Torque teno virus-induced aseptic meningitis has not been documented, although torque teno virus infections still remain under consideration for etiological agents. This study identified a torque teno virus sequence using next generation sequencing and immunoglobulin M response to the torque teno virus antigen, therefore, that would be a comprehensive diagnosis for torque teno virus infection.A 2-month-old Japanese boy was brought to our hospital because he was irritable, drowsy, and lethargic. He was admitted based on his test results which indicated the possibility of septic meningitis. He was started on treatment with high-dose antibiotics and steroids. On the third day of hospitalization, he became afebrile with improvement in his general status and was discharged on the sixth day. He had no developmental problems for up to 1 year after discharge. Metagenomic ribonucleic acid-Seq pathogen detection using next generation sequencing of a sample of his cerebrospinal fluid, which was collected at admission, revealed three short reads homologous to those in torque teno virus out of a total of 1,708,516 reads. This finding indicated that our patient was positive compared to the torque teno virus-negative cerebrospinal fluid samples (controls) from 13 other patients. The torque teno virus has been shown to have a whole genome sequence of 2810 nt by polymerase chain reaction. We prepared a recombinant GP2 antigen from torque teno virus and used it to study our patient’s anti-torque teno virus immune response. An anti-GP2 serum immunoglobulin M response was detected, providing further supportive evidence of torque teno virus infection.This case speculates that torque teno virus-induced aseptic meningitis has a good course. New technologies like next generation sequencing can help in the identification of such cases, and an accumulation of future cases is expected. Circoviridae and genus Anellovirus. TTV has a single-stranded circular DNA of approximately 3.8 kb. Human TTVs were first reported in 1997 [Torque teno viruses (TTVs) are ubiquitous viruses which are highly prevalent in several mammalian species and are currently classified into the family  in 1997 . Here weA 2-month-old Japanese boy (KS025) was brought to our hospital due to irritability, drowsiness, and lethargy. He had a cough for approximately 1 week and a fever for 3 days before admission. He was admitted because he was very pale, and the results of an examination showed the possibility of septic meningitis.Haemophilus influenzae type b (Hib), pneumococcal conjugate vaccine (PCV), Rotavirus (Rota), and hepatitis B virus (HBV) before admission. His brother and father also had a cough for 2 weeks. He had no relevant family history.He was born at 39 weeks, and his birth weight was 3788 g. At birth, he was admitted to the neonatal intensive care unit for 6 days due to transient tachypnea of the newborn. He had received one immunization each against 2), 98%. His body height was 65.0 cm and body weight was 5992 g. On physical examination, he appeared irritable and pale. His anterior fontanelle was not bulging, and there were no murmurs, rales, abdominal abnormal features, or exanthemas. The capillary refilling time was within 2 seconds.His vital signs at admission were as follows: body temperature, 37.5 °C; pulse rate, 156 beats per minute (bpm); respiratory rate, 30 breaths/minute; and blood oxygen saturation  and increased white blood cell (WBC) count. However, urine WBC levels were normal, and a chest radiograph showed no evidence of pneumonia. Cerebrospinal fluid (CSF) was remarkable for neutrophilic pleocytosis, although protein and glucose levels were within normal ranges Table . A brainBecause he was suspected of having septic meningitis because of above observations, he was given ampicillin (300 mg/kg per day), cefotaxime (300 mg/kg per day), and dexamethasone (0.15 mg four times a day). He became afebrile on the third day of hospitalization, and his general status improved. The antibiotic and steroid therapies were discontinued on the fourth day and, since his condition was stable, he was discharged on the sixth day. Bacterial culture tests were negative from two samples each of blood, urine, and CSF.After discharge using a sample of our patient’s CSF which was collected at admission, we performed metagenomic RNA(ribonucleic acid)-Seq pathogen detection by next generation sequencing (NGS) . Out of We prepared a His-tagged recombinant GP2  antigen from TTV using WEPRO7240H wheat germ extract and the Protemist® DT II robotic protein synthesizer . We tested our patient’s immune response to the specific GP2 antigen using the automatic Western blot detection system Wes  , thereby allowing the subsequent TTV infection to cause progressive neurological pathogenesis.The relationship that TTV establishes with the central nervous system (CNS) of the infected hosts is not clear, but it is suspected to increase the local expression of inflammatory mediators that may play a role in the pathogenesis of multiple sclerosis . It has Unlike cases in the past, using NGS we could detect TTV in this case of CNS infection. This case also shows that TTV-induced aseptic meningitis has a good course. New technologies like NGS can help in identifying cases of TTV as a causative virus, and we, therefore, expect an accumulation of such cases in the future.


Activation of antiviral innate immune responses depends on the recognition of viral components or viral effectors by host receptors. This virus recognition system can activate two layers of host defence, pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI). While ETI has long been recognized as an efficient plant defence against viruses, the concept of antiviral PTI has only recently been integrated into virus–host interaction models, such as the RNA silencing‐based defences that are triggered by viral dsRNA PAMPs produced during infection. Emerging evidence in the literature has included the classical PTI in the antiviral innate immune arsenal of plant cells. Therefore, our understanding of PAMPs has expanded to include not only classical PAMPS, such as bacterial flagellin or fungal chitin, but also virus‐derived nucleic acids that may also activate PAMP recognition receptors like the well‐documented phenomenon observed for mammalian viruses. In this review, we discuss the notion that plant viruses can activate classical PTI, leading to both unique antiviral responses and conserved antipathogen responses. We also present evidence that virus‐derived nucleic acid PAMPs may elicit the NUCLEAR SHUTTLE PROTEIN‐INTERACTING KINASE 1 (NIK1)‐mediated antiviral signalling pathway that transduces an antiviral signal to suppress global host translation. To cope with invading pathogens, plant cells evolved a sophisticated immune system under constant pressure for dominance over the pathogens’ virulence strategies, which in turn coevolved to escape the host recognition system  and Oilseed rape mosaic virus  and the potexvirus Potato virus X  depend on ligand (PAMP/DAMP)‐induced dimerization or oligomerization with coreceptors for activation  from the bipartite begomoviruses, et al., in vitro at conserved threonine residues, positions Thr‐468, Thr‐469 and Thr‐474 within the activation loop, and mediates substrate phosphorylation in vivo /Arabidopsis and Tomato yellow spot virus (ToYCV)/Tomato] indicates that virus infection both activates and suppresses NIK1‐mediated antiviral signalling  plants at the seven‐leaf stage were inoculated by biolistic delivery with infectious clones of CaLCuV DNA‐A, and DNA‐B and the accumulation of viral DNA was monitored by PCR  undergoes dsRNA ligand‐mediated dimerization and activation upon direct association of viral dsRNA produced during infection with its kinase domain  of plant viruses use the endocytic recycling pathway to move viral RNA– and viral DNA–protein complexes to the plasmodesmata for the MP‐assisted cell‐to‐cell spread of the virus (Lewis and Lazarowitz, Although this endocytic recycling pathway is an attractive hypothesis for virus perception by plant PRRs, similar to mammalian endosomal TLRs, it may not be possible to synchronize PTI activation as a primary, early response to pathogen attack, with the later event of virus movement to adjacent cells. Therefore, only with the identification of the origin and nature of virus‐derived PAMPs and the characterization of nucleic acid‐sensing PRRs will it be possible to address these possibilities for virus perception by plasma membrane‐anchored RLKs.Recent studies with SERK‐like coreceptors and known PRRs, which function in plant innate immunity, have demonstrated that the classical PTI is part of the antiviral defence arsenal of plant cells. Several components of classic PTI, including the co‐receptor SERKs, BAK1 and SERK1, and the MAPK4 negative regulator, in addition to common immune responses, co‐exist as part of antiviral PTI. As for viral PAMPs, dsRNAs isolated from virus‐infected plants have been demonstrated to induce typical PTI responses dependent on the coreceptor SERK1. Likewise, begomovirus‐derived nucleic acids were shown here to induce the NIK1‐mediated antiviral signalling in a NIK1‐ and NIK2‐dependent manner and were included as a piece of new complementary evidence for this review. However, the nature and origin of the nucleic acid PAMPs have not been deciphered, and the nucleic acid sensors have yet to be identified. Furthermore, as viruses are intracellular obligate parasites and may not have access to the apoplast, it remains to be determined how PRRs would sense viruses and viral PAMPS extracellularly. The identification of the origin and nature of virus‐derived nucleic acid PAMPs and the discovery of the cognate PRRs will shed light on the mechanism of antiviral PTI activation and its role in resistance against viruses.The authors declare no conflict of interest.


The purpose of this study was to assess the overall clinical results and range of motion (ROM) after total knee arthroplasty (TKA) in patients with preoperative stiffness. We also aimed to determine whether the severity or cause of the stiffness can affect the clinical outcome after surgery. This retrospective study included 122 knees (117 patients) with follow-up of more than 2 years . TKA was performed using posterior-stabilized, varus-valgus constrained (VVC), and hinged prostheses. To determine the effect of the severity of stiffness on the clinical outcome, the subjects were divided into two groups: the severe group  and the moderate group . Then, clinical results and ROM were compared according to the severity or cause of preoperative stiffness. After surgery, preoperative ROM  was improved . The severe group more frequently used the VVC or hinged prostheses (72% vs. 18%). Furthermore, the severe group had worse knee and function scores as well as more complications (33% vs. 13%), even though the severe group had a greater ROM increment (47° vs. 27°) after surgery. Patients with osteoarthritis and rheumatoid arthritis showed better ROM and clinical results compared to patients with infectious or traumatic arthritis. Although TKA in stiff knees can be successful, the results are inferior in knees with severe stiffness and knees with infectious or traumatic arthritis. The main goals of total knee arthroplasty (TKA) are pain relief and functional improvement . The majPostoperative ROM in patients with poor preoperative ROM may differ according to the severity of preoperative stiffness ,7. GenerAlso, the causative disease of end-stage knee arthritis can affect clinical outcome after TKA. The stiffness caused by mechanical impingement can be resolved during surgery. However, adhesion involving soft tissues caused by a previous infection or trauma may be more difficult to fix . ExtensiThe purpose of this study was to assess the overall clinical results and ROM after total knee arthroplasty (TKA) in patients with preoperative stiffness. We also aimed to determine whether the severity or cause of the stiffness can affect the clinical outcome after surgery.2. This study protocol was approved by the Institutional Review Board of the SMG-SNU Boramae Medical Center (16-2016-132). All patients gave their consent for using and assessing their data.This retrospective study included 122 knees with follow-up of more than 2 years. From March 2005 to March 2012, 1859 TKAs were performed at our institution. We defined preoperative stiffness as a ROM of knee joint ≤ 90° . Among tAll surgeries were performed using the same surgical protocol by a single senior surgeon (one of the authors), but various prostheses were used because of conditions of the knee joints and the surgeon’s preference. Spinal anesthesia was routinely tried for all patients, but in a subset of patients, general anesthesia was performed when the spinal anesthesia alone was not effective. A medial parapatellar approach with a tourniquet was used in most patients. However, a rectus snip was used to create a proper exposure in 27 knees (22%). The V-Y quadricepsplasty was used in only two (2%) knees, when a sufficient approach was not acquired using the rectus snip. No knee had a tibial tuberosity osteotomy. We used an intramedullary guide for resection of both the femur and tibia in all cases. If the knee was balanced and stable, a posterior-stabilized type implant was used. In contrast, if there was unacceptable soft tissue imbalance, a constrained prosthesis or a hinged prosthesis was used. All patellae were resurfaced and all components of the prosthesis were fixed with cement.The rehabilitation protocol was the same except for patients with a V-Y quadricepsplasty. Continuous passive motion was started 2 days after surgery and the patients were encouraged to perform quadriceps strengthening exercises. Compression stockings and a pneumatic compression device were applied for thrombo-prophylaxis. Ambulation with a walker was allowed on the second postoperative day, as tolerated. Patients received inpatient physical therapy once daily for 20 minutes. They were instructed in home-based rehabilitation by therapist including ice, controlling swelling, walking and ROM. After discharge, they continue home exercise. In patients who underwent V-Y quadricepsplasty, the ROM was restricted with a brace in extension for 4 weeks. The active extension exercise and straight leg raises were withheld for 4 weeks. We did not perform manipulation under anesthesia postoperatively.Data on clinical outcome were collected prospectively by a clinical investigator. Data on the causative disease of the end-stage arthritis and the prosthesis used were reviewed using the medial records. Clinical outcome including ROM, Knee Society (KS) knee score, KS function score, and Western Ontario and MacMaster (WOMAC) score were evaluated preoperatively and postoperatively at 6 months, 1 year and 2 years. The ROM of the knee joint was measured using a goniometer with the patient supine. Complications including wound necrosis, superficial infection, and deep infection were recorded. Recurrence of stiffness was defined as postoperative ROM ≤ 90° regardless of preoperative ROM.Statistical analyses were performed using SPSS for Windows , and P values of <0.05 were considered statistically significant. Pre- and postoperative ROM and knee scores were described using mean and range. The statistical significance of the difference between pre- and postoperative data was determined using a paired t-test. Most previous studies defined preoperative stiffness as when the ROM was less than 50°, while other studies used 90° to define preoperative stiffness ,7,15. ThClinical outcome after TKA in patients with stiff knees improved after surgery in terms of ROM and knee and function scores . PreoperTotal knee arthroplasty in the severe group used prostheses with more constraint and had worse knee and function scores as well as more complications even though patients in the severe group had a greater ROM increase after surgery. During surgeries, TKA in the severe group used VVC or hinged prostheses in 72% whereas TKA in the moderate group used these prostheses only in 18% of knees . In contTotal knee arthroplasty in patients with preoperative stiffness caused by osteoarthritis and rheumatoid arthritis showed better ROM and clinical results compared to patients with infectious or traumatic arthritis. Mean ROM was greater than 110° in patients with osteoarthritis and rheumatoid arthritis after surgery, whereas only 13% (3 of 25 knees) with infectious arthritis achieved a ROM greater than 110° . Clinica2 = 0.362) showed that preoperative ROM  and cause of stiffness  were significant factors related to postoperative ROM. In simple terms, patients who had better preoperative ROM showed greater postoperative ROM. Also, patients with OA and RA showed greater postoperative ROM than patents with infectious and traumatic arthritis  before TKA. We also hypothesized that clinical outcome would differ according to the severity and causative disease of preoperative stiffness. The principal finding of this study was that TKA substantially improved ROM and functional scores in the patients with preoperative stiffness. We also found that the severe stiffness group had more ROM gain than the moderate group. Nonetheless, the final ROM and clinical scores of the severe group were inferior, with a higher complication rate than in the moderate group. In addition, the stiffness caused by infectious and traumatic arthritis was associated with worse clinical outcomes after surgery. In multiple regression analysis, preoperative ROM and cause of stiffness were related to postoperative ROM. Our findings suggest that TKA can improve ROM and functional scores even in the presence of severe preoperative stiffness, but surgeons should be cautious about inferior outcome in patients with more severe preoperative stiffness and the stiffness caused by infectious and traumatic arthritis.Our findings are in line with the findings of previous studies showing that TKA improved ROM and functional scores in patients with poor preoperative ROM –18. McAuThe results of this study support the hypothesis that clinical outcome and ROM differ according to the severity of preoperative stiffness. In the previous comparison between stiff and flexible knees, mean ROM gain was greater in the stiff knees: an increment of 64.4% in stiff knees vs. 59.9% in the flexible knees . In the Our finding also affirms the hypothesis that clinical outcome would differ according to the cause of preoperative stiffness. Aglietti et al. compared ROM between patients with ankylosed knees caused by osteoarthritis and those caused by rheumatoid arthritis. The mean arc of motion was the same at 78° in both groups after TKA . In contThis study has several limitations. First, the study was retrospectively performed and there was no control group of flexible knees. However, we think this study can provide valuable information to readers by comparing knees with severe stiffness to knees with moderate stiffness, which are encountered more frequently during clinical practice. Second, the follow-up period of more than 2 years was relatively short. Thus, we were not able to examine complications such as aseptic loosening of the prosthesis or late prosthetic joint infection, which can develop in the longer term after surgery. However, we believe that the two-year follow-up was enough to present postoperative ROM and knee scores because the postoperative ROM rarely increases one year after surgery .Total knee arthroplasty improves ROM and clinical results after surgery in patients with preoperative stiffness. However, the clinical outcome was inferior in knees with more severe preoperative stiffness or stiffness caused by secondary arthritis such as infectious arthritis and traumatic arthritis. For those patients, special measures such as active physical therapy may be necessary. In addition, the findings of this study should be used to counsel patients before performing TKA in knees with preoperative stiffness.S1 AppendixThe entire dataset used in this study is presented in the file S1 Appendix.(XLSX)Click here for additional data file.


During 2014–2017, we isolated a novel orthobunyavirus from broiler chickens with severe kidney lesions in the state of Kedah, Malaysia; we named the virus Kedah fatal kidney syndrome virus (KFKSV). Affected chickens became listless and diarrheic before dying suddenly. Necropsies detected pale and swollen kidneys with signs of gout, enlarged and fragile livers, and pale hearts. Experimental infection of broiler chickens with KFKSV reproduced the disease and pathologic conditions observed in the field, fulfilling the Koch’s postulates. Gene sequencing indicated high nucleotide identities between KFKSV isolates (99%) and moderate nucleotide identities with the orthobunyavirus Umbre virus in the large (78%), medium (77%), and small (86%) genomic segments. KFKSV may be pathogenic for other host species, including humans. After inoculation, we checked eggs daily for 7 days and discarded those that died within 24 h. The chorioallantoic fluid was harvested aseptically from embryos that died after 24 h, and these embryos were examined for the presence of gross pathologic lesions. If at the end of the 7-day observation period no embryos had died, we removed the eggs with live embryos and kept them at 4°C for 24 h and then collected the chorioallantoic fluid for the next passage. We conducted 3 blind passages before considering a sample negative for pathogens.We collected samples of kidney and cecal tonsils from affected chickens in the field that died and submitted them to the Scientific Support and Investigation Laboratory of Ceva-Phylaxia  for detection of possible viral agents. The tissue samples were homogenized to give ≈10% (wt/vol) suspension in phosphate-buffered saline pH 7.2. The homogenized samples were centrifuged at 1,000 × The chorioallantoic fluid of the embryos showing pathologic lesions was inoculated onto LMH (chicken hepatocellular carcinoma) and Vero (African green monkey kidney) cell cultures and checked for cytopathogenic agents. LMH cells were also used for propagation and titration of the isolated virus and for reisolation of the virus from kidney samples from experimentally infected chickens.g for 5 min, and 200 μL of the supernatant was used for nucleic acid extraction by use of the ZiXpress32 Viral Nucleic Acid Extraction Kit and ZiXpress32 robot . Random primed reverse-transcription PCR (RT-PCR) was performed as described elsewhere . The resulting cDNA libraries were measured on Qubit 2.0 equipment by using the Qubit dsDNA BR Assay kit . The isothermal emulsion PCR that produced clonally amplified libraries was conducted according to the manufacturer’s protocol by using the IA 500 kit . Enrichment of the templated beads  and further steps of presequencing setup were performed according to the manufacturer’s 200-bp protocol. We strictly followed the sequencing protocol recommended for the Ion PGM Hi-Q View Sequencing Kit on a 316 v2 chip (Life Technologies).Supernatants of tissue cultures showing cytopathologic changes were centrifuged at 10,000 × https://www.nimagen.com) was used in the amplification reaction, and dye-labeled products were run on an ABI 3500 sequencer (Life Technologies).Sanger sequencing was used to validate results obtained by the viral metagenomics approach. Primer pairs were designed to amplify 0.8–1.2-kb fragments (not shown). The BrightDye Terminator Cycle Sequencing Kit . The minimal read length parameter was set to 40. The same software was used to assemble the genome sequence. After visually inspecting the sequence mappings, we created 1 consensus sequence for all 3 genome segments . Sanger sequencing outputs were analyzed by using BioEdit software (–http://www.ncbi.nlm.nih.gov/gorf/gorf.html).Sequence reads obtained by next-generation sequencing were trimmed by using CLC Genomics Workbench version 7 (http://www.pcrbio.com), 1.6 μL S gene specific forward (5′ GGGTAGCACTAGCATTTATCCA 3′) and reverse (5′ TGTAGACACCCACAAACGTATC 3′) primers (each from 10 μM stock solutions), 0.5 μL S gene specific probe (5′ [FAM] AGCTCACATACGAGGATACACAGGA [BHQ] 3′) from a 10 μM stock solution, 1 μL 20X RTase with RNase inhibitor, 0.3 μL nuclease-free water, and 5 μL RNA. The thermal profile of the qRT-PCR was as follows: reverse transcription at 50°C for 10 min terminated by heating to 95 °C for 2 min, followed by amplification performed in 40 cycles consisting of denaturation at 95°C for 5 s and hybridization and elongation at 60°C for 20 s. Throughout the study, we used a Mic qPCR cycler , kindly provided for the project by Péter Kisfali, Amplicon Ltd.).For assay development and quantification of the novel orthobunyavirus genomic RNA in organ samples of the experimental animals, we extracted viral RNA as previously described and designed primers and probes for the S genomic segment of the novel orthobunyavirus. The reaction mixture of quantitative RT-PCR (qRT-PCR) that we used was composed of 10 μL 2× qPCRBIO Probe 1-Step Mix ; QX-like, 4/91-like, and Massachusetts-like infectious bronchitis virus strains (Coronaviridae); representative isolates of avian metapneumovirus A, B, and C and Newcastle disease virus (Paramyxoviridae); isolates of influenza (H9N2) and (H5N8) viruses (Orthomyxoviridae); and Tembusu virus (Flaviviridae).To test the assay specificity, we used a number of viruses from our strain collection, including avian nephritis virus (10 50% tissue culture infective dose (TCID50) of the isolate by either the subcutaneous or oral route; for negative controls, we inoculated 5 chickens of the same age with phosphate-buffered saline pH 7.2. We kept all birds in isolators with negative pressure and monitored them daily for overt clinical signs and death for 26 days after inoculation.We tested the pathogenicity of 1 of the isolates (D2756/1/2014/MY) in 5-day-old broiler chickens purchased from a commercial hatchery in Hungary. We inoculated 10 chickens with 0.2 mL of 5.1 logWe recorded gross lesions of birds that died during the 26-day postinoculation period and collected samples from the spleen, liver, kidney, heart, pancreas, small intestine, bursa, and thymus and placed them in 10% buffered formalin for histologic analysis. We also collected tissue samples  from 5 chickens in the subcutaneously infected group and 4 chickens from the orally infected group that died and processed them for viral RNA detection by using qRT-PCR. We euthanized all remaining birds and inspected them for gross pathologic lesions, collecting samples for histologic analysis  and qRT-PCR .Tissue samples were processed according to standard histologic procedures. Samples were embedded in paraffin, cut in 5-μm sections, stained with hematoxylin and eosin or trichrome, and examined under light microscopy.The first outbreaks of the disease occurred during July–September 2014 and recurred during the same months each subsequent year. All cases were from broiler farms with open-sided houses in Kedah .Several chickens in the affected flocks became listless and exhibited signs of diarrhea. Affected birds died soon after the appearance of the first clinical signs. The disease sometimes occurred in birds ≈1 week of age but more routinely in those 2–4 weeks of age. Mortality rates varied within a wide range, from a low percentage to 30%–40% . The most striking lesions in dead chickens were pale, swollen kidneys with uric acid deposits, frequently localized in the tubules and ureters  and alsoFrom the kidney and cecal tonsil samples submitted from 1 affected chicken in 2014 and 3 affected chickens in 2017, we obtained 5 virus isolates after 1 blind passage in SPF embryos. Embryo deaths occurred 3–5 days after inoculation with the organ homogenates. The dead embryos exhibited severe subcutaneous edema, congestion, and hemorrhages on the skin. No hemagglutinating agents were observed when chicken red blood cells were used in analysis of allantoic fluid from dead embryos.10 TCID50/mL.Cytopathic effect was evident at 48–72 hours after inoculation of LMH and Vero cell cultures with the allantoic fluid from dead embryos. Elongated or rounded dark cells were seen in irregular clusters, expanding very fast to affect the whole cell sheet within 24–36 hours. The virus grown on LMH cell cultures reached a titer of 6.3–6.5 logViral metagenomics performed from 2 cell culture supernatants (D2756/1/2014/MY and D2756/2/2014/MY) identified sequence scaffolds with greatest BLAST hits to the orthobunyavirus Umbre virus (Turlock serogroup). The most 3′ and 5′ ends of the genomic RNA could not be determined with this method. To confirm the sequencing results obtained by next-generation sequencing, we also performed Sanger sequencing. The obtained nearly full-length genome sequences  showed >99% nt sequence identities between isolates D2756/1/2014/MY and D2756/2/2014/MY. When we compared sequences with Umbre virus, we identified 78% nt sequence identities along L, 77% along M, and 86% along S genomic segments.50/mL virus culture supernatant (or 0.03 TCID50 virus/5 μL RNA). The kidney samples submitted from the sick poultry in the field during 2014 and 2017 contained RNA copies corresponding to a viral titer of 9.00 × 104 to 4.81 × 105 TCID50 of infectious virus per gram of organ specimen. Phylogenetic analysis based on the partial amino acid sequences of proteins encoded by the L, M, and S segments showed that all 5 isolates detected from 4 different outbreak cases over a 3-year period were highly similar in the regions analyzed . From postinoculation day 3, chickens became listless and showed loss of appetite. Deaths occurred during postinoculation days 3–5 in the subcutaneously infected group and started 2 days later (postinoculation day 5) in the orally infected group. The mortality rate was higher for the group infected by the subcutaneous route .After clinical signs appeared, the chickens died within 12–24 hours. In those that died, gross lesions were mainly confined to the kidneys, which were pale and swollen with distended ureters filled with uric acid , panel AIn the organs collected from the experimentally infected chickens that died, the predominant histologic lesions were in the kidney, liver, heart, and lymphoid organs. The most consistent lesions were tubulonephrosis with occasional tubular epithelium necrosis; intraglomerular urolithiasis and multifocal interstitial lympho-histiocytic infiltration , panel AHistopathologic changes were nearly completely missing in the birds sampled at the end of the experiment. Only some focal interstitial nephritis, hepatitis, and myocarditis could be found in a few birds.High viral loads in the different organ samples of dead chickens were similar, irrespective of the route of infection and time of death . These rWe found the causative agent of the disease outbreak of severe kidney damage among broiler chickens in Malaysia in 2014 to be a newly emerging orthobunyavirus. On the basis of the main pathologic features of the disease seen in the field and after experimental infection, we propose that the virus be designated Kedah fatal kidney syndrome virus (KFKSV) of broilers.Orthobunyavirus genus (Analysis of the near full-genome sequence of 2 isolates, D2756/1/2014/MY and D2756/2/2014/MY, demonstrated that the virus is similar to Umbre virus, which belongs to the Turlock serogroup of the Culex mosquitoes and birds (Umbre virus was isolated during the 1950s from Orthobunyavirus genus are of major veterinary and public health concern (Several viruses within the The D2756/1/2014/MY isolate of KFKSV was highly pathogenic in young broiler chickens, causing mortality rates of 50%–70% and severe gross and histopathologic lesions in the kidneys and liver after experimental infection. The gross and histopathologic changes caused by the isolate were similar to those found in field-infected chickens. The virus could be detected from all tested internal organs of dead chickens, including intestines, but the spleen samples collected from asymptomatic chickens after recovery were also positive for KFKSV 3 weeks after infection. Therefore, investigation of virus persistence and transmission is needed.There are no commercially available or standardized tests for diagnosing bunyavirus infections in poultry. Establishing a diagnosis requires submitting samples to specialized reference laboratories. KFKSV can be isolated and grown in chicken embryos and can be propagated in LMH and Vero cells. When reported disease and clinical history of a flock includes kidney lesions, diagnostic laboratories can use the molecular diagnostic method we describe in this article to check submitted materials for this newly discovered emerging species of orthobunyavirus. Similar kidney lesions could be observed after infection with avian infectious bronchitis virus, astrovirus, and leucocytozoonosis. Diagnosis of these infections is generally based on detection by RT-PCR and sequencing, virus isolation, and demonstration of an immune response by virus neutralization or enzyme immunoassay.For determining a possible viral origin of an idiopathic disease, the viral metagenomics technique is useful, as is demonstrated by our discovery of this novel orthobunyavirus. The potential of this virus to spread to other animal species or humans cannot be excluded. Therefore, studies in which additional data are collected to elucidate the pathogenic potential of this virus in avian and other animal species should be initiated. Studies to scrutinize its potential as a public health hazard should also be considered. Because of the zoonotic nature of some orthobunyaviruses closely related to KFKSV (


Each dilution tested reduced the infectious EBOV/Mak titer by ~5 log10 within one min. Detectable virus was still present after an 0.5-min exposure, but each DAL dilution caused >4 log10 reduction within this time. Detection of virus below the limit of detection of the TCID50 assay was assessed by inoculating flasks of Vero E6 cells with undiluted neutralized sample and evaluating the cultures for cytopathic effect after 14 days incubation. No infectious virus was detected with this non-quantitative method in samples subjected to DAL for 5 or 10 min, regardless of the dilution evaluated. The rapid and substantial inactivation of EBOV/Mak by DAL suggests that use of this hygiene product could help prevent the spread of Ebola virus disease during outbreaks.The efficacy of Dettol Antiseptic Liquid (DAL) for inactivating Ebola virus (Makona C07 variant) (EBOV/Mak) within an organic load in suspension was evaluated per ASTM E1052-11. Three DAL lots were evaluated at dilutions of 1:10, 1:20, and 1:40, with contact times of 0.5, 1, 5, and 10 min. Approximately 7 log As of December 11, 2018, the World Health Organization reported that the Ebola outbreak in the North Kivu and Ituri provinces of the DRC has included a total of 505 cases, with 457 confirmed and 48 probable, and has resulted in 296 deaths2. Overall, to date there have been 34 Ebola virus disease outbreaks, 18 of which have involved Zaire ebolavirus since the initial emergence of this strain in 1976. This most recent outbreak, and the outbreaks occurring between 2014–2016, have refocused efforts of public health agencies such as the World Health Organization2 on identifying approaches to reduce the spread of the disease from community to community and from nation to nation. Ebola virus disease is included in the World Health Organization’s List of Blueprint Priority Diseases3, a list of diseases for which: “… given their potential to cause a public health emergency and the absence of efficacious drugs and/or vaccines, there is an urgent need for accelerated research and development…”3.The Ebola virus continues to re-emerge in lethal outbreaks, the most recent occurring in the Democratic Republic of the Congo, Africa in May 20184. It has also been shown that fomites in the vicinity of infected patients may be contaminated with Ebola virus RNA5. Environmental persistence of infectious EBOV Makona (EBOV/Mak) suspended in an organic soil load have been reported at eight days from experimentally contaminated surfaces6. An important intervention approach might therefore involve the use of an effective virucidal agent for disinfecting surfaces and spills potentially contaminated with Ebola virus, thereby mitigating the risk of transmission to healthy individuals, including health-care providers.It is known that the Ebola virus may be transmitted by contact with infected corpses, infected environmental surfaces (fomites), and secretions and excretions from infected individualsFiloviridae family, and being an enveloped virus should be relatively susceptible to a variety of microbicidal inactivation approaches. In view of the lethality of the virus, however, the United States Centers for Disease Control and Prevention (CDC) has provided the following guidance7: “… selection of a disinfection product with a higher potency than what is normally required for an enveloped virus is being recommended at this time. EPA-registered hospital disinfectants with label claims against non-enveloped viruses  are broadly antiviral and capable of inactivating both enveloped and non-enveloped viruses.” Per the United States Environmental Protection Agency (EPA), in order to claim efficacy of a product for an emerging enveloped virus, the product needs to be approved by EPA for inactivating at least one large or one small non-enveloped virus8.There are relatively little suspension inactivation data for the Ebola virus itself. The efficacies of microbicides (disinfectants and antiseptics) for inactivation of Ebola virus typically been determined through evaluation of the inactivation, by such products, of appropriate surrogate viruses such as bacteriophages, enveloped viruses , or non-enveloped viruses such as caliciviruses or picornaviruses. The Ebola virus is a member of the Efficacy testing of microbicides through the study of inactivation of surrogate non-enveloped viruses theoretically should ensure their efficacy for inactivation of the Ebola virus. However testing conducted specifically with the high-risk pathogens including viruses is also needed, to provide assurance to critical facilities and personnel. In the present effort, efficacy studies were performed at the Canadian Science Centre for Human and Animal Health Biosafety Level 4 (BSL-4) facility.9. It is also used for personal hygiene purposes, and as a microbicide for decontaminating environmental surfaces, objects, and equipment. The microbicidal active ingredient in DAL is 4-chloro-3,5-dimethylphenol (chloroxylenol), also known by its non-systematic name para-chloro-meta-xylenol or PCMX10. The concentration of the active in DAL is 4.8% (weight to volume) PCMX. Three concentrations each of DAL lots 15083E, 16004E, and 16005E were evaluated for inactivation of EBOV/Mak suspended in an organic soil load using the method specified in the ASTM 1052-11 international standard11. This test method was developed by the American Society for Testing and Materials International (ASTM) to standardize the evaluation of virucidal activity of microbicidal products in suspension. Organic soil loads are added to the study design in order to better model viral inactivation by microbicides in relevant matrices such as human sputum or blood. Use of hard water as diluent was included in the study design to simulate water hardness in the field. Four contact times  were evaluated in triplicate for each of three independent lots of DAL. In addition to the methodology described in the ASTM standard, we also evaluated any residual infectious virus following exposure to DAL through inoculation of 500 µL of undiluted neutralized test sample into T-75 flasks of Vero E6 indicator cells. This was done to evaluate the possibility of virus being present at levels lower than the limit of detection of the tissue culture infectious dose50 (TCID50) assay performed in Vero E6 cells per the ASTM standard.In the present study, we evaluated the efficacy of a commercially available hygiene product, Dettol Antiseptic Liquid (DAL), for inactivating EBOV/Mak is suspension. Dettol is used in Europe, Africa, and Asia in homes and healthcare settings for various first aid antiseptic purposes, including wound cleansing13, prevented inactivation of the virus. As shown in Supplemental Figs P < 0.05) differences in the viral titers obtained for the virus positive controls, the virus + DAL dilution + neutralizer, and virus + neutralizer conditions were observed when 1× Letheen broth was evaluated. The disinfectant neutralizing agent that was used in each of the inactivation efficacy studies described below was 1× Letheen broth.During the evaluation of possible neutralizing agents, it was determined that 100% fetal calf serum (FCS) and 100% virus culture medium (VCM) failed to adequately terminate the viral inactivating effects of DAL. On the other hand, 1× Letheen broth in VCM (10× Letheen broth diluted 1:10 in VCM), added to the DAL dilutions prior to introduction of the EBOV/Mak in tripartite soil load8 log10 TCID50/mL in tripartite soil load was exposed to the various DAL dilutions and contact times using the methodology depicted in Fig. 10 reduction values for each time point were calculated by subtracting the titers obtained for the DAL test conditions from the titers of the corresponding positive virus controls.Three different lots of DAL were evaluated at three dilutions of DAL each  in triplicate. Contact times of 0.5, 1, 5, and 10 min at ambient temperature were evaluated in a BSL-4 facility. An initial EBOV/Mak titer of 1.7 × 1010 EBOV/Mak titers measured for the positive virus control condition  and the post-exposure titers are displayed in Fig. 10) of EBOV/Mak to the limit of detection of the assay (1.8 log10 TCID50/mL virus) was observed for all replicates and contact times.For the 1:10 dilution of the three DAL lots at 0.5, 1, 5, and 10 min contact time, the mean ± standard deviation values for the log10 EBOV/Mak titers measured for the positive virus control condition  and the post-exposure titers are displayed in Fig. 10) of EBOV/Mak to the limit of detection of the assay (1.8 log10 TCID50/mL virus) was observed for all replicates following contact times of 5 min or greater.For the 1:20 dilution of the three DAL lots at 0.5, 1, 5, and 10 min contact time, the mean ± standard deviation values for the log10 EBOV/Mak titers measured for the positive virus control condition  and the post-exposure titers are displayed in Fig. 10) of EBOV/Mak to the limit of detection of the assay (1.8 log10 TCID50/mL virus) was observed for all replicates following contact times of 1 min or greater.For the 1:40 dilution of the three DAL lots at 0.5, 1, 5, and 10 min contact time, the mean ± standard deviation values for the log50 assay (1.8 log10 TCID50/mL) was evaluated by inoculating T-75 flasks of Vero E6 cells with 500 µL of undiluted neutralized test sample after each contact time and then evaluating the cultures microscopically for viral cytopathic effects (CPE) after 14 days of incubation. The results of this flask safety testing for the 1:10, 1:20, and 1:40 DAL dilutions (n = 1 flask per condition and DAL lot) are displayed in Table The possible presence of infectious EBOV/Mak below the limit of detection of the TCIDThe results of the flask safety testing for the 1:40 DAL dilution (n = 1 flask per condition and DAL lot)  for disinfection of infected excreta and corpses. Reproducible inactivation of viruses by chlorine is dependent upon free chlorine concentration, which is dependent both upon the chlorine demand of the inactivation solution as well as the free chlorine concentration of the bleach solution being used. The efficacy of free chlorine for inactivation of EBOV/Mak in sterilized municipal wastewater was investigated by Bibby et al.16. The inactivation resulting from 1 ppm chlorine at 20 °C was incomplete but substantial . Higher chlorine concentrations  resulted in complete inactivation of the EBOV/Mak (4.2 log10 reduction for 0.3 min contact time).In view of the paucity of available information on suspension inactivation of Ebola virus and other hemorrhagic fever viruses, the CDC has recommended17 and for disinfecting surgical instruments18. It is an active ingredient used in a number of household microbicidal products, including DAL. As with other phenolics, the mechanism of viral inactivation for PCMX is likely related to disruption of lipid envelopes19. The virucidal effectiveness of PCMX formulations is therefore expected to be greatest for enveloped viruses.Bleach, while an effective microbicide, is not practical for use on intact or wounded human skin. In addition, as mentioned above, the free chlorine levels of commercial bleach products may vary and effect the final free chlorine of the final inactivation solutions. On the other hand, the halophenolic compound PCMX, which is the active microbicidal ingredient in DAL, is suitable for use for preoperative skin antisepsis20. As is implicit in this guidance, the inactivation afforded by 0.1% Triton X-100 is not to be considered complete and, in fact, van Kampen et al.21 have shown that the percentage of serum in a test sample determines the extent of inactivation. In a sample matrix consisting of 98% fetal bovine serum, there was no statistically significant log10 reduction of Ebola virus resulted from a 1-hour exposure to 0.1% Triton X-100. A similar result was obtained when 0.1% sodium dodecyl sulfate was used as the inactivating agent. In samples containing lower percentages  of fetal bovine serum, the inactivation caused by these agents was >3 log10, but still not complete. The inability of Triton X-100 at up to 1% to completely inactivate Ebola virus in blood serum has also been reported by Burton et al.22 and Colavita et al.23.It has been proposed that the detergent Triton-X 100 be used at 0.1% to decontaminate clinical specimens  in order to reduce  the titer of Ebola virus in such specimens10 reduction value. For instance, the US Environmental Protection Agency (EPA) stated in its 2012 disinfectant product guidance24 that “The product should demonstrate complete inactivation of the virus at all dilutions. If cytotoxicity is present, the virus control titer should be increased to demonstrate a ≥3 log10 reduction in viral titer beyond the cytotoxic level.” For disinfectants that are non-cytotoxic to the cellular infectivity assays used for demonstrating efficacy, a 4-log10 reduction in viral titer is considered to be effective. These EPA requirements for demonstrating acceptable efficacy were recently modified in the 2018 revision25. In the revised guidance, a valid test requires (1) that at least 4.8 log10 of infectivity per carrier be recovered from the dried virus control film; (2) that a ≥3 log10 reduction in titer must be demonstrated in the presence or absence of cytotoxicity; (3) if cytotoxicity is present, at least a 3 log10 reduction in titer must be demonstrated beyond the cytotoxic level; and (4) that the cell controls be negative for infectivity. In the revised guidance, therefore, an efficacious product does not need to demonstrate complete inactivation at all dilutions.Efficacy for viral inactivation is typically expressed in terms of an expected log10 reduction value must, however, be considered against the backdrop of the mortality associated with the virus, the estimated infectious dose, and the expected viral load within a contaminated bodily fluid. The mortality associated with Ebola virus disease is high. For example, the overall case fatality rate for outbreaks occurring between 1976 and 2017 has been calculated as 67%26. The infectious dose of Ebola virus is believed to be relatively low in humans (between 1 and 10 infectious units)27. The median plasma viral load in confirmed Ebola virus disease patients during the 2014–2015 Sierra Leone outbreak was found to be 6.7 log10 genomic copies per mL (range: 5.5 to 7.8 genomic copies/mL)28. The average peak blood serum titer in cases from the 2000–2001 Uganda outbreak with a fatal outcome was 3.4 × 109 genomic copies per mL, corresponding to 3.4 × 105 to 3.4 × 106 infectious units per mL29. Human-to human transmission of Ebola virus disease has been attributed to direct contact with infected blood (including reuse of contaminated needles) or other secretions or bodily fluids of infected persons, and to contact with infected corpses31. For example, infected patients have been reported to produce large volumes (5 L or more) of watery diarrhea over a period of 7 days or longer32. The point of this discussion is to stress that the Ebola virucidal efficacy needs to be as high as possible, and certainly should be greater than the 3 or 4 log10 reduction routinely expected of microbicidal products.The significance of Ebola virucidal efficacy determined in terms of a log10 reduction in titer, as typically is done in demonstrating virucidal efficacy for microbicidal products. In addition, however, studies were conducted in a manner intended to assure that no infectious virus remained following application of the microbicidal product. The latter studies, referred to here as flask safety testing, was based on the assumption that any residual infectious viral particles following exposure to DAL should be able to infect the susceptible Vero E6 cells in the inoculated T-75 flasks and to amplify over a 14-day observation period, causing detectable viral CPE in the flasks. The greatest assurance of complete inactivation of EBOV/Mak was demonstrated for contact times of 5 min or greater (DAL dilutions of 1:20 and 1:40) or 1 min or greater in the case of the 1:10 DAL dilution. Under these conditions, over 5 log10 reduction in titer of infectious Ebola virus in tripartite soil load was demonstrated in the quantitative TCID50 assay and no detectable virus was detected in the non-quantitative flask safety tests.Efficacy and lot-to-lot consistency of commercially available DAL for inactivating Ebola virus in suspension in the presence of a tripartite organic load has been demonstrated in the present studies. Due to the lethality of the Ebola virus and the low infectious dose for humans noted above, the studies were performed to determine log50 assay and the flask safety testing performed for this DAL dilution made in hard water indicate that over 7 log10 inactivation of EBOV/Mak was achieved under these conditions.These results suggest that DAL used at a 1:10 dilution in water should afford complete and rapid (within 1 min contact time) inactivation of EBOV/Mak in suspensions at ambient temperature. The data from the TCID2 in Dulbecco’s modified Eagle medium  supplemented with 10% FCS (Gibco) and 10 units/mL penicillin/streptomycin (Gibco). Ebola virus (Makona C07 variant) (Ebola virus/H. sapiens-tc/GIN/2014/Makona-C05) was obtained from a clinical isolate. All manipulations involving EBOV/Mak were carried out in a BSL-4 laboratory at the Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada.African green monkey Vero E6 cells (ATCC CRL-1586) were maintained at 37 °C/5% CO10/mL by TCID50 assay, with titer calculation following the Reed-Muench method33.A characterized stock of EBOV/Mak was prepared by infecting ten T-75 flasks of African green monkey Vero E6 cells (ATCC CRL-1586) at ~80% confluency at a multiplicity of infection (MOI) of 0.01. At approximately 9 days post-infection, marked cytopathic effects were evident in the Vero E6 cells, at which time the flasks were frozen at −70 °C. The flasks were thawed the following day and the conditioned medium from the flasks was clarified by low-speed centrifugation (4500 × g) for 10 min. The supernatants were pooled and overlaid onto 20% w/v sucrose cushions prepared in Tris- NaCl-EDTA buffer. After centrifugation at ~130000 × g for 2 h, the resulting viral pellets were resuspended in VCM (DMEM containing 2% FCS and 10 units/mL penicillin/streptomycin) overnight at 4 °C. The resuspended virus was pooled and aliquoted into usable amounts and frozen at −70 °C until needed. Stock virus titers were determined to be >9 log12  on the day of assay performance. The resulting solutions were inverted to mix and used within 2.5 to 4 h of preparation.Dilutions of DAL lot# 15083E, 16004E, and 16005E were prepared in hard water4 to 106 TCID50 per mL with 10 µL virus evaluated per control in replicates of three. The candidate reagents were evaluated for neutralization efficacy (by scoring wells for CPE) and cytotoxicity to Vero E6 cells  using the following conditions:A neutralization assay was performed to evaluate the ability of candidate neutralizing reagents to neutralize the virucidal effects of DAL post-exposure to the neutralizers. The reagents evaluated included 100% FCS, VCM, 1× Letheen broth in VCM . Ebola virus (Makona C07 variant) was diluted to approximately 10Cells were cultured in VCM and used as a baseline of comparison for evaluation of cytotoxicity or CPE.0 (undiluted) to 10−3. Neutralizer dilutions were added (50 µL) to Vero E6 cells (n = 5 replicates per dilution). Cells were scored for cytotoxicity 14 days post-inoculation.The candidate neutralizers to be evaluated were diluted in VCM using a 10-fold serial scheme from 1013  to 990 µL of VCM. Final concentrations in the control were: virus (102 to 104 TCID50/mL), BSA (0.0025%), tryptone (0.0035%), and mucin (0.0008%). The positive control were diluted in VCM using a ten-fold dilution scheme from 100 (undiluted) to 10−3, and 50 µL of the resulting solutions were added to Vero E6 cells. Cells were scored for CPE 14 days post-inoculation.The virus control was prepared by adding 10 µL of EBOV/Mak in a tripartite soil load0 (undiluted) to 10−3, and 50 µL of the resulting solutions were added to Vero E6 cells. Cells were scored for cytotoxicity and CPE 14 days post-inoculation.To account for the effect of the neutralizer acting on the virus, neutralizer + virus controls were prepared. These were prepared exactly the same way as the positive virus control, except that the 10 µL of virus in tripartite soil load was added to 990 µL of candidate neutralizer instead of VCM. The neutralizer + virus controls were diluted in VCM using a ten-fold dilution scheme from 100 (undiluted) to 10−3, and 50 µL of the resulting solutions were added to Vero E6 cells. Cells were scored for cytotoxicity and CPE 14 days post-inoculation.To demonstrate the efficacy of the candidates for neutralizing viral inactivation by DAL, 50 µL of diluted DAL were added to 940 µL of the candidate neutralizers. Shortly thereafter, 10 µL of virus in tripartite soil load (concentrations of components given above) were added and the resulting mixtures , were diluted in VCM using a ten-fold dilution scheme from 1011. Stocks of EBOV/Mak in tripartite soil load were prepared on the day of assay as follows. A single tube of stock virus was removed from frozen storage and mixed with a tripartite soil load13 . The virus in tripartite soil load (10 µL) was added to 90 µL of diluted DAL or to 90 µL VCM (positive virus control). The resulting solutions were mixed and held at room temperature for 30 seconds, 1 min, 5 min and 10 min contact times. At the end of each exposure time point, the DAL was neutralized by adding 900 µL of prepared 1× Letheen broth to the test solutions and pipetted repeatedly to mix. A 500-µL portion of each neutralized test solution was diluted in VCM using a ten-fold dilution scheme, and 50 µL of the resulting dilutions were added to 96-well plates containing Vero E6 cells monolayers (n = 5 replicates per dilution). After a 45-min adsorption period, 150 µL of VCM were added to each well. The Vero E6 cell monolayers in the wells were scored 14 days post-infection for CPE and virus titers (TCID50) were calculated according to the Reed-Muench method33.Inactivation efficacy testing for DAL was performed in suspension studies Fig.  conducte2 for 14 days and then scored for presence or absence of CPE.Since the undiluted post-neutralization DAL displayed cytotoxic effects in the Vero E6 cells and therefore could not be evaluated for viral inactivation, an alternative method (flask safety test) was employed to evaluate the undiluted test solutions for the presence of any remaining infectious EBOV/Mak. In this test, 500 µL aliquots of undiluted neutralized inactivation test solution were added to single T-75 flasks  of Vero E6 cells monolayers at ~80% confluency, along with 20 mL of VCM. The flasks were incubated at 37 °C/5% COIn addition to the efficacy test conditions described above, a single cytotoxicity control was performed in parallel with each assay. This was done to control for observable CPE or cytotoxicity caused by the neutralizer and DAL dilution being tested. This involved combining 900 µL of 1× Letheen broth and 100 µL of diluted DAL. A 500-µL portion of the resulting solution was diluted in VCM using a ten-fold dilution scheme, and 50 µL of the resulting dilutions were added to 96-well plates containing Vero E6 cells (n = 5 replicates per dilution). The Vero E6 cell wells were scored for cytotoxicity 14 days post-inoculation.50 titers for positive virus controls and neutralized DAL test conditions were determined by the method of Reed and Muench33. The log10 reduction values achieved by the various DAL lots and exposure time points were calculated by subtracting the post-virucidal efficacy test of DAL, log10 TCID50 values from the log10 titers obtained for the corresponding positive virus controls. Statistical comparison of the mean (n = 5 replicates) viral titers obtained in the neutralization effectiveness studies (Supplemental Fig. t-test, with statistical significance set at P < 0.05.To score the test plates post-incubation, TCIDSupplemental information


Cimex lectularius, is an obligatory blood-feeding ectoparasite that requires a blood meal to molt and produce eggs. Their frequent biting to obtain blood meals and intimate association with humans increase the potential for disease transmission. However, despite more than 100 years of inquiry into bed bugs as potential disease vectors, they still have not been conclusively linked to any pathogen or disease. This ecological niche is extraordinarily rare, given that nearly every other blood-feeding arthropod is associated with some type of human or zoonotic disease. Bed bugs rely on the bacteria Wolbachia as an obligate endosymbiont to biosynthesize B vitamins, since they acquire a nutritionally deficient diet, but it is unknown if Wolbachia confers additional benefits to its bed bug host. In some insects, Wolbachia induces resistance to viruses such as Dengue, Chikungunya, West Nile, Drosophila C and Zika, and primes the insect immune system in other blood-feeding insects. Wolbachia might have evolved a similar role in its mutualistic association with the bed bug. In this study, we evaluated the influence of Wolbachia on virus replication within C. lectularius.The common bed bug, Wolbachia-cured and a line of Wolbachia-positive C. lectularius a virus-laden blood meal, and quantified the amount of virus over five time intervals post-feeding. The antibiotic rifampicin was used to cure bed bugs of Wolbachia.We used feline calicivirus as a model pathogen. We fed 40 bed bugs from an established line of Wolbachia-positive and Wolbachia-cured groups.There was a significant effect of time post-feeding, as the amount of virus declined by ~90% over 10 days in both groups, but no significant difference in virus titer was observed between the Wolbachia, and our conclusions underscore the need for future research.These findings suggest that other mechanisms are involved in virus suppression within bed bugs, independent of the influence of  Cimex lectularius, is an obligate blood-feeding ectoparasite that has undergone a global resurgence in the last two decades . Recentviews in –5 makes views in . Highly views in . The proTrypanosoma cruzi, the etiological agent of Chagas disease, are hemimetabolous, so each instar and all adults require at least one blood meal to develop and reproduce. Such frequent re-feeding contaminates the indoor environment with histamine [T. cruzi [Bartonella quintana, the etiological agent associated with Trench fever [Bartonella [Burkholderia multivorans in bed bugs.Bed bugs, like the related triatomine bugs that transmit istamine  and coulistamine –12). Hepistamine , 13, 14,T. cruzi , and Barch fever , but a srtonella . HoweverRhodnius [Glossina [Wolbachia with fruit flies and mosquitoes. Wolbachia protects Drosophila against virus-induced mortality for Drosophila C virus (DCV) and Flock house virus (FHV) [Wolbachia also stimulates immune gene expression in several mosquito species [Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes for viruses such as Dengue, Chikungunya, West Nile, and Zika [Wolbachia in Drosophila and mosquitoes might be limited to RNA viruses [Microbe-microbe interactions with respect to pathogen suppression have been studied in various blood-feeding insects such as the kissing bug Rhodnius , 19, tseGlossina , 21, andGlossina ), as welGlossina , 24), anus (FHV) , 26. Wol species –31, and and Zika –38. Deng viruses , 40.Cimex lectularius harbors Wolbachia as its primary endosymbiotic nutritional mutualist that biosynthesizes B vitamins for its nutritionally deficient host [Wolbachia could influence the vector competence of C. lectularius through mechanisms involving interactions with the host or with ingested pathogens, thus preventing certain viruses from replicating within the host. This may explain in part why bed bugs are not a major disease vector for arboviruses. The objective of this study was to compare virus titer in Wolbachia-positive and Wolbachia-free C. lectularius at several time intervals after ingesting a virus-laden blood meal.ent host , 42 in aent host . AdditioC. lectularius was collected in Winston Salem, NC in 2008 and fed defibrinated rabbit blood  in an artificial feeding system, as described by Sierras & Schal [Wolbachia-free (Wb–) colonies and it was maintained at 27 °C and a photoperiod of 12:12 . These two colonies were fed weekly on defibrinated rabbit blood supplemented with the antibiotic rifampicin (10 µg/ml blood) and the Kao and Michayluk B Vitamin Solution (10 µl/ml blood) . Note however that this vitamin solution differs substantially from the Lake & Friend [The Winston-Salem (WS) strain of  & Schal . Ten adu& Friend  solution& Friend . RearingC. lectularius were free of Wolbachia, a comparison to the WS-Wb+ normal strain was conducted. After several filial generations, six adults were randomly selected from each of the two antibiotic-treated and vitamin supplemented colony jars (n = 12) and six from the untreated WS strain. Total genomic DNA was extracted using the DNeasy Blood and Tissue kit  with a modified purification of total DNA from animal tissues (spin-column) as per manufacturer’s protocol. Individual bed bugs with heads removed (to minimize interference from eye pigments) were homogenized in the 1.5 ml microcentrifuge tube using a sterile plastic pestle and then digested overnight (~ 24 h) in 180 µl of ATL buffer solution, 20 µl of proteinase K, and 4 µl of RNase in a 56 °C water bath. Following initial digestion, samples were vortexed for 15 s, 200 µl of AL buffer was added, and then incubated in a 70 °C water bath for 10 min. After incubation, 200 µl of 96% ethanol was added, the mixture was then pipetted onto the DNeasy Mini spin column, and the DNA was bound, washed, and eluted into 200 µl of AE buffer as outlined in the protocol. An additional wash with AW2 buffer was included to further remove salts. Samples were stored at − 20 °C until polymerase chain reaction (PCR) for Wolbachia was conducted.To verify that antibiotic-treated colonies of Wolbachia and measure presence or absence of Wolbachia. The Wolbachia-specific primers INTF2-FWD and INTR2-REV, adopted from Sakamoto & Rasgon [Wolbachia 16S gene that produced a 136 bp amplicon. The GoTaq Green Master Mix  and nuclease-free water were used for all reactions at the following concentrations and volumes: 12.5 µl of 2× Master Mix, 2.5 µl of 10 µM INTF2-FWD, 2.5 µl of 10 µM INTR2-REV, 5 µl of template DNA, and PCR-grade nuclease-free water was added to achieve a final reaction volume of 25 µl. Reactions were performed using an MJ Research thermocycler  with the following protocol: 95 °C for 2 min  × 36 cycles, and 72 °C for 5 min. A no-template control was used in the PCR reactions as well. A 2.0% agarose gel was used to separate the 136-bp amplicon using a 100=bp DNA ladder and GelRed nucleic acid stain , and visualized with a ChemiDoc-It TS2 imaging system .Conventional PCR was conducted to amplify a specific gene target within & Rasgon , targeteWolbachia in each individual bed bug was obtained with a droplet digital PCR (ddPCR) system  and protocol from Fisher et al. [Wolbachia-specific primers, TaqMan probes, and the ddPCR Supermix for Probes (Bio-Rad) into PCR-ready samples. Primers for a ribosomal protein (RPL18) specific to C. lectularius were used as the reference gene due to its stability [Wolbachia, a 5′ HEX fluorophore for C. lectularius, and 3′ Iowa Black® FQ quenchers with internal ZEN quenchers  specific to each target. Primer and probe sequences are listed in Table Absolute quantification of r et al. . Bed bugtability ; they protein RPL8 specifi+ and Wb– lines. The bed bug/Wolbachia ddPCR assay comprised 22 µl of 1× Droplet Supermix (Bio-Rad), 5 µl of genomic DNA isolated from a bed bug, 2 U of MseI restriction enzyme , 500 nM each of forward and reverse primers and 250 nM each of FAM- or HEX-labeled TaqMan probes for bed bug and Wolbachia sequences, respectively. Then the 22 µl of PCR mixtures were partitioned into an emulsion of ~ 20,000 droplets using a QX200™ AutoDG Droplet Digital PCR™ system (Bio-Rad). The PCR was performed on a T100 Thermal Cycler using the following protocol: 95 °C for 10 min and  × 40 cycles, and 98 °C for 10 min. Post PCR, droplets were analyzed on the QX200 Droplet Reader. Absolute DNA copy numbers of bed bug and Wolbachia sequences in a sample were calculated on the Poisson distribution using the Quantasoft software version 1.7.4 (Bio-Rad). Previously confirmed Wb+ bed bug DNA sample and Wb– bed bug DNA sample were included in each experiment as positive and negative controls. No-template control was also included in each experiment to ensure no non-specific amplifications. To estimate the limit of detection of the ddPCR assay, serial dilutions  of a DNA sample in water were prepared and repeated three times.The ddPCR reaction was optimized using extracted bed bug DNA from Wb+), antibiotic-vitamins (Wb–), and Wb– maintained for 90 days on vitamin-supplemented blood without antibiotic. The latter (vitamin-only) group was removed from antibiotic 90 days prior to inoculation with virus.The experiment evaluated virus titers over time in three cohorts of bed bugs: control , and incubated 16 h at 37 °C in 5% CO2, which resulted in virus-induced destruction of ~ 90% of the monolayer.Feline calicivirus (FCV) was chosen as the inoculum due to its environmental stability and feasibility as a viral pathogen. Feline calicivirus is a (+)ssRNA virus that is one of the primary causes of respiratory infections in felines. Virus was grown in existing Crandell Reese Feline Kidney (CrFK) cell line at the North Carolina State University College of Veterinary Medicine Clinical Virology Laboratory and stored at − 80 °C in 2 ml aliquots per established protocols. The FCV stock was produced by first removing the growth medium from confluent CrFK monolayers in 75 cm7 CCID50/ml) per 1 ml of blood. The CCID50 is the 50% cell culture infective dose, as defined below. Bed bugs were starved 7 days prior to feeding FCV-laden blood. Individuals that did not feed or only partially fed were removed and discarded. Vials were kept thereafter at room temperature under a sterile laminar flow hood and 12:12 (L:D) photoperiod.In each feeding, 40 individual bed bugs were chosen randomly, placed in 7 ml glass vials with screened-caps, and fed as previously described for 15 min on fresh defibrinated rabbit blood supplemented with 1 ml FCV (10rpm for 1 min). A volume of 220 µl of the supernatant was pipetted into a new 3.5 ml tube with 2.2 ml of DMEM, and serial 10-fold dilutions (10−1 to 10−6) were performed to obtain virus titration. A total of 100 µl of each dilution was placed in 4 wells  of a flat-bottomed, 96-well plate, and 100 µl of CrFK cells was added to each well. Each plate contained a row of wells as a cell control with no virus. Plates were incubated at 37 °C for 5 d [Bed bugs were killed at the following time intervals: 5 hours (h), 24 h, 4 days (d), 7 d, and 10 d post-feeding. Three randomly chosen bed bugs were removed from each cohort, sexed, and surface sterilized with 0.05% NaClO and 70% ethanol. Surface-sterilized bugs were homogenized in 3.5 ml round-bottom polystyrene tubes  in 0.5 ml of Dulbecco’s Modified Eagle’s Medium (DMEM)  with sterile plastic pestles and the tubes were centrifuged (8000×  for 5 d –51 and t50) endpoint dilution assay was used to quantify the amount of virus required to kill 50% of infected CrFK cells as described by Reed & Muench [In each virus dilution, the percentage of dead (infected) cells was visually determined for each well. To measure the infectious virus titer, the 50% cell culture infective dose . A P-value < 0.05 was considered significantly different. A General Linear Model Repeated Measures analysis (Wilks’ Lambda {λ}) was also conducted in SPSS to identify any effect of time, treatment group, replicate, and interactions between these variables on virus titer.Differences in the mean virus titer in CCIDWolbachia. As well, the colony removed from antibiotics and maintained on vitamin-supplemented blood contained no Wolbachia  and measured values  matched well with high reproducibility even at extremely low concentrations . No Wolbachia or bed bug DNA was detected in the no-template controls.The conventional PCR results confirmed that the bed bug colony treated with rifampicin and supplemented with B vitamins contained no hia Fig. . The ddPolbachia  Absolutefor 90 d . The ddP4 virus in a single feeding. Live virus was detected in relatively large amounts in all treatment groups at all time intervals up to 10 d  = − 4.724, P = 0.0179).We estimated that adult bed bugs ingested between 4-6 µl of blood meal , which F = 36.48, P = 0.0271; ƞ2 = 0.986), with an average of 90.7% decline across all three treatments after 10 d, ranging from 96.5% decline in the Wb+ group, 92.2% in the Wb– group, and 83.4% decline in the Wb– 90 d group  = 0.359, P = 0.575) or replicate on FCV titer  = 0.127, P = 0.884), and there was no effect of time*treatment  = 6.48, P = 0.1381, ƞ2 = 0.928), or time*replicate  = 1.73, P = 0.3143, ƞ2 = 0.775). No infection was observed in any of the cell line controls in any of the three bed bug groups at any sampling time interval. We observed no mortality in any of the FCV-infected bed bugs that were left in the vials after feeding on FCV-supplemented blood.There was a significant decline in FCV titer over time after the blood meal  as a model pathogen in our investigation because it is environmentally stable, it represents RNA viruses, and it could be transmitted by bed bugs in residential settings. Since FCV is not a human pathogen, it served as a useful surrogate for other more infectious ssRNA virus without placing laboratory workers at risk of infection. Our results showed that FCV did not replicate within to occur , 57. Impia Table .Wolbachia-cured (Wb–) group was treated with antibiotic during weekly blood meals before the experiment started. Therefore, this group was also expected to suffer from an altered gut microbiome, whose possible interactions with and effects on FCV are not known. Another Wolbachia-cured group (Wb– 90 d) was weaned off rifampicin 3 months before the experiment started. We expected this group to be less affected by the antibiotic, and its gut microbial community might have recovered during the three months, which represented less than two generations. This group, however, was not different from the other two groups, including the Wolbachia-containing group (Wb+) which was never exposed to rifampicin, suggesting that neither Wolbachia nor the gut microbiome influenced the FCV titers.It is important to note that secondary effects from the rifampicin treatment could have affected the interaction of the bed bug with FCV. The Wolbachia influence on FCV titers might be related to minimal interactions between the symbiont and the virus. Factors related to the physiological conditions of the midgut, inability of FCV to permeate midgut barriers, and host immune responses may minimize these interactions. Wolbachia’s intracellular sequestration within the bed bug bacteriome might further diminish contact between these two microbes.The absence of a As blood is ingested, FCV would interact with a wide range of bed bug salivary proteins that are secreted to counteract the vertebrate host’s hemostasis . The bed bug genome revealed expanded families of salivary apyrases, nitric oxide carriers, and members of the Ap4a_hydrolase family . Bed bugAfter the bed bug ingests a blood meal containing FCV, the virus must interact with the insect alimentary canal, penetrate the hemocoel, and for effective transmission with subsequent blood meals the virus needs to replicate in the salivary glands or other tissues associated with the mouthparts. Alternatively, if the virus survives passage through the alimentary canal, it can be transmitted in feces, though this pathway is considered less efficient. Mildly acidic to neutral midgut pH (5–7) is ideal for a wide range of microorganisms, but in most insects the midgut is alkaline (pH ≥ 8) and typically unfavorable for most microorganisms , 60. AduC. lectularius, R. prolixus, and Triatoma infestans all have a modified PM effectively known as a ‘plexiform surface coat’ type PM that is permeable to digestive enzymes [We did not determine whether FCV was able to cross the midgut barrier and enter the bed bug hemocoel. Many insects, including blood feeders, form a peritrophic membrane (PM) around the food bolus during or shortly after ingestion. The PM is a physical barrier that protects the midgut lumen. A pathogen or parasite must penetrate the PM and invade the midgut tissues before it can cross into the hemolymph. Although the PM is absent in most Hemiptera , C. lect enzymes . The PM  enzymes . Several enzymes . The mos enzymes , and mos enzymes . To perm enzymes . Once cr enzymes .C. lectularius by way of hemocytes, activation of proteases, production of antimicrobial peptides, or immune signal transduction pathway activation  by the fat body that could lower FCV titer. The genome of C. lectularius has revealed members of all these pathways, as well as the RNA interference pathway [Wolbachia.If FCV crossed the PM into the hemolymph, the next host defense would be a systemic immune response by  pathway  and tran pathway . It is g pathway , and bed pathway . We did Wolbachia has been shown to play important roles in mediating host-microbe interactions. In Drosophila, higher Wolbachia densities correlate with greater antiviral protection [Culicoides midges, BTV replicated significantly in all cell lines examined from BTV-infected Drosophila melanogaster reared without Wolbachia [Wolbachia also mediates immunocompetence in isopods [Wolbachia-virus interactions, Wolbachia primes the mosquito innate immune system [Wolbachia naturally occurs, the presence of Wolbachia has little to no influence on resistance to or suppression of viruses [otection , 69, andolbachia . Wolbach isopods . In somee system , 31, 36, viruses , 73.Wolbachia within the host is relevant to its involvement in pathogen suppression. In many insects, Wolbachia is systemically distributed either throughout the body or in specialized but highly diffuse tissues . In C. lectularius on the other hand, Wolbachia resides exclusively in a bacteriome of both sexes, in association with the gonads [Wolbachia could respond to immune challenges by remotely signaling to the fat body and hemocytes, its location in the gonad-associated bacteriome would make such a signaling pathway less likely than in mosquitoes, flies and isopods.The localization of e gonads  and woulC. lectularius and other cimicids conceivably may have a greater significance from a veterinary medicine perspective. Other species of cimicids may be epidemiologically important in diseases of birds and bats yet to be investigated [Oeciacus vicarius can vector several arboviruses [Plasmodium, Leishmania, Yersinia, and numerous bacterial species, immune responses by bed bugs when challenged with a pathogen remain poorly understood.Bed bugs do not appear to be competent vectors for ssRNA human viruses associated with disease in humans, and their status as a medically important vector of disease remains uncertain. However, bed bugs could be more important vectors of dsDNA viruses such as hepatitis B virus (HBV). Moreover, stigated . The swaoviruses , and canoviruses . Commercoviruses , and theWolbachia on virus titer in Cimex lectularius. Our results indicate that Wolbachia does not play a role in ssRNA virus suppression in bed bugs, in contrast to its involvement in several other hematophagous insects. These results offer further supporting evidence that bed bugs are likely not competent vectors of ssRNA viruses, adding feline calicivirus to the list of viruses examined thus far. Our conclusions underscore the need for future research to include (i) quantification of virus titers in various body compartments, particularly the hemolymph and salivary glands; (ii) hemocoel injections of virus for titer comparison in Wolbachia-free and normal bed bugs; and (iii) investigation of the ability of the bed bug to transmit the virus upon re-feeding.To our knowledge, this is the first study to evaluate the influence of


The developed nano-porous supports have enabled us to reproducibly produce silica-based separation membranes with high permeace and selectivity .Silica-based membranes show both robust properties and high-permeability, offering us great potential for applying them to harsh conditions where conventional organic membranes cannot work. Despite the increasing number of paper and patents of silica-based membranes, their industrial applications have yet to be fully realized, possibly due to their lack of technologies on scaling-up and mass production. In particular, quality of membrane supports decisively impacts final quality of silica-based separation membranes. In this study, therefore, we have developed mass producing technologies of nano-porous supports  with surface center pore size distribution of 1–10 nm, which are generally used as supports for preparing separation membranes with a pore size of less than 1 nm. The developed mass production apparatuses have enabled us to reproducibly produce nano-porous silica-based supports with high permeance (e.g., N Silica-based membranes, such as SiOembranes , SiO2–Zrembranes ,4,5, sho−6 mol m−2 s−1 Pa−1 in most cases, such as hydrogen separation and water separation. Generally, silica-based molecular sieve separation layers with their pore size of less than 1 nm are formed on the surface or inside of nano-porous supports with a pore size of 1–10 nm. In order to obtain high-flux (more than 10−6 mol m−2 s−1 Pa−1) and high-selective membrane with their pore size of less than 1 nm, the permeances of the nano-porous supports with their pore size distribution of 1–10 nm need to be more than 10−5 mol m−2 s−1 Pa−1. In addition, effects of permeation pathway through defects, such as pinholes or cracks of the nano-porous supports should be minimized to obtain reasonable separation performance after membrane separation layer coated.Despite the increasing number of papers and patents of silica-based membranes, their industrial applications have yet to be fully realized, possibly due to lack of technologies on scaling-up and mass production. In particular, the quality of membrane supports decisively impacts final quality of silica-based separation membranes. Considering industrial applications and their economic feasibility of ceramic-based separation membranes, the permeance of target separation molecules are expected to be more than 10The purpose of this study is to develop mass producing technologies of industrially applicable, highly permeable, and defect-free nano-porous supports for producing silica-based membranes. In this study, firstly, highly permeable silica-based nano-porous supports were developed by controlling size of nano-particles deposited on the surface of the supports. Secondly, an example of our mass-producing apparatus for the nano-porous supports was introduced. Finally, the developed nano-porous supports were tested to prepared silica-based separation membranes for hydrogen separation.In order to effectively develop technologies on scaling-up and mass production of silica-based supports and membranes, we focused on clarifying correlations among synthesis conditions, structural properties, and gas permeation properties. Materials and methods of synthesis, structural characterization, and gas permeation tests are shown below.2 [2–ZrO2 (Si/Zr = 1) [Silica-based nano-porous supports and membranes were formed on α-alumina symmetric porous tubes  by sol-gel method, based on the procedure reported by the research group of Hiroshima University . First, /Zr = 1) . The col/Zr = 1) . The par2 or SiO2–ZrO2 were coated on the α-alumina intermediate layers at 180 °C and fired at 550 °C repeatedly for synthesizing silica-based nano-porous supports. BTESE-derived organo-silica membranes were synthesized based on the procedure reported by the research group of Hiroshima University [2–ZrO2 nano-porous supports at 145 °C with a piece of wet cloth and fired at 350 °C for 7 min in N2 repeatedly for synthesizing gas separation membranes. This procedure was repeated 4 times to obtain BTESE membranes with less defects. The SiO2–ZrO2 nano-porous supports used for synthesizing the BTESE membranes had high permeance  with an average pore size of about 1–2 nm, as shown later. In this study, the hot coating method  was emplIn order to enhance the productivity, a flow-type apparatus was prototyped. In the flow-type apparatus, substrates samples are designed to be circulated from preheating chamber at 180 °C for 10 min → hot coating device → firing chamber at 550 °C for 10 min → cooling chamber for 4–6 min. The residence times in each of the chambers were controlled by adjusting the feeding speed of belt conveyors. The flow-type apparatus has enabled the coating cycle to repeat continuously and automatically. Compared with the batch-type apparatus , the flow-type apparatus (automatic operation) in 2, SiO2–ZrO2, and organo-silica synthesized were measured by a dynamic light scattering (DLS) analyzer  and a transmission electron microscope (TEM). The structures of synthesized silica-based nano-porous supports were observed by a scanning electron microscope (SEM). The sizes of colloidal sols of SiOTwo types of gas permeation tests, a nano-permporometory ,7 and siThe synthesized silica-based nano-porous supports were evaluated by a nano-permporometory, which is suitable for investigating pore size distribution in the range of 1–10 nm. The synthesized silica-based membranes were evaluated by single-gas permeation tests, which are suitable for investigating membranes with their pore size of less than 1 nm. 2. In the gas permeation tests, as-made nano-porous supports or membranes  were placed inside the module cell and sealed with silicon caps at both ends. The effective membrane was 143 cmK, υ, σ, and θ are Kelvin diameter, molar volume, surface tension, and contact angle, respectively. In this study, N2 (non-condensable gas) and water (condensable vapor) were used, assuming cosθ = 1 in calculation of Kelvin diameter of SiO2 and SiO2–ZrO2-based nano-porous supports because both supports were hydrophilic, probably due to immediate formations of –OH at surface of the supports. The nano-permporometry measurements were performed at 25 °C.The pore size distribution of synthesized silica-based nano-porous supports was evaluated by a nano-permporometory. In this method, non-condensable gas permeances through a porous sample are measured with increasing relative pressure of condensable vapor in a stepwise manner. The contributions of gas permeations via a specific size of pores are estimated by blocking their nanopores with a vapor condensate. The diameters of the pores blocked by the condensate as a function of vapor partial pressure are estimated using the well-known Kelvin Equation (1)The schematic diagram of the nano-permporometry measurements and their experimental conditions are shown in 2 to remove chemisorbed water molecules. Samples were attached in a glass module and sealed with silicon caps. Pure gases of H2 (0.29 nm), CH4 (0.38 nm), or SF6 (0.55 nm) were fed to the outside of a cylindrical membrane at 4–20 kPa. The gas permeation area was kept at 100 °C by a thermostatic chamber. The permeance of gases was measured by a bubble film meter.Single-gas permeation tests were conducted after membranes were preheated at 350 °C in NIn this section, results of characterization of coating nano-particles, synthesized silica-based nano-porous supports, and membranes are shown in this order.In this section, characterization results of nano-porous supports and membranes synthesized using the batch-type apparatus (as shown in 2-based nano-sols of Sample-2 (5–15 nm) and Sample-4 (2–10 nm) in this order. Probably due to the limitation of resolution of the SEM, no clear boundary between the silica layers with different particle sizes were observed from the SEM observation.2-based nano-porous supports). Based on the results of nano-permporometry measurements, distributions of gas permeation ratio through each of permeation path with different pore sizes were calculated and summarized in Nano-permporometry profiles of nano-porous supports prepared by coating nano-sol samples with different particle sizes are shown in 2 permeance of more than 10−5 mol m−2 s−1 Pa−1) minimizing effects of membrane defects less than 0.1% of total flux. Different from the manually operative batch-type apparatus, the automatic operative flow-type apparatus seems suitable for mass production of the nano-porous supports and membranes. 2-based nano-porous supports, SiO2–ZrO2-based nano-porous supports are less stable in a strong acidic condition but more stable against water and alkali. Therefore, development of SiO2–ZrO2-based nano-porous supports are also indispensable for applying them to conditions where pure SiO2-based materials are unsuitable. SiO2–ZrO2-based nano-porous supports are often used as intermediate layers for preparing organo-silica membranes for gas separation [Compared with pure SiOparation , probabl2–ZrO2-based nano-porous supports prepared by coating nano-sol samples with different particle sizes Based on the results of nano-permporometry measurements, distributions of gas permeation ratio through each of permeation paths with different pore sizes were calculated and summarized in 2–ZrO2, 2–10 nm), Sample-6 , and Sample-7  in this order. Different from the other samples (Sample 1 to 6), Sample-7 (BTESE derived organo-silica) showed water-repellent property. Since the top surface of the organo-silica membranes seem hydrobobic with their pore size of less than 1 nm, single-gas permeation tests through the membranes were employed. The results of single-gas permeation tests are listed in The BTESE-derived organo-silica membranes were synthesized by multi-coatings of nano-sols of Sample-5 , minimizing effects of large pores  less than 0.1% of total flux, as shown in 2 permeance of about 5 × 10−6 mol m−2 s−1 Pa−1 and H2/SF6 permeance ratio of more than 2000) were reproducibly produce, as shown in 4 (0.38 nm) and little permeation of SF6, the effective pore sizes of the membranes were estimated to be 0.4–0.5 nm. Since the type of organo-silica sol employed in this study was BTESE, the network structure of BTESE(–Si–C–C–Si–) would allow permeation of smaller molecules, or H2 (0.29 nm), N2 (0.36 nm), and CH4 (0.38 nm), and block permeation of larger molecules, SF6 (0.55 nm) [2 permselectivity for silica-based membranes and zeolite membranes, Kanezashi et al. [The developed nano-porous silica-based supports showed high permeance  membranes synthesized. Compared with γ-alumina intermediate layered supports [2–ZrO2 intermediate layered supports developed in this study were more than ten times higher, resulting in more than ten times higher H2 permeances of BTESE-derived silica membranes. In this study, we have succeeded in scaling-up the high quality BTESE-derived organo-silica membranes reported by the research group of Hiroshima University [We assume the use of highly permeable nano-porous supports greatly contributed to the high Hsupports ,13, the iversity  from theConsidering mass production of silica-based membranes, both sol-gel methods ,2,3,4,5 2 permeance of more than 10−5 mol m−2 s−1 Pa−1), minimizing effects of membrane defects less than 0.1% of total flux. The developed nano-porous supports have enabled us to reproducibly produce silica-based separation membranes with high permeance and selectivity . In additions, a flow-type sol-gel method was developed for the mass productions of silica-based nano-porous supports and membranes.In this study, we have successfully scaled-up high quality silica-based nano-porous supports and membranes by controlling sizes of nano-particles coated. The developed nano-porous silica-based supports have showed high permeance (e.g., N


As a result, the NVES associated with afforestation was 55.9% less than the NVES of natural forests. In some regions, NVES was negative because of the huge costs of human‐made plantations, high evapotranspiration rates , and low forest survival rates. To maximize the ecological benefits of conservation, it is necessary to account for as many costs as possible so that management decisions can be based on NVES, thereby helping managers choose projects that maximize both economic and ecological benefits.Land degradation is a global problem that seriously threatens human society. However, in China and elsewhere, ecological restoration still largely relies on a traditional approach that focuses only on ecological factors and ignores socioeconomic factors. To improve the effectiveness of ecological restoration and maximize its economic and ecological benefits, a more efficient approach is needed that provides support for policy development and land management and thereby promotes environmental conservation. We devised a framework for assessing the value of ecosystem services that remain after subtracting costs, such as the opportunity costs, costs of forest protection, and costs for the people who are affected by the program; that is, the net value of ecosystem services (NVES). To understand the difference between the value of a resource and the net value of the ecosystem service it provides, we used data on VES, timber sales, and afforestation costs from China's massive national afforestation programs to calculate the net value of forest ecosystem services in China. Accounting for the abovementioned costs revealed an NVES of ¥6.1 × 10 Article impact statement: Accounting for the effects of costs on the value of ecosystem services provides a net value that more strongly supports policy development. Since the end of the 20th century, the number of studies of ecosystem services has grown exponentially  so as to identify the true net benefits of projects  under China's national afforestation policy with the NVES of natural forests, we used data from Niu et al.  and WangThe main ecosystem services in their study were water conservation, biodiversity protection, carbon fixation, oxygen generation, soil conservation, maintenance of soil fertility, air purification, and nutrient accumulation  associated with these benefits, which include direct costs (Cd), opportunity costs (Co), and risk costs (Cr) associated with these benefits. We defined an NVES that represents the real benefits (including income) after accounting for these costs:Cd is the direct cost of ecological conservation and restoration, Co is the opportunity cost of using resources for afforestation rather than for alternative uses , and Cr is the cost entailed by risks. We defined these risks as the actual management costs to protect against insects, diseases, and wildfire.When we estimated the benefits  provided by a forest ecosystem in a given situation, we also considered the costs , and from not using the water (Cw) for other, nonforest, economic objectives:We converted all values to 2014 values based on the government's official mean annual inflation rate of 4.9% for the study period. To simplify the calculations, we defined http://www.tdzyw.com). Our approach is coarse grained, and we hope future researchers will use finer‐grained data and the values for additional combinations of land uses.We obtained the average land rent in every province from a Chinese real‐estate market database (Cr) is what must be paid for forestry management to prevent natural disasters, including wildfires and outbreaks of plant diseases and insect pests. We based Cr on government statistical data on expenditures for the control of insects, diseases, and wildfire , from China's annual forestry statistical yearbooks, and China's first to eighth national forest resource inventory bulletins SFA . The amoBecause many of the areas where afforestation has been conducted are arid to semiarid, water is a precious resource in these areas. We therefore used potential evapotranspiration to represent water consumption by human‐made and natural forests based on 7 previously developed evapotranspiration models. All 7 models were tested by Chen et al.  to confiV is the value of the resource , P is the per capita water availability in the province, and b and a are curve‐fitting parameters. We multiplied the available water  by the 3), which has the most expensive water in China, was Ɏ1.2 m−3 in 2014 based on data from the South‐to‐North Water Diversion Project , represented the lowest price in China, and this value was Ɏ0.17 m−3 , Co , and Cr , annual NVES totaled Ɏ6.1 × 1012 RMB for forest ecosystem services in 2014, which is 35.9% less than the value calculated without accounting for costs. As a result, the annual net benefit of the afforestation areas was Ɏ15.9 × 103 ha−1, which is 55.9% less than that of natural forest. Even when we included income from timber production (China's policy prohibits timber harvesting in most of the country), the net benefits were only Ɏ6.4 × 1012 for China's forest ecosystem (both forest types combined) in 2014, which is 33.0% less than the VES.China's natural and artificial forests covered 69.2 × 10Survival of the artificial forests was greatest in southeastern China, where water is generally abundant and the warm climate favors tree growth Fig. a. The pr6 km2, equivalent to 34.8% of the country's land area . The annual NVES of natural forest decreased from Ɏ4.1 × 1012 in 1952 to Ɏ2.6 × 1012 in 1993 and then increased to Ɏ5.5 × 1012 in 2014 (Fig. 12 in 2014, which was 68.8% less than the VES value calculated using the traditional approach (Table From 1952 to 2014, China's afforestation program affected a total of 3.4 × 10Land degradation is a global environmental problem that seriously threatens humans (Sivakumar The traditional way of assessing the VES ignores many costs and therefore does not accurately reflect the real benefits of ecological projects. As a result, it provides inaccurate data to support land‐use planning (Wegner & Pascual Our results show the importance of basing land‐use policy and assessments of ecosystem services on detailed cost and benefit information, and this will be important anywhere in the world. Land managers must quantify the impacts of changes in land use on the provision of ecosystem services and understand the values and flows of benefits to nearby and distant human populations (Naidoo & Iwamura Managers must account for the trade‐offs revealed by our results to maximize society's total long‐term benefits, which include the values of goods and ecosystem services, for optimal land use NRC . Because