantified by PCR or by virus isolation;The sampling interval of virus shedding is described or;Peak virus shedding is described or can be determined from the data.Studies that did not meet all the mentioned criteria did not advance to the second screening. If it was unclear from the abstract whether all criteria were met, the study did proceed to the next screening to ensure high sensitivity. The purpose of the second level screening was to identify studies of sufficient quality that quantify AIV shedding. Full texts of studies remaining after first screening were screened for the following criteria: A study was included if all criteria were described. Studies that did not meet all criteria were considered as qualitatively insufficient or irrelevant. Screening was executed by one reviewer, however, in the case of doubt a second reviewer was consulted.If all criteria of the screening were met and the article passed quality assessment, data was extracted and added to a database. This database was developed by two reviewers and one reviewer executed the data extraction.50, EID50-equivalents, TCID50, TCID50-equivalents, plaque forming units (PFU), viral RNA copies or cycle thresholds (ct). Studies reporting EID50 or TCID50 equivalents used a standard curve, based on calibration controls containing known amounts of virus, for conversion of ct to EID50-equivalents or TCID50-equivalents.Qualitative and quantitative data were both extracted. Qualitative data, which was extracted for each experiment, included: (1) AIV subtype (including the clade if applicable); (2) infected poultry species ; (3) pathotype (HPAIV or LPAIV); (4) age at infection (in days); (5) inoculation route and dose; (6) type of samples and (7) host origin of the AIV (the species from which the AIV originates). Daily shedding levels were extracted as quantitative data, which were used to determine peak shedding, average shedding and shedding duration. The geometric mean was used to represent average virus shedding, because a geometric mean is less sensitive to outliers than an ordinary arithmetic mean. If data was only reported in figures, the PlotReader tool  was usedThe analysis consisted of two parts: A qualitative and a quantitative analysis .The qualitative analysis identified and described different biological and experimental characteristics assumed to influence AIV shedding e.g., poultry species, age of poultry, inoculation routes, type of samples that were collected, outcome units and virus subtypes used in the included studies. In addition, the qualitative analysis shows the heterogeneity in the experimental approaches observed in the selected studies.50 (virus isolation) or EID50-equivalents  were assessed. Studies that reported outcomes in threshold cycle (ct), TCID50, PFU and ELD50 were not included in the meta-analysis. Studies from which data from individual animals could not be extracted were also not included. Reported data of virus isolation and real time-PCR results were first assessed for similarity and were found to be highly similar [10 EID50 to minimize variance between studies. Geometric means of virus shedding levels were calculated for every observation.The quantitative analysis described the relationship between several explanatory variables and virus shedding levels and duration of AIV. Data used to characterize virus shedding patterns were: (1) geometric mean virus shedding per unit sample , (2) peak virus shedding titer and (3) duration of virus shedding in days. To combine and compare virus shedding levels across studies, only studies that reported virus shedding levels in either EID similar . Virus sp < 0.05, with significant variables and variables showing a confounding effect being kept in the final models. Variables considered biologically relevant were also kept in the model, regardless of their p-value. Goodness of fit of the models was assessed by inspecting residuals.Generalized linear multivariable mixed models (GLMM) were used to model the association between virus shedding levels (geometric mean or peak shedding) and the variables that could have an effect on shedding patterns (explanatory variables). In these models, peak or geometric mean shedding were the response variables. Virus subtype, inoculation route, poultry species, etc. and possible interactions between these variables were included as explanatory variables  the model fit, assessed by evaluating the agreement between the fitted survival curve and the non-parametric (Kaplan Meier) survival estimates and (2) by competing different parametric models (models using different distributions) using the Akaike information criterion (AIC) and choosing the models with the lowest AIC. Similar to the model of virus shedding levels, a backwards elimination procedure was used. Only data where the complete shedding period was observed (followed up daily or every two days) or where data was right censored (last test positive before the experiment was terminated) were included. For contact infected animals, it was assumed that they were infected the day before they tested first positive ,27 in eiThe last search in the electronic databases was performed on 19th July 2017. During the literature search, 2798 citations were retrieved in total. The database PubMed provided 1826 citations and the search in Ovid, with searching in three databases simultaneously, resulted in 952 citations . Twenty 50 and EID50-equivalent were the most used units to describe viral shedding. Overall, 30 different subtypes were examined where H5N1 was the most studied subtype (n = 49). H5N1 represents the most studied HPAIV, whereas H9N2 was the most studied LPAIV.The qualitative analysis was performed for 117 studies. 10 EID50/mL) and duration of shedding (days). Combined data from these studies resulted in a total of 322 and 381 experimental animals, infected with HPAIV and LPAIV respectively, providing information for the analysis. Data from 71 studies were used for the meta-analysis . These dp < 0.001) in poultry  infected with LPAIV than HPAIV, with an estimated average difference in mean and peak shedding of 1.82 and 3.14 log10 EID50/mL of the sample, respectively  was significantly lower . Ducks shed via the respiratory route higher amounts of virus (p < 0.001) and via the cloacal route lower amounts of virus than chickens . This association was not found in ducks, and available data did not allow us to make this assessment for turkeys. Age had a significant effect on shedding, with young poultry shedding less of the virus than adult poultry (p < 0.001). There were no significant associations found for the inoculation routes  or the type of chickens (broiler vs. layers) with shedding levels. Differences were also observed between virus subtypes, with differences observed between viruses having different hemagglutinin (H5 or H7) and between viruses having the same hemagglutinin , whilst quails had a higher respiratory shedding level (p = 0.007). Pigeons had lower respiratory and cloacal shedding levels than chickens (p = 0.042). Virus origin had the same effect on shedding for LPAIV as observed for HPAIV, with chickens infected with virus isolated from chickens shedding higher levels of virus (p < 0.01). In contrast to HPAIV, there was a positive association between the inoculation route and shedding of LPAIV, with aerosol (only chickens), intranasal and oropharyngeal inoculated poultry shedding higher levels of virus than contact infected poultry. There were no significant differences in shedding levels between other inoculation routes and contact infected poultry or age and type of chickens for LPAIV. As for HPAIV, virus subtype had a significant effect on shedding (shedding  and this5 to 106 EID50), hence it was only possible to assess this relationship only for chickens. For HPAIV infections, doses used ranged from 102 to 108 EID50. There was a positive association between inoculation dose and shedding when doses were above 105 EID50, the higher the dose, the higher the shedding . In both species the shedding duration of HPAIV via the respiratory route is similar to that via the cloacal route. LPAIV behaved differently from HPAIV: The duration of respiratory virus shedding in ducks was not different to that in chickens (p > 0.05) but the duration of cloacal shedding appeared to be longer in ducks than in chickens (p = 0.08). As for turkeys, cloacal shedding appeared to be longer than chickens (p = 0.05). There were no significant differences in the duration of virus shedding via both respiratory or cloaca routes for other species. However, data were limited, thus limiting power, and resulting in large confidence intervals for the estimates  had a significant influence on shedding of both HPAIV and LPAIV only for chickens. It was also observed that the inoculation route had no significant influence on shedding of HPAIV whilst the inoculation route did have an influence on shedding of LPAIV.50 or EID50 equivalents (the most common outcome units observed in this review), would improve comparability of the studies. We would encourage experimentalists to strive for standardization of the experimental method, although we acknowledge that the experimental methods sometimes must vary due the objective of the study, e.g., to identify infection routes [With many AIV subtypes and different poultry species being at risk, challenge studies contribute to improving our understanding of AIV infections in poultry by providing observations that taken together could lead to robust identification of risk factors associated with infection. However, our review highlights an observed large heterogeneity in experimental methods and outcome units used, limiting comparability of experimental results and therefore the confidence in conclusions drawn when considering the evidence of similar experiments together. In detail, 46 out of 117 studies selected for the qualitative analysis could not be included in the quantitative analysis, hence limiting the power of our quantitative analysis and the range of variables that could be assessed . Some level of standardization, such as reporting shedding levels in say EIDn routes ,31,32. In routes  to reporOne of the variables that was analyzed in the quantitative analysis was the pathogenicity of the virus. It was shown that poultry shed more of the virus after inoculation with HPAIV compared to LPAIV. This high HPAIV shedding caused a high viral load in the environment, which could make indirect virus transmission between flocks or farms more effective, due to a higher chance for virus contamination of materials, for example, trucks or boots. The higher virus shedding by infection with HPAIV could be an explanation for the enhanced transmissibility of HPAIV H7N1 compared to the LPAIV variant as described in .5 EID50 results in a linear increase in virus shedding. Spekreijse et al. [5 EID50 could be used in future challenge studies to infect all experimental units but to avoid affecting the mean virus shedding level.In addition to the pathogenicity of the virus, the effect of experimental methods, such as inoculation dose, on shedding was included in the quantitative analysis. The inoculation dose could only be assessed for chickens. For HPAIV it appears that using inoculation doses above 10e et al.  previouse et al. . Based o6 EID50/mL [In contrast to HPAIV, no significant association between the inoculation dose and LPAIV shedding was found. It might be expected that for LPAIV the inoculation dose is more critical compared to HPAIV as it is less virulent and pathogenic, a relatively low dose of HPAIV might be sufficient for infection, whilst infection with LPAIV requires a higher dose to achieve virus shedding in chickens ,37. At lEID50/mL ,37) may As for the inoculation route, no differences in shedding were found between the intranasal or intrachoanal routes of inoculation and contact infected poultry for HPAIV. These findings appear to be in line with the study by Pantin-Jackwood et al. , which wIn addition to the expected differences in virus shedding between various experimental methods, it was expected that virus shedding would differ among poultry species in both shedding route and shedding levels. Traditionally it is thought that viral replication of AIV in ducks occurs primarily in epithelial cells of the intestinal tract, and high concentrations of virus are shed in the feces, whereas virus replication is primarily respiratory in chickens ,40,41,420) for a LPAIV H7N1 virus in both species: The R0 in turkeys was 15.3 (11.8–19.7) [0 was 3.8 (1.3–6.3) [According to the analysis, turkeys shed also higher levels of LPAIV via the cloaca than chickens. Due to this higher level of virus shedding, more of the virus will be present in the environment of turkeys  and therefore the virus transmission is probably higher. The higher viral load in the environment of turkeys, in combination with the higher susceptibility of turkeys for LPAIV ,44, and .8–19.7)  whilst i1.3–6.3) . The inc50) and lethal (BLD50) doses were not significantly different for broilers of five, eight and 30 weeks after inoculation with a HPAIV H5N2. Thus, for this virus there was neither a difference in susceptibility nor virus shedding between the different age groups of chickens. Most of the data for this assessment were for chickens with a limited number of observations coming from other species, hence these results were mainly representative for chickens and additional research (data) is needed to be able to confirm this observation for other poultry species. For LPAIV no significant differences in virus shedding were found between young and adult chickens (this assessment was done only for chickens). The finding for LPAIV in chickens was comparable with the results of the study of Lavoie [Data for the variable “age” was difficult to obtain for all poultry species and ages, hence our age findings were mainly applicable to young and adult chickens. Virus shedding was expected to decrease with age (except for very old poultry), as animals become fully immunocompetent and viral pathogenicity in immunocompetent hosts is decreased. However, we observed that young (non-reproductive) animals shed significantly lower amounts of HPAIV than adults (reproductive). A study here reviewed  showed tf Lavoie , in whicf Lavoie  detectedf Lavoie  detectedThe authors were aware of limitations and some potential sources of bias in this review, which must be taken into account when drawing conclusions. During summarizing and analyzing data, the observations of the same subtype, regardless of their origin, were pooled. Combining these observations was a potential source of bias. Differences in virus shedding patterns might exist between different strains of the same subtype, which could be the case for example for the HPAIV H5N1 strains . Hence t50 or EID50 equivalents, will improve the comparability of individual studies. When studies are more comparable, the formal assessments for variables that could not be performed during this meta-analysis because of data limitations could be performed in the future. Using guidelines for reporting animal experiments, such as the ARRIVE guidelines, will improve the quality of reporting. The quantitative analysis showed that some experimental variables affect the virus shedding patterns for HPAIV, LPAIV or both. For this reason, all variables should be considered carefully before starting a challenge experiment and here we identified some variables that could lead to some level of standardization . This review could be used as a background for the design of future challenge studies for AIV.To conclude, this review showed the large heterogeneity in the experimental methods of challenge studies. More standardization in the experimental methods and reporting, for example reporting the outcome unit in EID


Despite extensive research, inconsistencies between studies remain a challenge, and the distribution and genetic characteristics of DNVs need to be precisely evaluated. To address this issue at the whole-genome scale, a large number of DNVs identified from the whole-genome sequencing of 1,902 healthy trios  from the Simons Foundation for Autism Research Initiative study and 20 healthy Korean trios were analyzed. These apparently nonpathogenic DNVs were enriched in functional elements of the genome but relatively depleted in regions of common copy number variants, implying their potential function as triggers of evolution even in healthy groups. No strong mutational hotspots were identified. The pathogenicity of the DNVs was not strongly elevated, reflecting the health status of the cohort. The mutational signatures were consistent with previous studies. This study will serve as a reference for future DNV studies.The implications of germline  De novo variants (DNVs) are mutations that are not inherited from parents but arise from mutational events during gametogenesis and embryogenesis. DNVs are believed to be a source of genetic variation at the population scale and can be used for studying evolutionary processes [rocesses . They arrocesses -5.–8 per base, resulting in approximately 74 DNVs per person [Studies using family whole-genome sequencing (WGS) revealed that the average de novo substitution rate per generation ranges from 1.0–1.5 × 10r person . Many faThe identification of DNVs is challenging because high-coverage WGS data of probands and parents are required for reliable DNV detection. Compared to inherited variants, DNVs are rare and require a large cohort to obtain enough statistical power to detect reliable patterns within DNVs . FurtherThe genetics of autism spectrum disorder has been extensively studied, leading to the identification of many disease-related genes -11. Recehttp://ginet.kr, #10050164) maintained by KOBIC.The trio-based genome data for DNV calling were approved for use and downloaded. The Korean Bioinformation Center (KOBIC) cohort data of 65 individuals (20 families) were downloaded in Variant Call Format from the Genome InfraNet (The Simons Foundation for Autism Research Initiative (SFARI) cohort data were obtained from Supplementary Table 2 of a previously published article  in whichA total of 15 trio and 5 quartet families  were identified from the KOBIC cohort. From the quartet samples, only one sibling was included for further analysis. Variants that were present in the probands and were not present in both parents were selected. The following filtering criteria were used: QUAL > 200, DP > 20 and custom-defined GQ values. Annotation was performed with Variant Effect Predictor . VariantThe mutational spectrum of the DNVs and the contribution of 30 well-known COSMIC  mutationVariants in the gnomAD  version A total of 455 and 115,870 DNVs were called from the KOBIC and SFARI cohorts, respectively. Most of the DNVs were located in intronic and intergenic regions , as thesThe sequence context of DNVs shapes mutation rates. The mutational signature was originally used for the modeling of mutational processes in a somatic mutation analysis of cancers ,28, whicAdditionally, we reconstructed our mutational spectrum with 30 well-known signatures curated by COSMIC and quantified the contribution of each signature. Signatures 1, 5, and 16 contributed the majority of the signatures, contributing 32%, 25% and 31% of the total, respectively. These findings are consistent with a previous report  that demA rainfall plot was employed to visualize mutational hotspots , and ourNext, we subjected various annotated genetic elements to DNV burden analysis. Genic regions including 3′-UTRs, 5′-UTRs, exons and introns were enriched, as shown by the ratio of the observed count to the expected counts exceeding 1.0. In contrast, intergenic regions were depleted of DNVs . RegionsRAPSN , which is known to cause myasthenic syndrome with an autosomal recessive pattern, was functionally assayed [The prediction of DNV pathogenicity quantified by the CADD score  showed a assayed -33. Alth assayed  distribuHere, we analyzed the distribution and genetic patterns of 116,325 DNVs derived from 1,922 healthy individuals. The mutational signatures were consistent with previously reported signatures. We could not identify strong mutation hotspots in our cohort. Notably, the DNVs were enriched within elements with potential functionality, such as genic regions and regulatory regions, but depleted in intergenic regions and regions that are tolerant to copy number changes. This observation was unexpected since the carriers of these DNVs are healthy and are not expected to display enrichment in functional regions of the genome. This enrichment was not strong enough to be differentiated by the CADD scores. Due to the discrepancies in the sizes of the cohorts , their ethnicity compositions and the sequencing procedures applied, direct comparison between the two databases is challenging. However, regarding the consistency of the mutational spectrum and the signatures of the SFARI DNVs with previous studies, the DNVs from KOBIC are expected to follow the patterns of the DNVs from SFARI with a larger cohort size and DNV validation. Further research involving epigenetic signatures and individualized mutational cluster analysis may elucidate the factors affecting the germline mutation rate, leading to better identification of disease-associated DNVs and an improved understanding of human genome evolution.


Accordingly, PIKfyve inhibitors are now in clinical trials as anti-cancer drugs. Among those, apilimod is the most promising, yet its potency to inhibit PIKfyve and affect endomembrane homeostasis is only partially characterized. We demonstrate here for the first time that apilimod powerfully inhibited in vitro synthesis of PtdIns5P along with that of PtdInsP2. HPLC-based resolution of intracellular phosphoinositides (PIs) revealed that apilimod triggered a marked reduction of both lipids in the context of intact cells. Notably, there was also a profound rise in PtdIns3P resulting from arrested PtdIns3P consumption for PtdInsP2 synthesis. As typical for PIKfyve inhibition and the concomitant PtdInsP2 reduction, apilimod induced the appearance of dilated endomembrane structures in the form of large translucent cytoplasmic vacuoles. Remarkably, bafilomycin A1 (BafA1) fully reversed the aberrant cell phenotype back to normal and completely precluded the appearance of cytoplasmic vacuoles when added prior to apilimod. Inspection of the PI profiles ruled out restoration of the reduced PtdInsP2 pool as a molecular mechanism underlying BafA1 rescue. Rather, we found that BafA1 markedly attenuated the PtdIns3P elevation under PIKfyve inhibition. This was accompanied by profoundly decreased endosomal recruitment of fusogenic EEA1. Together, our data demonstrate that apilimod inhibits not only PtdInsP2 but also PtdIns5P synthesis and that the cytoplasmic vacuolization triggered by the inhibitor is precluded or reversed by BafA1 through a mechanism associated, in part, with reduction in both PtdIns3P levels and EEA1 membrane recruitment.PIKfyve, an evolutionarily conserved kinase synthesizing PtdIns5P and PtdInsP Natural soybean PtdIns was from Avanti Polar Lipids, Inc. (USA). Polyclonal anti-PIKfyve antibody was previously described [Apilimod {3-methyl-2-[6-(4-morpholinyl)-2-[2-(2-pyridinyl)ethoxy]-4-pyrimidinyl] hydrazone, benzaldehyde}, obtained from Axon Medchem LLC (USA), and YM201636 {furopyrimidin-2-yl)phenyl)-3-pyridinecarboxamide]}, purchased from Symansis NZ , were used as recommended by the manufacturers. BafA1 was purchased from Enzo Life Sciences, Inc., USA. Thin layer chromatography (TLC) 20 x 20 cm glass plates  and an HPLC 5-micron Partisphere SAX column were from Whatman. Methylamine (40% w/w solution in water), n-propoanol and tetrabutylammonium bisulfate (TBAS) were from Sigma-Aldrich, USA. Glucose- and inositol-free DMEM was prepared in house in sterile distilled deionized water from amino acids and vitamins purchased from Gibco Laboratories  or Sigma , and inorganic salts from various commercial sources.  were first preincubated for 15 min (37°C) in the presence of varying concentrations of BafA1 or apilimod  or only vehicle . The kinase assay was carried out for 15 min at 37°C with 15 μM ATP and [γ-32P]ATP (30 μCi) in a 50 ul volume and terminated with 200 μl 1N HCl. Lipids were then extracted with chloroform, spotted on a TLC glass plate, and resolved by a chromatographic solvent system of n-propanol/2 M acetic acid (65:35 v:v). Generated radioactive products were detected by autoradiography and quantified with ImageJ software .These were performed following previously published protocols . Brieflymyo-[2-3H]inositol following our previous protocols [2 atmosphere. The medium was then replaced with fresh starvation medium supplemented with 25 μCi/ml myo-[2-3H]inositol  and cells were labeled for 25 h. Cell treatments were performed in the same labeling medium for 1–2 additional hours as specified in the figure legends. The incubation time was sufficient to reach a condition of 90–100% isotopic equilibrium  across the lipid pools as calculated from the PIP/PI ratios in both HEK293 cells and podocytes as described previously [3OH/1M HCl (1:1 v:v)] in the presence of 5 mM EDTA and 5 mM tetrabutylammonium sulfate], deacylated at 54°C for 60 min with methylamine reagent . Glycerophosphoinositol phosphates (GroPInsP) were analyzed by HPLC (Waters 5215) on a 5-micron Partisphere SAX column eluted as previously described [3H] radioactivity after the addition of scintillation mixture. Data evaluation and documentation was performed by Microsoft Excel. Individual peak radioactivity was quantified by area integration and presented as a percentage of the summed radioactivity from the [3H]GroPIns3P, -4P, -5P, -P2, and -P2 peaks .Cells were labeled with rotocols , 37, 41,eviously , 42. Celescribed , 41, 43.PIKfyve were fixed with 3% paraformaldehyde for 30 min and then visualized by confocal microscope. For EEA1 immunofluorescence microscopy, HEK293 cells, seeded on glass coverslips were fixed, permeabilized with saponin and then stained with anti-EEA1 as described ATP and a native PtdIns substrate from soybean, which supports production of both PtdIns5P and PtdInsP2 as we previously established inositol and examined the PI profiles by our well-characterized protocols for HPLC-based inositol headgroup separation, attaining a clear-cut detection and quantification of PtdIns5P along with PtdInsP2 and the other PIs inositol metabolic labeling, podocytes were treated for 40 min with apilimod (100 nM), when lipids were extracted, deacylated and analyzed by HPLC. Similar to our results in HEK293 cells we observed that apilimod treatment markedly reduced steady-state levels of PtdIns5P, along with those of PtdInsP2 P2 and PtdIns5P production.To reveal whether this powerful reduction of PtdIns5P, along with PtdInsPe kidney  to a simA phenotypic hallmark of PIKfyve perturbation is the formation of multiple cytosolic vacuoles as identified by us and confirmed by other investigators , 15, 18.We have previously established that short pretreatment of COS7 cells with low doses of BafA1 (5–15 nM) completely precluded the appearance of cytoplasmic vacuoles under PIKfyve inhibition by YM201636 . Likewis2 are markedly reduced, i.e., >2-fold below those of controls inositol. At the end of the labeling period, cells were treated with BafA1 (15 nM) or vehicle for 40 min prior to apilimod (100 nM) or vehicle addition for 60 min. Under these conditions, BafA1 completely precluded the appearance of cytoplasmic vacuoles (see 2 and elevated PtdIns3P vs. control (only DMSO) at a level, very similar to that shown in 2 not only remained reduced by BafA1 pretreatment but even exhibited a trend, although not statistically significant, for a further decrease Pcontrols , 30. Thecontrols , 18 or ooles see . Quantitdecrease . Likewisapilimod . Strikin control . Quantit control . In factrophages . TogethePIKfyve, that binds PtdIns3P-enriched endosome membranes with high affinity inositol labeling experiments in HEK293 cells. At the end of the labeling protocol, cells were first treated with BafA1  followed by YM201636 . Lipids were then extracted, deacylated and analyzed by HPLC. As illustrated in 2 and PtdIns5P and they both remained equally reduced following BafA1 treatment. Conspicuously, however, whereas steady-state levels of PtdIns3P increased ~1.6-fold above controls by YM201636, this rise was only by ~1.15-fold if BafA1 was added prior to YM201636 P2 but also for PtdIns5P synthesis. The IC50 for both products was estimated to be in the subnanomolar range P2 levels did not occur P2 synthesis under PIKfyve perturbation inositol. Cells were than treated in the same labeling medium with vehicle  or BafA1 (15 nM in DMSO) for 40 min when additional vehicle (0.1% DMSO) was added for 60 min. Lipids were extracted, deacylated and subjected to HPLC separation. Shown are representative HPLC [3H]GroPInsP profiles from control (left panel) and BafA1-treated (right panel) cells (A) and quantification of bafilomycin-dependent changes in steady-state levels of PtdIns3P, PtdIns4P, PtdIns5P, PtdInsP2 and PtdInsP2 from 3 independent experiments (B). Apparent is the lack of changes in steady-state levels of PtdIns3P, PtdIns4P, PtdInsP2 and PtdInsP2 by BafA1. For reasons that remain to be clarified in future studies, there was a 40% decrease in steady-state levels of PtdIns5P compared to the control that received only vehicle. Note that BafA1 did not alter low levels of PtdIns5P reduced upon apilimod treatment (see (A and B): HEK293 cells cultured in complete media grown to 90–100% confluence run in parallel with apilimod and BafA1+apilimod samples shown in ment see .(PDF)Click here for additional data file.


English has been and continues to be extensively used as a common language in much of the political discourse and argumentation that occurs worldwide, with the vast majority of scientific papers and academic conferences/meetings also using English. It follows, then, that academic papers written in other languages would surely be less well represented, having fewer opportunities to be read and evaluated globally. This may also link to various difficulties in the promotion of epidemiology and other scientific fields. In practice, an epidemiologist may consider publishing papers in English for several reasons: (1) The research deserves to be read by a global audience, as it is novel and has implications in other areas or has universally applicable findings and policy implications; (2) publishing papers in English is required to advance one’s career; or (3) the researcher is a native English speaker or someone who likes writing in English.Every epidemiologist knows the heroic story of John Snow and the pioneering work he undertook to control the cholera outbreak that occurred in London in 1854. The fact that it happened in an English-speaking country would seem to be a major reason why this story has become famous globally, despite the events unfolding in a small district in downtown London. Or, to put it another way, if a modern-day epidemiologist comes from a non-English speaking area and sees no merit in any of the reasons listed above for publishing in English, then regardless of the potentially wider impact of the research, he/she may well choose to only write in his/her own native language, which would be a rational and reasonable choice. Writing in one’s own language should have clear advantages: low financial, mental, and time costs associated with writing, while having the potential to make an important contribution locally.Despite this, in the current era of globalization, important epidemiologic data on any emerging disease, especially if it is contagious like Ebola, is likely to be published in English. However, the situation was very different in previous decades; that is, epidemiological research was much more of a local activity than it is today. Hence, it is highly likely that there are vast numbers of epidemiologic papers that are not written in English, but that, nevertheless, deserve to be published in English and read by a global audience given their potential contemporary relevance. Moreover, it is likely that much of this research may even exist from many years ago, given the almost total absence of scientists publishing epidemiological research in English until only recently in many countries across the world.schistomatosis japonica was eradicated in the Kofu basin, Yamanashi, Japan belong in this category: 100 years of multidimensional interventions, including ways to ensure a sanitary environment and behaviors, research on civil engineering, medication, community organizing, and policy initiatives are extraordinarily interesting and have many potential implications not only for epidemiology and public health but also for the fields of medicine, governmental policy, engineering, cultural science, equity, and justice. These materials could have a significant impact in terms of global health, especially as a case study in disease eradication. However, information written in English about such topics is still very limited.Some of these papers may have important public health implications even now. For example, early-stage epidemiologic surveys of a local disease outbreak may be especially valuable, particularly if the disease subsequently spreads to many areas of the world in later years. A good example of this comes from Japan, with epidemiological research on diseases related to industrial pollution, including Minamata disease (organic mercury poisoning), Itai-itai disease (cadmium intoxication), and Yokkaichi asthma . Other potentially valuable papers are those that describe the history of how local disease was eradicated. For example, epidemiologic papers on the story of how With these thoughts in mind, I, as an editorial board member of the Journal of Epidemiology (JE), asked all the Associate Editors if they knew of “any papers, written in Japanese or other non-English languages that deserved to be translated into English, so that they could be read by a global audience”. Since that time, several candidate papers have been suggested. Subsequently, the editorial board of JE decided to launch a Reprints of Pioneering Papers series in order to spotlight these papers. In this series, we will publish reprints of these groundbreaking papers translated into English together with an invited commentary by an epidemiologist eminent in that specific field of research, providing a more comprehensive explanation of the topic and its implications for the current era.In relation to this, we now invite readers of JE to nominate possible papers to be included in this series. Those who have potential papers in mind should contact the JE editorial office (edit@jeaweb.jp) via email, while providing a copy of the suggested paper and a brief explanation of why the paper is pioneering and has potentially important implications today (this text should not exceed 300 words). If the proposed paper is not written in Japanese and there is no supporting information in either Japanese or English , please also attach a summary of the paper written in Japanese or English.1 Nonetheless, together with his colleagues, Kitamura ruled out various hypotheses on the cause of the disease that were suggested by the local authorities at that time, including that the disease was infectious, congenital, and caused by polluted drinking water or malnutrition.The first paper in this series is about Minamata disease, a disease caused by industrial pollution that emerged in the early stage of Japan’s post-war economic boom in the late 1950s. The outbreak of this disease, which is caused by chronic organic mercury poisoning, occurred in two bay areas: Minamata (Kumamoto Prefecture) and Niigata (Niigata Prefecture), resulting in 2,280 officially recognized fatalities. The legal dispute about what took place and its effects is still ongoing with the current discussion focusing on second-generation victims (those who were exposed in utero) and the discrimination being experienced by them. The seminal paper by Shoji Kitamura, who was Professor of Public Health at Kumamoto University at that time, presents statistical findings from his epidemiological research and draws a firm conclusion about the cause of the emerging disease: that it was poisoning due to eating polluted fish, despite the fact that, just like in John Snow’s time, no one, including Kitamura himself, knew that the disease was caused by organic mercury.2I feel certain that the readers of JE will find this research and the story of it just as exciting and informative as reading about John Snow’s work for the first time, as the paper by Professor Kitamura and his coworkers together with Takashi Yorifuji’s accompanying commentary provide a unique window into a world of, as yet, little-known but potentially valuable research.