Coltivirus, Colorado tick fever virus (CTFV), was discovered in 1943 and is the most common tick-borne viral infection in the Western US. Despite its long history, very little is known about the molecular diversity of viruses classified within the species Colorado tick fever coltivirus. Previous studies have suggested genetic variants and potential serotypes of CTFV, but limited genetic sequence information is available for CTFV strains. To address this knowledge gap, we report herein the full-length genomes of five strains of CTFV, including Salmon River virus and California hare coltivirus (CTFV-Ca). The sequence from the full-length genome of Salmon River virus identified a high genetic identity to the CTFV prototype strain with >90% amino acid identity in all the segments except segment four, suggesting Salmon River virus is a strain of the species Colorado tick fever coltivirus. Additionally, analysis suggests that segment four has been associated with reassortment in at least one strain. The CTFV-Ca full-length genomic sequence was highly variable from the prototype CTFV in all the segments. The genome of CTFV-Ca was most similar to the Eyach virus, including similar segments six and seven. These data suggest that CTFV-Ca is not a strain of CTFV but a unique species. Additional sequence information of CTFV strains will improve the molecular surveillance tools and provide additional taxonomic resolution to this understudied virus.The type species of the genus  Reoviridae is comprised of two subfamilies, Spinareovirdae and Sedoreoviridae [Coltivirus is one of nine genera in the subfamily Spinareroviridae, with two tick-borne viruses, Colorado tick fever virus (CTFV) and Eyach virus, representing the first viruses to comprise the genus [Coltivirus have genomes made of 12 segments of double-stranded RNA, encoding for 13 viral proteins (VP) 1–12 [Dermacentor andersoni, is common [Ixodes species tick [The family oviridae . The genhe genus . VirusesVP) 1–12 . Colorads common ,6. Addits common ,8. Eyachies tick  and was ies tick . In conties tick , TarumizCTFV is the most commonly reported tick-borne viral disease in the Western US. Cases occur in the early spring and summer months when ticks are most active. CTFV causes a febrile illness, often with a biphasic fever and leukopenia. Up to 30% of the cases require hospitalization . Life-thAll protocols followed manufacturers’ recommendations unless otherwise noted.v/v dimethyl sulfoxide and incubated 1.5 h at 65 °C to denature double stranded RNA [Viruses sequenced in the study were provided by the Arboviral Diseases Branch, Division of Vector-Borne Diseases, Centers for Disease Control and Prevention’s Arbovirus Reference Collection (ARC). Isolate details are described in nded RNA . RNA wasThe cDNA was generated from extracted RNA using random primers provided in the Ovation RNA-seq system V2 kit . Sequencing libraries were prepared from the cDNA using the Ion Xpress plus fragment library kit , barcoded with Ion Xpress barcodes (Thermo Fisher), and quantified using the Ion library TaqMan Quantitation kit (Thermo Fisher). Sequencing templates were prepared using the Ion One Touch 2 system and Ion Hi-Q View OT2 kits (Thermo Fisher). Whole genome sequencing was performed on the Ion Torrent Personal Genomics Machine system using the Ion Hi-Q view sequencing kit and 318 v2 chips (Thermo Fisher), sequencing two libraries per chip.Sequences (fastq) with quality phred Q > 20 were uploaded into CLC Genomic workbench v20 (Qiagen). Genomes were assembled via de novo assembly with a bubble size of the average read length (150 bp). The longest contigs with deepest coverage were submitted for BLAST analysis through NCBI. Consensus sequences for each segment were extracted from CLC genomic workbench and used for reference guided assembly in SeqMan NGen v17 . Whole genomes were obtained for each virus with a minimum of 20× coverage, and a minimum average of 8.9 read depth per nucleotide. Putative open-reading frames were identified using EditSeq (DNASTAR). Terminal ends were determined for all sequences using 5′ RACE System 2.0 kits (Thermo Fisher) and 3′ poly(A) tailing kit , followed by 3′ RACE kit (Thermo Fisher) using gene specific primers . RACE DNA templates were cloned with TOPO-TA sequencing kits (Thermo Fisher) and sequenced by capillary sequencing on an ABI 3130 instrument (Thermo Fisher) using the primers provided in the TOPO-TA kits. All sequences generated in this study have been deposited into GenBank .https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 29 January 2021)). Nucleotide sequences comprising the open-reading frames for each protein were codon aligned using the Clustal W function of Mega 7 [www.phylo.org (accessed on 1 February 2021)) [Conserved domains were identified in each segment using CD-search through NCBI (f Mega 7 . Percentf Mega 7  executedy 2021)) . Maximumy 2021))  and FigTp < 0.05 using the Bonferroni adjustment and detected by three or more models using Recombination Detection Program (RDP) v 4.9 [Reassortment was evaluated by concatenating the open-reading frames of 12 segments representing the complete genomes. Only reassortment events associated with a full segment of the concatenated genome that had P) v 4.9  were conP) v 4.9 .w/v) Agarose  and 3% (v/v) bicarbonate  were added to each well, and plates were incubated at 37 °C. 3 mL of secondary overlay of Ye-lah media with 0.5% (w/v) agarose, 3% (v/v) bicarbonate, and 2% (v/v) neutral red  was applied after an appropriate number of days to allow for differences in viral growth: 5 days for Eyach virus, 4 days for Salmon River, 4 days for CTVF-Ca, and 2 days for CTFV. Plaque counts were then noted on the following day. Titers were determined by taking the reciprocal of the highest serum dilution that reduced plaque counts by 90%, as indicated by the back titration of each virus.Positive control hyperimmune acidic fluids were diluted 1:5 in a total of 120 µL, followed by a serial 2-fold dilution with BA-1 media. Dilutions were then challenged with 100 plaque forming units of virus in a total of 60 µL, and incubated 1 hr at 37 °C. Following incubation, 100 µL of the virus with control mixture was added to a confluent Vero Cell monolayer on a 6-well plate  and incubated at 37 °C for 1 hr. Following incubation, 3 mL of yeast extract-lactalbumin (Ye-lah) overlay media with 0.5%  and was also cross-neutralized by Salmon River virus antiserum, although at a dilution >4-fold less than homologous neutralization (PRNT90 1:320). Salmon River virus was neutralized equally by antiserum generated against CTFV Florio and Salmon River virus (PRNT90 1:640). Salmon River virus was also cross-neutralized by antiserum produced against CTFV-Ca, but at a very low end-point titration (PRNT90 1:20). Moreover, CTFV-Ca was only neutralized by homologous antiserum (PRNT90 1:160) and CTFV-Ca antiserum did not cross-neutralize CTFV Florio or Salmon River virus. Eyach virus was cross-neutralized at very low dilutions by antiserum generated against Salmon River and CTFV Florio (PRNT90 1:10) but was not neutralized by antiserum from CTFV-Ca.Plaque reduction neutralization testing was completed for CTFV, Salmon River virus, and CTFV-Ca to understand the serological relationships . CTFV FlColtivirus has expanded in recent years to include newly described viruses as new species ; however, very little full-length sequence information has been available for the known viruses in the CTFV species complex, specifically CTFV, Salmon River virus, and CTFV-Ca. This study was initiated to improve the current knowledge of CTFV diversity and expand the known database of sequence information. We were able to generate full-length sequence information for two isolates of CTFV, two isolates of Salmon River virus, and the only known isolate of CTFV-Ca. Collectively, the data presented herein highlight a previously underappreciated diversity of CTFV and suggest the placement of Salmon River virus as a strain of CTFV, while CTFV-Ca likely represents a unique coltivirus species.The genus Lepus californicus) and given the designation S6-14-03 [Coltivirus.California hare coltivirus was isolated in California in 1976 from a hare (S6-14-03 . PreviouS6-14-03 , and, heS6-14-03 . The phyS6-14-03 ,27. In oS6-14-03 . CollectS6-14-03 . HoweverColtivirus, Salmon River virus could be considered a strain of the species Colorado tick fever Coltivirus.Salmon River virus was isolated from an ill person in Idaho in 1990. Presently, Salmon River virus is an unclassified coltivirus . The fulThe highest genetic diversity between the CTFV strains was observed within segment four. The nucleotide diversity in segment four is supported by the unique phylogenetic placement of the CTFV strains compared to other segments, as well as RDP analysis, suggesting this segment was subject to reassortment. Our observation of segment reassortment is supportive of previous studies suggesting the reassortment of multiple CTFV segments in the field ,29. A loOf unique interest, the sequences derived in this study display sequence variation from the CTFV strains previously sequenced and deposited into public databases. The sequences derived from CTFV Florio prototype virus obtained from the Arbovirus Reference Collection differed in segments two and eleven from the published reference sequences . Prior t


Chaoborus americanus. Here we take advantage of the geographical isolation in this species to investigate the diversity and persistence of its inherited virome. We identify eight novel RNA viruses from six families and use small RNA sequencing in reproductive tissues to provide evidence of vertical transmission. We report region-specific virus strains that mirror the continental phylogeography of the host, demonstrating that members of the inherited virome have independently persisted in parallel host lineages since they last shared a common ancestor in the Mid-Pleistocene. We find that the small interfering RNA pathway, a frontline of antiviral defense in insects, targets members of this inherited virome. Finally, our results suggest that the Piwi-mediated RNA silencing pathway is unlikely to function as a general antiviral defense in Chaoborus, in contrast to its role in some mosquitoes. However, we also report that this pathway generates abundant piRNAs from endogenous viral elements closely related to actively infecting inherited viruses, potentially helping to explain idiosyncratic patterns of virus-specific Piwi targeting in this insect.Inherited mutualists, parasites, and commensals occupy one of the most intimate ecological niches available to invertebrate-associated microbes. How this transmission environment influences microbial evolution is increasingly understood for inherited bacterial symbionts, but in viruses, research on the prevalence of vertical transmission and its effects on viral lineages is still maturing. The evolutionary stability of this strategy remains difficult to assess, although phylogenetic evidence of frequent host shifts and selective sweeps have been interpreted as strategies favoring parasite persistence. In this study, we describe and investigate a natural insect system in which species-wide sweeps have been restricted by the isolation of host populations. Previous work identified evidence of pronounced mitochondrial genetic structure among North American populations of the phantom midge,  Common Aedes mosquitoes, a second RNAi pathway mediated by Piwi family argonautes generates abundant small RNAs 25–30 nt in length from virus targets  derived from inherited viruses, are processed by the piRNA pathway following their acquisition, and may occasionally mediate interactions between Piwi proteins and exogenous viruses.The phantom midge, erbodies . While terbodies . We prevociation . In the 2.2.1C. americanus in the Western and Midwestern United States and Canada, including Michigan, Wisconsin, Minnesota, Montana, Utah, Idaho, Washington, and British Columbia (C. americanus sequence entries in the Barcode of Life Database (BOLD), which add considerable representation in the Midwest and East, while continuing to support near complete genetic isolation among regions . Two observations support the interpretation that isolation is not due to geographic distance alone. First, the region covered by the Midwest and East is expansive yet contains a weak genetic structure. Second, the genotypes from the Rocky Mountains region group with Alaskan genotypes rather than those in nearby Western British Columbia and Washington. Meanwhile, the Utah populations are closest in proximity to Montana and Idaho, yet these are among the most divergent pairwise genetic comparisons in our dataset. Together, these observations suggest physical barriers or habitat deserts have limited the dispersal of C. americanus across the North American continent, resulting in long-term maintenance of isolated mitochondrial lineages.We collected larval phantom midges from permanent and semi-permanent freshwater ponds across the species range. We sampled fifty-nine locations and identified sixteen ponds with larval Columbia . DespiteColumbia . To incr2.2Chaoborus americanus, we sequenced ribosome-depleted total RNA libraries from eight larvae collected in British Columbia, Washington, and Wisconsin. We also searched a previously published metagenomics assembly from a pool of five larvae collected in Alaska. We identified eight novel virus genomes, summarized below  genome of approximately 12.7 kb was assembled from hosts in all regions. Homologs of the N, P, M, G, and L genes are present, but no putative accessory ORFs are present between these genes  assembled as single contigs from hosts in British Columbia and Washington. The two genomes share 96.8 per cent nucleotide identity and the RdRps share 37 per cent amino acid identity with the most closely related viruses in public databases. Like the xinmovirid, all five genes characteristic of members of the Order Mononegavirales are present and no putative accessory ORFs >50 amino acids in length were identified  was assembled from two read sets acquired from one site in Washington and from Utah. The virus genomes each assembled into two fragments—one encoding putative N, P, M, and G genes flanked by untranslated regions (UTRs), and the other a full-length L gene with UTRs , was identified in larvae from all regions. Previously, the complete genome of this phasmavirid was reported in Alaskan C. americanus and a partial sequence was reported from British Columbia . In addition to six complete Niukluk virus genomes from RNASeq data sets, we generated partial L, M, and S segment sequences from fifteen larvae in Utah, British Columbia, and Wisconsin by Sanger sequencing. Phylogenetic analysis of the twenty-one full or partial virus genomes showed well-supported clades corresponding to host mitochondrial clades -like piwi gene and a single Aubergine-like piwi gene . Those derived from the negative strand show a strong U1 bias, while those derived from transcripts (or antigenomes) show a strong A10 bias . These d2.8C. americanus RNAseq assemblies showed similarity to virus genomes but appeared incomplete or otherwise unlikely to be attributed to exogenous viruses. First, a nearly-complete NP segment similar in sequence to Byreska and Byreldi virus is present in a subset of C. americanus larvae from which the other five genome segments are absent. While potentially explained as a defective viral genome, small RNAs mapping to this orphan NP sequence were also detected in one of the adult small RNA datasets and are heavily enriched in the 25–30 nt size range . Therefore, as reported for mosquitoes and other insects, nonretroviral EVEs expressed from the Chaoborus genome are processed into small RNAs through the piRNA pathway.Three contigs in the ze range . Phylogeze range . Putativze range . This seze range , also yize range . For allThe structure of the Niukluk virus S segment EVE was previously shown to include a partial duplication of the viral nucleoprotein gene. In our present assemblies, we found the EVE covers the full coding sequence of the S segment , and lik3.In this paper, we show the stable persistence of inherited RNA viruses within parallel host lineages over evolutionary timescales. Using total and small RNA sequencing, we identified eight novel RNA viruses and confirmed their infection in larvae, adult tissues, and ovaries or eggs. Our small RNA profiling analyses implicate the siRNA pathway in host defense. EVEs derived from ancestors of two of these viruses are expressed and interact with components of the piRNA pathway, yet both viruses persist across the host species range. Further studies should examine whether EVE-derived piRNAs meaningfully contribute to controlling inherited virus replication beyond the mosquitoes , particuAedes aegypti and Galbut virus of Drosophila melanogaster, are inherited from both parents but are not acquired by ingestion .While most invertebrate viruses are known from genome sequences alone, transmission modes and fitness effects have been recently investigated for mosquito-associated lineages of several widespread invertebrate virus groups. For example, partitivirids related to Ezimos virus, Verdadero virus of ngestion . Aedes aeography . Yet, geChaoborus in which pronounced effects of Pleistocene glaciation are evident in mitochondrial genetic structure . Before sequencing libraries were built, ribosomal RNA depletion was performed on samples for total RNA-Seq using RiboMinus reagents for eukaryotes and bacteria (Invitrogen). Barcoded RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs). Small RNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set (New England Biolabs). After cDNA synthesis, small RNA libraries were size-selected from a 5 per cent polyacrylamide gel. We excised and purified fragments 140–160 bp in length, corresponding to insert sizes of 19–39 bp for siRNAs and piRNAs. Small RNAs were sequenced using 75 bp single end Illumina NextSeq sequencing at HudsonAlpha Discovery, Alabama USA. Total RNA-Seq libraries were sequenced using 150 bp paired end Illumina sequencing at Novogene Corporation, California USA.3.1.3de novo assembled with Trinity 2.8.4  to remove Chaoborus sp. entries that grouped outside the monophyletic C. americanus clade recovered, with Chaoborus flavicans used as the outgroup, and (2) to reduce sequence redundancy when three or more identical sequences were present from a single collection location.3.2.1C. americanus (Supplementary Tables S2 and S3). cDNA synthesis for screens was done using Superscript III Reverse Transcriptase (Invitrogen) and primed with random hexamers (IDT). Primers used for PCR are listed in Supplementary Table S5. A touchdown program was used for all PCR reactions and consisted of an initial denaturation step of 95°C for 120 s, followed by 10 cycles of 30 s at 95°C, 30 s at 54°C (−0.6°C/cycle), and 60 s at 72°C, then 30 cycles of 30 s at 95°C, 30 s at 48°C, and 60 s at 72°C. For reactions generating long Niukluk virus fragments for Sanger sequencing the 60 s extension was increased to 120 s. Recombinant Taq DNA polymerase and PCR reaction buffer were sourced from ThermoFisher (EP0402) and dNTPs from Invitrogen (10297117). All custom primers used in this study were synthesized by IDT. Sanger sequencing was used to supplement Niukluk virus genomes acquired through RNA-Seq and focused on increasing sampling in British Columbia and Wisconsin. Amplicons were sequenced for the L (∼900 bp), M , S (∼950 bp), and host mitochondrial COI (∼730 bp).Niukluk virus screens were performed on aliquots of total RNA extractions from each of 353 3.2.2Game of Thrones.All novel viruses in this study were assigned names derived from the High Valyrian language from the fantasy series, veac018_SuppClick here for additional data file.


The correlation coefficient R values of nine bulls between IVF and AI methods were 0.73 and 0.97 for the conventional frozen and sexed frozen semen, respectively. The average conception rate of three bulls undergoing AI was 69.5% and 64.2%, 61.8% and 58.8%, and 48.2% and 46.2% in first-, second-, and third-born cows when conventional frozen and sexed frozen semen were used, respectively, which showed a positive correlation with the fertilization rate in the same parity. We propose an evaluation standard to assess the fertilization ability of bulls based on their IVF test results, which is categorized into three grades: grade one, normal fertility bull with an AI conception rate of 40%±5% and IVF rate of 45% to 60%; grade two, higher fertility bull with an AI conception rate of 50%±5% and IVF rate of 61% to 80%; and grade three, highest fertility bull with an AI conception rate of 60%±5% and IVF rate of >80%.These findings reveal that IVF results can be used as a breeding index for bulls to evaluate their AI conception ability, which may shorten the time required to select bulls for breeding. In coelopment –8. The celopment .AI and IVF are widely used treatments for assisted reproduction. AI is the main breeding technology for large-scale cattle breeding, which has been promoted by sexed semen technology in recent years ,11. ImprThe Institutional Animal Care and Use Committee of Inner Mongolia University approved the experimental protocol employed in this study (SYXK2014-0002).All reagents were purchased from Sigma-Aldrich Co , unless indicated otherwise.Nine Holstein bulls aged 3 to 5 years were randomly selected from the bull station of Inner Mongolia Saikexing Reproductive Biotechnology  Co., Ltd. The registration numbers of the bulls are provided in 2 and 100% humidity. Bull spermatozoa were thawed in a water bath set at 38°C and washed twice in Brackett and Oliphant (BO) medium [in vitro sperm functional assessments [2. The COCs that matured in vitro were washed thrice and placed in 50 μL drops of pre-equilibrated IVF medium covered with warm mineral oil in a 35-mm culture dish (30 COCs per drop). Fifty tiny drops of supernatant solution containing sperm (minimum concentration of 1.0×107 spermatozoa/mL) were then added to the drops that contained COCs and were incubated at 38.5°C under conditions of 5% CO2 and high humidity for fertilization [Ovaries were obtained from cows at a local slaughterhouse and were transported to the laboratory at 35°C in 0.9% NaCl containing 70 mg/mL kanamycin. Cumulus-oocyte complexes (COCs) were aspirated from medium-sized follicles (approximately 5 mm in diameter) with a 10-mL disposable syringe. Only COCs surrounded by a compact cumulus mass with an evenly granulated cytoplasm were harvested and then washed thrice in a maturation medium. A total of 60 to 80 COCs were transferred into each well of a Nunc 4-well dish containing 500 μL pre-equilibrated maturation medium TCM199) –17 at 38) medium . Centrifessments . BO’s feessments  was plac9 –17 at 2 and 100% humidity for 48 h to assess fertilization as indicated by embryo cleavage. Cleaved 2-cell embryos were continuously cultured for 7 days to observe blastocyst formation [At 6 h after coculturing, COCs were washed and transferred (30 to 50 COCs per well) to a Nunc 4-well dish containing 500 μL embryo culture medium  covered ormation . The sexormation . For eac7 sperm) 10 to 12 h after the estrus period (4 to 6 h before or after ovulation). After 35 days of AI, B ultrasonic detection was used to confirm the conception of the cows. AI treatment for all recipients of 1 to 3 parities were performed by same AI and veterinary working group.Heifers aged 13 to 15 months, weighing approximately 350 kg, and cows with 2 to 3 parity with a regular estrus period of 60 to 90 days after delivery were selected for AI following healthy examination, and also we avoid normally hot summer of July to August and cold winter of December to January, to eliminate the differences due to the climate influence. Then, AI with conventional frozen or sexed frozen semen was performed in three 5,000 large-scale Holstein farms. A pedometer computer information monitoring system was used to confirm the estrus and ovulation periods of the cow. The ovulatory side was determined by rectal examination, and the uterine horn was injected with semen  with high IVF rates were selected to produce conventional frozen and sexed frozen semen on a large scale. AI conception rates of 23,610 cows with different cow parities were compared . The aveThe software SPSS17.0 was used to analyze the variance and to test the significant difference and correlation between the IVF and AI conception rates of the bulls . ConsequSome studies suggest that the outcome of IVF is useful for predicting the AI conception rate of bull semen and the ability to support early embryo development ,23; howeIn this study, the correlation between the AI conception and IVF rates were investigated by using conventional and sexed frozen semen from nine bulls. The average rates of IVF and AI conception were approximately 48.4% to 95.8% and 43.3% to 58.4%, respectively, when using conventional frozen semen. The average rates of IVF and AI conception were approximately 48.9% to 88.6% and 40.3% to 65.2% respectively when using sexed frozen semen. Based on these results, we found that the IVF rates, which were compartmentalized with different fertility abilities, were positively correlated with AI conception rates in each of the nine bulls by using either conventional frozen semen  or sexed frozen semen , including AI recipient changes with different AI recipient parities.Considering the rates of IVF and embryonic development and the sex ratio, we conclude that the IVF rate in both conventional and sexed frozen semen can be used as a criterion for evaluating the bull AI conception ability. Thus, we propose an evaluation standard for the fertilization ability of bulls as per their IVF test results. This evaluation standard of the bull AI conception ability is categorized into three grades : grade oThe IVF can be used as a bull breeding index to evaluate bull AI conception, which was classified into three grades, namely, normal fertility, higher fertility, and highest fertility. It provides a more useful index than the genome-wide detection of Holstein bull breeding ability.


Furthermore, the study found that conductivity-temperature dependence followed the Vogel-Tammann Fulcher equation. From that, it could be clearly observed that 30 wt% TBAI indicated the lowest activation energy of 6.947 kJ mol−1. By using the fitting method on the Nyquist plot, the number density, mobility and diffusion coefficient of the charge carrier were determined. The charge properties were analysed using the dielectric permittivity, modulus and dissipation factor. Apart from this, the stoke drag and capacitance were determined.Jatropha oil-based polyurethane acylate gel polymer electrolyte was mixed with different concentrations of tetrabutylammonium iodide salt (TBAI). The temperature dependences of ionic conductivity, dielectric modulus and relaxation time were studied in the range of 298 to 393 K. The highest ionic conductivity of (1.88 ± 0.020) × 10 Jatropha curcas plant that are easily cultivated1, as well as being hardy, and non-edible3 in nature with a high yield of oil per unit hectare6 help spearhead the research into discovering more applications in the field of electrochemistry8.Concern for the environment and the dependency on fossil fuel for producing chemical feedstock has researchers focusing their effort into developing materials which use green and sustainable materials. Sustainably grown crops such as 10. Being in a gel like state, it combines the advantage of a solid electrolyte such as high safety as there is no leakage, with low flammability, flexibility, processability and thermal stability12 at the same time offering some of the benefits of a liquid electrolyte without the drawbacks. As the electrolyte is sticky in nature, it does not spill easily a like liquid electrolyte. It also can operate at higher temperatures14 as it does not face an issue with solvent evaporation such as occurs with a liquid electrolyte17. It is also flexible in nature which offers great mechanical properties over a solid electrolyte14. It also generally has a higher ionic conductivity compared to a solid electrolyte19.Deriving a polyurethane acrylate (PUA) polymer electrolyte from jatropha oil produces a gel like material which is soft and sticky. A gel based bio polymer electrolyte provides a fair amount of advantage compared to their liquid and solid electrolyte counterparts20. Other than that, PUA has a unique structure in which it has hard and soft segments that are arranged in an alternative pattern on the same chain23. With this unique property, PUA acts as a suitable candidate for electrochemical devices27.Due to the advantages mentioned above, jatropha oil-based PUA is used as the host polymer for preparing the gel polymer electrolyte. This is because PUA has high cross-linking density, solvent resistance, good mechanical properties and excellent adhesion2 gel polymer electrolyte must be conducted. The PUA-TBAI-I2 gel polymer electrolyte performance can be gauged by extensive study of its electrochemical properties such as the behaviour of the charge carriers, dielectric permittivity, ionic conductivity, electrical modulus, relaxation time, number density, including the mobility and diffusion coefficient of the charged carriers. To gauge the feasibility of the Gel Polymer Electrolyte (GPE) for industrial usage, the above electrochemical properties can also be measured at various temperatures.However, it is known that the ionic conductivity of gel polymer electrolytes is still lower than liquid electrolytes. Therefore, further in-depth study for the feasibility of PUA-TBAI-I−/I3− redox shuttle. TBAI salt was chosen as the primary salt in this gel polymer electrolyte system. This was due to its low lattice energy of 320.8 kJ mol−128. The low lattice energy makes it easier to dissociate into ions compared to other iodide salts. The higher degree of dissociation of iodide ions increases the number density of the charge carriers which also directly increase the mobility of the charge carriers. This results in the increase of ionic conductivity of the GPE. This phenomena has been reported by Bandara et al.29. Their sample containing 0.1 g polyacrylonitrile (PAN): 0.4149 g ethylene carbonate (EC): 0.3848 g propylene carbonate (PC): 0.0696 g TBAI: 0.0048 g iodine (I2) recorded an ionic conductivity of 2.14 × 10–3 S cm−129. Chowdhury et al.30 reported the highest room temperature ionic conductivity at 3.46 × 10–3 S cm−1 for their 0.27 g PAN:1.02 g EC: 1.02 g PC: 1 g TBAI: 0.069 g I2 gel polymer electrolyte. The values of ionic conductivity in30 increased about eleven orders of magnitude compared to pure PAN which was ≤ 10–14 S cm−131. Another example was reported by Theerthagiri et al.32. Their sample of polyvinylidene fluoride (PVDF): poly(methyl methacrylate) PMMA: EC: dimethylformamide (DMF): 60% TBAI: I2 polymer electrolyte achieved an ionic conductivity value of 5.11 × 10–3 S cm−1 at 303 K. Tiong et al. reported the same findings33. Their gel polymer electrolyte with a composition of 7.02 wt% PVA: 7.02 wt% polyethylene glycol (PEO): 30.11 wt% EC: 30.00 wt% dimethyl sulfoxide (DMSO): 24.08 wt% TBAI: 1.66 wt% I2 recorded the highest ionic conductivity of 5.50 × 10–3 S cm−1 at room temperature. From the literature above, it is known that TBAI salt is suitable to be used as a primary salt in a PUA gel polymer electrolyte system.In this work, a PUA host polymer was added with various concentrations of tetrabutylammonium iodide (TBAI) salt in order to enhance the ionic conductivity. TBAI salt was used together with iodine to form the redox I34. During the epoxidation process, the jatropha oil was pre-mixed with formic acid under mechanical stirring using an overhead stirrer at 313 K. Hydrogen peroxide was added slowly into the mixture using a dropping funnel to prevent over heating of the mixture solution. The mixture was then heated to 333 K while being stirred continuously for 6 h. After 6 h, the mixture was left to cool down to room temperature before transferring it to a separating funnel. The mixture was allowed to settle in the separating funnel and the aqueous layer was removed. The remaining organic layer was rinsed with warm distilled water several times to remove any excess acid. Thus the epoxidised jatropha oil (EJO) was produced.Jatropha oil-based PUA was synthesised by a three-step process: epoxidation, hydroxylation and the introduction of the acrylate group in the urethanation processAfter that, the hydroxylation process was carried out. Some 133 g of methanol, 0.93 g of sulphuric acid and 15 g of distilled water was pre-mixed and stirred for about 15 min at 313 K. Then, 150 g of EJO was added to the mixture and was heated up to 338 K for another 30 min. The polyol was allowed to cool down to room temperature before it was poured into a separating funnel. After that, the excess methanol and distilled water in the polyol was removed by using a vacuum rotary evaporator until a clear yellow polyol was obtained.Polyol, TDI, HEMA and DMF as solvent were used in order to prepare polyurethane acrylate. An amount of 20 g of Polyol and 10 ml of DMF were mixed at 323 K, then 8.57 g of TDI was added drop wise into the mixture using a dropping funnel. The mixture was then heated up to 333 K for about 2 h. During this process, 10 ml of DMF was added into the mixture. After that, the mixture was allowed to cool to 313 K and 6.38 g of HEMA was added drop wise. The mixture was heated up again to 323 K for another 1 h. At the same time, another 5 ml of DMF was added. This was done to reduce the viscosity of the mixture and the PUA gel polymer electrolyte was successfully produced.2 gel polymer electrolytes were prepared with the selected ratio as listed in Table 2 chips  were then added into the mixture and stirred overnight. The electrolytes were placed in a desiccator for 24 h before being used for characterisation.PUA-TBAI-I2 GPE was sandwiched in air between two stainless steel electrodes with a 2.011 cm2 working area equipped with a 0.26 cm thickness separator and then the assembly was placed into the coin cell and clamped. The coin cell was placed in the oven using controlled heating for the conductivity-temperature studies. A small sine wave with voltage of 10 mV with a frequency range of 50 Hz to 1 MHz was applied across the sample. A current flow in the sample was recorded and the impedance data was calculated from it. Nyquist plots were plotted based on the real (Zr) and negative imaginary (− Zi) impedance data. The intercept of the plot on the real axis corresponded to the bulk resistance (R). At lower temperatures, R was obtained from the intersection of semicircle and the spike. At higher temperatures, the semicircle disappeared as the resistance of the sample became smaller and smaller until only the spike was observed. Then, the R was determined from the intercept on the Zr axis. The ionic conductivity (σ) of the electrolyte was then calculated by using the equation below36:EIS measurements were carried out to study the resistance and capacitance properties of the electrolytes. A HIOKI IM3570 Impedance Analyser was used to perform the measurements at various temperatures ranging from 298 to 393 K. The PUA-TBAI-It is sample thickness and A is the electrolyte/electrode contact surface area.In the above equation, Zr and − Zi impedances are as follows:For various temperatures, the equations for k2–1 is the electrical double layer capacitance at the electrode/electrolyte interface. P2 is the angle between the Z′ axis and the spike line. ɷ is the angular frequency corresponding to the minimum imaginary impedance. From the fitting parameters, n, µ, and D could be determined using Eqs. .4.4k2–1 isThe value of the diffusion coefficient could then be calculated by using the Stokes–Einstein equation:The formula for Stoke drag is shown as below:Kb is the Boltzmann constant, T is the absolute temperature, η is viscosity, r is radius and v is velocity. In this work, v can be ignored as v is kept constant.By substituting Eq.  into Eq.2 GPE, the complex permittivity was studied. The equations below were used to calculate the complex permittivity of the electrolyte38:To further understand the ionic conductivity behaviour in PUA-TBAI-Iω = 2πf where f is the frequency in Hz. For complex electrical modulus studies, the real electrical modulus, Mr and imaginary electrical modulus, Mi can be achieved by using the following equation:Here –4 S cm−1 using an R value of (922 ± 2) Ω. On the other hand, 30 wt% TBAI salt showed the lowest R value of (686 ± 7) Ω and a calculated ionic conductivity value of (1.88 ± 0.020) × 10–4 S cm−1. This was due to R which was inversely proportional to the ionic conductivity as in Eq.  × 10–4 S cm−1 for 10 wt% TBAI salt to (1.88 ± 0.020) × 10–4 S cm−1 for 30 wt% TBAI salt. This suggests that the n, µ and D values increased as the amount of TBAI salt added into the PUA gel polymer electrolyte increased from 10 to 30 wt%.Here From Eq. , the vallot Fig.  based onn; (1.68 ± 0.071) × 1021 cm−3 increased to (1.93 ± 0.042) × 1021 cm−3. The value of µ; (5.21 ± 0.223) × 10–7 cm2 V−1 s−1 increased to (6.24 ± 0.117) × 10–7 cm2 V−1 s−1., while the value of D increased from (1.34 ± 0.057) × 108 cm2 s−1 to (1.60 ± 0.030) × 108 cm2 s−1. However, a TBAI salt loading of 40 wt% and above showed a lower ionic conductivity compared to the 30 wt% TBAI salt. For instance, the 50 wt% TBAI salt sample showed an ionic conductivity of (1.47 ± 0.019) × 10–4 S cm−1. This was mainly due to the decrease of the µ value of (2.66 ± 0.075) × 10–7 cm2 V−1 s−1 and the D value of (0.68 ± 0.019) × 108 cm2 s−1 as compared to the 30 wt% TBAI salt. As the number density of the charge carriers increased, more salt dissociation occurred. The increase of salt dissociation tended to cause the µ and D values to decrease as a blocking effect occurs due to the presence of too many charged carriers in the electrolyte system. Ion-ion interaction occurred and this favoured the formation of ion clusters41. This has been shown in a study conducted by Kadir et al.42 where it was mentioned that the rate of ion dissociation was favourable at a concentration range of around 10 wt% to 30 wt%. At higher concentrations of salt, ion association took place and neutral charges were formed which in return reduced the mobility of free ions and resulted in a decrease in ionic conductivity. Another example was spotted in the report of Addullaziz Abdukarimov et al.43 who reported that as the amount of TPAI salts in PEO: PC: EC: DMF: I2 liquid electrolyte increased, the ionic conductivity increased up to 8.98 ± 0.23 mS cm−1 for a sample containing 19.61 wt% TPAI as it had the lowest R value (14.2 Ω). Addullaziz Abdukarimov et al. also stated that the value of the ionic conductivity could be enhanced by increasing the number density of the free ions in the liquid electrolytes.In comparison with the 10 wt% TBAI salt sample and the 30 wt% TBAI salt sample, the value of Thus in this work, the R value is expected to decrease with the increase of TBAI salt. The increase of the number density and the mobility of free ions also contributed to the increase of ionic conductivity. As more TBAI salt was added to the GPE, ion dissociation increased accordingly which caused an increase in free ions present in the PUA polymer matrix. Due to the increase of free ions in the polymer matrix, this could lead to an increase in ion mobility which in return increased the ionic conductivity of the gel polymer electrolyte.44. At low temperature, the Nyquist plot showed a combination of a semicircle and spike. As the temperature increased, the semicircle which appeared at lower frequencies disappeared and left only a spike at higher frequencies.The ionic conductivity of the electrolyte with respect to different temperatures ranging from 298 to 393 K was conducted in this study. The Nyquist plot acquired from the study was fitted using the best equivalent circuit model. The Nyquist plot obtained could appear in three forms: (i) a semicircle predominantly at low frequency, (ii) a spike at high frequency or (iii) a combination of both semicircle and a spike2 gel electrolyte system at various temperatures is illustrated in Fig. The ionic conductivity of the PUA-TBAI-IA is the pre-exponential factor, Ea is pseudo-activation energy, kb is the Boltzmann constant, T is absolute temperature and To is the ideal glass transition temperature . Coupled with Fig. Ea can be estimated from the gradient of Eq.  and anions (I−) once they receive enough energy to break the bonds. This will produce more free ions available in the PUA-TBAI-I2 gel polymer electrolyte. This reason explains the increase of the number density of charge carriers with temperature as showed in Fig. Figure To and Ea values drop gradually from 10 wt% TBAI salt to 30 wt% TBAI salt. This was due to the movement of the free ions. The free ions move with acceleration from one site to another within the polymer matrix. Due to this, the 30 wt% TBAI salt recorded the lowest activation energy and glass transition temperature. Beyond 30 wt% TBAI salt, the Ea value increased from 6.947 to 14.183 kJ/mol for the sample containing 50 wt% TBAI salt.In Table + cations and I− anions.This is because at higher TBAI salt concentrations above 30 wt% TBAI salt, the mobility of charge carriers starts to decrease. As seen in Fig. 48 as well as the kinetic energy of the ions will be reduced. This causes the free ions in the PUA polymer to move with difficulty and required more energy. Thus, high activation energy was required. This is supported by Fig. The higher rate of collisions between the cations and anions will result in a higher chance of them forming a neutral charged pair. In other words, the TBAI salt prefers to remain in its salt form and not in its dissociated ion pair form. This will indirectly result in reduced mobility charged carriers and the polymer chain will become rigidApart from this, the stoke drag, ots Fig. . The Stoots Fig. . Other t2 GPE, the complex permittivity was studied. The two main components in complex permittivity are the dielectric constant  was assigned to increase the charge carrier density. Another example was the addition of ethylene carbonate (EC) into PVA-LiBr-H2SO4 by Sheha56. The author said that the increase of dielectric constant was able to increase the storage charge in the electrolyte system. This was due to the ion dissociation in the EC plasticised polymer system as EC has a higher dielectric constant value and it causes more salts to be dissociated. It is best to described why 57. Ion polarisation occurring with the formation of ion pairs is more favourable compared to the formation of free ions. When free ions are in an excess amount, the free ions will begin to interact with each other and form neutral charge pairs. Therefore, the mobility as well as ionic conductivity will reduce as shown in Table Figure entclass1pt{minimaentclass1pt{minima58. This meant that no extra ion diffusion occurred in the direction of the electric field as there was not enough time to accumulate at the electrolyte/electrode interface59. Under those circumstances, the Furthermore, a graph of log dielectric loss  is discussed. Figure 2 GPE at selected temperatures (303 K to 393 K). It can be observed clearly that both 62:The dielectric properties can vary due to the frequency applied, temperature, structural characteristics or other factorsn is charge density, U is dissociation energy, kb is the Boltzmann constant and T is the absolute temperature. From Eq. . The relaxation time of PUA-TBAI-I2 GPE systems are tabulated in Table δ. As the peak shifted toward higher frequencies, it was observed that the relaxation time was reduced67. From Fig. –5 s) as well as highest in ionic conductivity which was (1.88 ± 0.020) × 10–4 S cm−1 at room temperature.Figure 2 GPE at selected temperatures and relaxation time at Table –5 s at a temperature of 303 K to 0.56 × 10–5 s at a temperature of 318 K. Hence, this proved that the relaxation time was reduced as the temperature increased.Figure 2 showed promising results and that it was feasible to be used as an alternative green and sustainable electrolyte. The GPE system also managed to perform on par with other green and sustainable polymer electrolytes. The 30 wt% TBAI recorded the highest ionic conductivity at all temperatures tested, starting from room temperature of 298 K up to 393 K compared with the other ratios prepared in this work. This showed that a high amount of TBAI salts were not necessary to obtain greater ionic conductivity as there were diminishing returns beyond the 30 wt% TBAI. The mobility and diffusion coefficient of the charge carriers also corresponded to the positive result of the ionic conductivity. The 30 wt% TBAI also reported the lowest pseudo-activation energy compared to other samples at 6.947 kJ mol−1. The values of the dielectric constant, dielectric loss, electrical modulus, dissipation factor, stoke drag and the capacitance were also reported in this work accordingly and affirmed the great performance of the 30 wt% TBAI-PUA polymer in terms of electrochemical properties.In this study, jatropha oil based PUA gel polymer electrolyte enhanced with TBAI-I


PurposeThis study aimed to determine whether prone cross-body adduction (superman stretch) improves range of motion (ROM) more than the sleeper stretch.MethodsCollegiate overhead athletes were randomized to either a sleeper group or a superman stretch group. ROM measurements were collected before and after stretches by three orthopedic surgeons.ResultsWe assessed a total of 212 shoulders. Both stretches demonstrated significant improvements in ROM, except horizontal adduction, which only improved in the superman stretch group.ConclusionsThe superman stretch may be superior in producing immediate improvements in horizontal adduction when compared to the traditional sleeper stretch. Throwing athletes subject their shoulders to repeated forces, which leads to several adaptive changes . One resThese adaptive changes act as the catalyst for pathology, as the majority of shoulder injuries occur at the limits of ER -2. RepeaThe sleeper stretch is commonly used in throwing athletes, as it isolates the soft tissue restraints in the posterior shoulder. Laudner et al. investigated the use of the sleeper stretch in collegiate baseball players and found that it led to significant improvements in shoulder IR and horizontal adduction . The strPatient selectionInstitutional Review Board approval was obtained by our institution. Asymptomatic, in-season division III collegiate overhead athletes, including baseball/softball, volleyball, and tennis, were enrolled prospectively and randomized at our institution. Exclusion criteria included patients with a history of prior surgery, evidence of hyperlaxity (defined as Beighton score of ≥6), or initial IR greater than 70°. Once enrolled, patients were randomized to either the sleeper stretch cohort or the superman stretch cohort, with both shoulders allocated to the same stretching protocol. Allocation was done using a computer-generated block randomization sequence, with the study examiners blinded to patient allocation. Due to the nature of the stretches, it was impossible to blind the patients to their allocation.Measurements and instrumentationMeasurements were performed by one of three examiners, all of whom were fellowship-trained orthopedic surgeons, and certified athletic trainers were used for patient positioning and stabilization. The examiner used a digital goniometer  to measure shoulder IR and ER and glenohumeral horizontal adduction. For IR, the participant was examined in a supine position, with the shoulder and elbow in 90° of abduction and flexion, respectively, and with the humerus supported to ensure a neutral horizontal position (level with the acromion process). A certified athletic trainer stabilized the scapula by applying pressure to the anterior acromion, with the participant passively internally rotated until the termination of humeral rotation. The digital goniometer was aligned with the ulna, providing an angle between the forearm and a perpendicular plane to the examination table.For ER, the participant was examined in the supine position, with the shoulder and elbow in 90° of abduction and flexion, respectively, and with the humerus supported to ensure a neutral horizontal position (level with the acromion process). A certified athletic trainer passively externally rotated the humerus, and with the other hand, stabilized the scapula by applying pressure to the anterior acromion until termination of humeral rotation. The digital goniometer was aligned with the ulna, providing an angle between the forearm and a perpendicular plane to the examination table.For glenohumeral horizontal adduction, participants began in a supine position, with both shoulders flat against a standard examination table. A certified athletic trainer stood at the head of the examination table and positioned the test shoulder and elbow into 90° of abduction and flexion, respectively. The certified athletic trainer stabilized the lateral border of the scapula by providing a posteriorly directed force. With the other hand, the certified athletic trainer held the proximal portion of the participant’s forearm just distal to the elbow and passively moved the humerus into horizontal adduction, where the examiner then measured the amount of motion present. The digital goniometer was aligned with the ventral midline of the humerus to complete the measurement. The angle created by the end position of the humerus to 0° of horizontal adduction was recorded as total glenohumeral horizontal adduction motion.Procedure descriptionsBefore performing the randomized stretch, each participant had a baseline ROM assessed using the digital goniometer, as described above by one of the examiners. Following baseline measurements, participants were moved to a separate room and were supervised by a certified athletic trainer while performing their assigned stretch for three rounds of 30 s on each arm, with allocation and pre-stretch ROM measurements blinded to the examiner who is performing the measurements. After completing three rounds of stretches, participants were brought back to the examination room, where the same examiner performed the ROM measurements again using the digital goniometer. Both cohorts were provided the same but separate instruction by a certified athletic trainer and provided a picture demonstrating either the sleeper or superman stretch to provide a consistent description of the respective stretches. In addition, the stretch was demonstrated to the patients by the certified athletic trainer.The sleeper stretch participants were placed in a side-lying position, with the scapula stabilized against the table. The elbow was placed in 90° of flexion, and the participant’s contralateral arm was used to rotate the arm toward the table with maximum IR. This position was held for 30 s. This stretch protocol was repeated three times for each side in succession, with rest for one side occurring, while the other was being stretched. The superman stretch participants were placed prone on the athletic trainer’s table. The ipsilateral shoulder was flexed 90° and adducted horizontally across the torso, and the contralateral shoulder was flexed forward 180°. This position was held for 30 s. This stretch protocol was repeated three times. The sleeper stretch and superman stretch can be seen in Figures Statistical analysist-tests were conducted to determine whether there were any significant differences in baseline and post-stretch ROM values. Two-sample t-tests were carried out to determine whether there were any significant differences in ROM values for the sleeper stretch and superman stretch groups. Effect sizes for the comparative analysis were calculated using Hedges’ g-test. A p-value <0.05 was considered statistically significant. A power analysis found that 200 shoulders required enrollment to achieve a power of 80%.Degrees of IR, ER, the total arc of motion, and horizontal adduction before and after stretching were reported as mean and standard deviations. Paired Patient enrollment, exclusion, and randomization can be seen in Figure p=0.02). Significant increases in all ROM parameters included in the study were found for both groups, except for horizontal adduction in the sleeper stretch group .Standard errors of the mean (SEMs) for each ROM value were calculated using the recorded pre-stretch values. SEM values for IR, ER, the arc of motion, and horizontal adduction were 1.06º, 0.86º, 1.40º, and 0.70º, respectively. This translated to minimum detectable changes of 2.94º, 2.38º, 3.88º, and 1.94º, respectively. Table p=0.002). This difference in horizontal adduction had a medium effect size (0.457). All other ROM measurements had small effect sizes between groups.Table Athletes who perform repetitive overhead movements at the extremes of glenohumeral motion place immense repetitive stresses on the shoulder joint and soft tissues . PatholoOur study investigated the effect of the superman stretch and sleeper stretch on ROM and found that both stretches led to significant time-zero ROM improvements. Only horizontal adduction was significantly improved by the superman stretch compared to the sleeper stretch. Our measurement technique demonstrated similar SEM values to prior studies utilizing similar stretches for IR, ER, the arc of motion, and horizontal adduction . PreviouWe found that the sleeper stretch effectively increases IR, ER, and the total arc of motion, consistent with previous findings on the utilization of this stretch -9. We fuIn our practice, we utilize the superman stretch due to its ability to maintain scapular stabilization passively using the patient’s body weight. This is similar to the bodyweight assistance provided in the sleeper stretch technique. With the superman stretch, once the arm is positioned, the patient’s only variable to consider is the degree of body rollover. In comparison, while performing the sleeper stretch, patients must consider proper arm positioning, degree of body rollover, and the proper application of IR force by the contralateral arm. These variables make this technique more difficult to perform without proper supervision. Additionally, the superman stretch resulted in greater horizontal adduction compared to the sleeper stretch in our study. This degree of improvement is likely to be clinically significant, as Camp et al. described that throwing athletes had a 4.5º horizontal adduction deficit and that this deficit may be associated with increased risks of injury .We are aware of several limitations with our study design. Age and sex were not collected, so it is unclear whether there may have been any significant differences between the two groups. However, patients were randomized to each group, they were asymptomatic, and only one ROM value differed at baseline between groups. Hand dominance was not considered, as stretches were performed on both sides of the participants. Additionally, this study only evaluated ROM before and immediately after the performance of the stretch. For this reason, the results of this study cannot be used to make conclusions regarding the long-term effects of these stretches. However, we believe this helped minimize the bias introduced by external stretching programs or sports practices that would be impossible to control for in a long-term study. Finally, the ROM measurements were recorded at a single time point and were not re-assessed by a second examiner.In conclusion, our study demonstrated that both the superman and sleeper stretches led to significant immediate improvements in ROM. Furthermore, we found that the superman stretch may be superior in improving immediate horizontal adduction. The results suggest that the superman technique should be considered for stretching routines in overhead athletes.


Mareca penelope) and barnacle geese (Branta leucopsis). This study determined that 2020 H5N8 virus had a high level of attachment to the intestinal epithelium (enterotropism) of dabbling ducks and geese and retained attachment to airway epithelium (respirotropism). Natural HPAI 2020 H5 virus infection in Eurasian wigeons and barnacle geese also showed a high level of neurotropism, as both species presented with brain lesions that co-localized with virus antigen expression. We concluded that the combination of respirotropism, neurotropism, and possibly enterotropism, contributed to the successful adaptation of 2020/2021 HPAI H5 viruses to wild waterbird populations.Highly pathogenic avian influenza (HPAI) outbreaks have become increasingly frequent in wild bird populations and have caused mass mortality in many wild bird species. The 2020/2021 epizootic was the largest and most deadly ever reported in Europe, and many new bird species tested positive for HPAI virus for the first time. This study investigated the tropism of HPAI virus in wild birds. We tested the pattern of virus attachment of 2020 H5N8 virus to intestinal and respiratory tissues of key bird species; and characterized pathology of naturally infected Eurasian wigeons ( Highly pathogenic avian influenza (HPAI) outbreaks have become increasingly frequent in wild bird populations and have caused mass mortality in many wild bird species ,6,7,8,9.Mareca penelope) and tufted ducks (Aythya fuligula) accounted for the highest rates of mortality [Branta leucopsis) and graylag goose (Anser anser), experienced among the highest rates of mortality [In Europe, outbreaks coincide with the autumn migrations of wild birds, a time when many long-distance migratory waterbirds congregate to overwinter or stop-over around water-rich areas . The twoortality . The 202ortality ,7,8,9. Fortality ,7,8,9,13Anas platyrhynchos) were reported dead in Europe during surveillance [Both experimental studies and field observations identified wild ducks, and in particular dabbling species such as Eurasian wigeons and mallards, as the most suitable vectors for the long-distance spread of HPAI viruses ,2,14,15.eillance ,7,8,9. Ieillance .In this study, we investigated whether 2020 H5N8 virus followed the trend of its predecessor, 2016 H5N8 virus, of increased enterotropic potential. We hypothesized that enterotropism is likely to be maintained, even enhanced, especially in bird species such as the mallard and Eurasian wigeon that are hypothesized as being vectors for long-distance viral dispersion. Furthermore, enterotropism could be also evident in new host species, such as graylag goose and barnacle goose, with the advantage of virus perpetuation in these globally abundant host populations and their movements.Our study is based on the hypothesis that the 2020 HPAI H5N8 virus was more enterotropic than the 2016 HPAI H5N8 virus, both in established and in new host species. To investigate this hypothesis, we determined the pattern of attachment of the 2020 H5N8 virus to intestinal and respiratory tissues of key species of wild ducks and geese, as well as chickens, and then compared it with the patterns of attachment of older HPAI viruses such as H5N1, 2014 H5N8, and 2016 H5N8 viruses. Furthermore, we examined wild Eurasian wigeons and barnacle geese that died during the 2020/2021 epizootic and determined virus distribution, tissue tropism and associated lesions in their major organs. Because the barnacle goose is a new host species for HPAI virus, associated pathological findings in this species have not yet been described. Because HPAI virus-infected geese have been reported to show mainly neurological clinical signs  , we wereOur study consisted of two parts. First, we performed virus histochemical analysis of four HPAI viruses to compare the pattern of attachment of these viruses in the respiratory and intestinal tracts of five bird species. We were particularly interested to determine whether 2020 H5N8 virus attached similarly to the 2016 H5N8, 2014 HPAI H5N8 and 2005 H5N1 viruses to the digestive tract of three wild duck species  and one key wild goose species (graylag goose). Second, we examined the carcasses of 14 wild birds (5 Eurasian wigeons and 9 barnacle geese) that were found dead during the 2020/2021 H5N8 HPAI epidemic in the Netherlands and that tested positive for H5 HPAI virus, to characterize the virology, pathology, and cell type tropism of this virus infection in different organs. The Eurasian wigeons were freshly dead at the time of necropsy; the barnacle geese were at different stages of autolysis, ranging from freshly dead to mildly autolyzed. We were particularly interested to determine whether 2020 H5N8 HPAI virus had similar tropism as 2016 H5N8 HPAI virus, from the 2016/17 epidemic, for the digestive tract of wild birds. The techniques of investigation were similar to Caliendo et al., 2020 [g. The viruses were individually concentrated by centrifugation of the cleared supernatants in filter tubes  for 40 min at 4000× g at 4 °C. The concentrated virus was inactivated by dialysing against 0.1% formalin for 3 days at room temperature (RT). After inactivation, the virus solution was dialysed against phosphate-buffered saline solution (PBS) and complete inactivation was confirmed by passaging twice on MDCK cells. Virus was labelled by adding an equal volume of 0.1 mg/mL of fluoresceinisothiocyanate (FITC)  in 0.5 M bicarbonate buffer (pH 9.5) for 1 h at RT while constantly stirring. Labelled virus was dialysed against PBS in order to lose all unbound FITC. The concentration of the different virus suspensions used for virus histochemistry was standardized at 50 hemagglutination units/100 μL (HAU) using hemagglutination assay with turkey red blood cells.The following four virus isolates were used: 2005 HPAI virus H5N1 (A/Turkey/Turkey/1/05), 2014 HPAIV H5N8 (A/Eurasian wigeon/Netherlands/emc-1/2014), 2016 HPAI virus H5N8 (A/Eurasian wigeon/Netherlands/19/2016), and 2020 HPAI virus H5N8 (A/Eurasian Wigeon/Netherlands/3/2020). The viruses were individually passaged in Madin-Darby canine kidney (MDCK) cells. After 2–3 days, the supernatant was harvested and cleared of cell debris by low-speed centrifugation for 20 min at 1455× n = 3), Eurasian wigeon (n = 3), mallard (n = 3), graylag goose (n = 3) and chicken  (n = 2). These tissues came from the Erasmus MC tissue bank, and were from healthy animals that showed no abnormalities or histological lesions. From the digestive tract of the same birds, tissues selected were duodenum, jejunum, ileum and colon. Sections of jejunum and ileum of graylag goose were not available for virus histochemistry because of limitations of sampling due to high workload during HPAI outbreak. From the respiratory tract, tissues selected were trachea, primary bronchus, secondary bronchus, tertiary bronchus or parabronchus, air capillaries and air sacs.Tissue sections of the following species were used: tufted duck . The signal was amplified using a tyramide amplification system . Peroxidase was revealed with 3-amino-9-ethylcarbazole (Sigma-Aldrich) resulting in a bright red precipitate. Tissues were counterstained with hematoxylin and embedded in glycerol-gelatin . Omission of the FITC-labelled virus was used as a negative control.The slides were assessed with light microscopy to estimate the abundance of viral attachment to epithelial cells and scored as follows: attachment to <1% of epithelial cells (−), attachment to ≥1 and <10% of epithelial cells (±), attachment to ≥10% and <50% of epithelial cells (+), and attachment to ≥50% of epithelial cells (++). Finally, the median score was determined for each species at the different anatomical sites. Sections were examined without knowledge of the identity of the birds.The carcasses of 14 wild birds (5 Eurasian wigeons and 9 barnacle geese) had been collected in the Netherlands in November and December 2020. Pharyngeal and cloacal swabs were collected from each bird for virus diagnostics, using sterile cotton swabs placed in 1 mL of virus transport medium. All the birds tested positive for HPAIV H5 2020 by real-time reverse-transcription PCR (RRT-PCR) assays in oropharyngeal and/or cloacal swabs as described previously .At postmortem examination, the following tissues, when available, were examined: brain, lungs, air sacs, pancreas, liver, stomachs (proventriculus and ventriculus), small intestine , large intestine , kidney, adrenal gland, spleen and heart. Duplicate tissue sections were collected for virus detection and kept at −80 °C until analysis, or were fixed in 10% neutral-buffered formalin and embedded in paraffin. Organs of barnacle geese were not available for virology. For virus detection, tissue samples were first weighed and then homogenized with a FastPrep 24  in Hank’s balanced salt solution and centrifuged briefly before dilution in lysis buffer for RNA extraction ,15. ExtrIn intestinal epithelia, 2014 H5N8, 2016 H5N8, and 2020 H5N8 had overall higher attachment than H5N1 .In comparison among host species, virus attachment to intestinal epithelia was low in tufted duck, intermediate in Eurasian wigeon and graylag goose, and high in chicken, mallard. The level of attachment of 2020 H5N8 per host species was: in mallard, similar to 2016 H5N8 and higher than H5N1 and 2014 H5N8; in Eurasian wigeon, similar to 2014 H5N8, higher than H5N1 and slightly higher than 2016 H5N8; in tufted duck lower than 2014 H4N8, 2016 H5N8 and H5N1; in graylag goose, slightly higher than 2014 H5N8, 2016 H5N8 and H5N1, because of the higher attachment to the duodenum; in chicken, all the viruses had the same, high level of attachment.In respiratory epithelia, the virus attachment of each virus was generally high in all species, especially for trachea, primary and secondary bronchus .The level of attachment of 2020 H5N8 was lower than 2016 H5N8 in secondary bronchi of tufted duck and air capillaries of graylag goose; and higher than 2016 H5N8 in the air capillaries of chicken.For Eurasian wigeons, pharyngeal and cloacal swabs were positive in all five tested birds .Viral RNA was detected in all the organs tested , meaning that the virus had spread systemically.For barnacle geese, cloacal swabs were more frequently positive than pharyngeal swabs , but, when both positive, they overall contained a similar amount of virus.Grossly, the main pathological changes consisted of multifocal necrosis in liver and pancreas; pin-point hemorrhages in the brain; sub-pericardial hemorrhages; and multifocal pulmonary. Histologically, lesions were detected in the liver, brain, kidney, lung, heart .In liver and pancreas, lesions were characterized as multifocal, mild-to-moderate necrosis; these necrotic foci co-localized with presence of viral antigen. In the brain, lesions were characterized as multifocal encephalitis with foci of gliosis, neuronal degeneration, and necrosis; abundant viral antigen was present in the nucleus and cytoplasm of several neurons in these lesions. In kidney, lesions were characterized as mild interstitial nephritis, with no presence of viral antigen. In the heart, lesions were characterized as multifocal to focally extensive myocardial necrosis; few myocardial cells in these lesions presented viral antigen. Mild-to-moderate inflammatory changes and infiltration with mononuclear cells were observed in lung, and few epithelial cells in these lesions presented viral antigen. There were no histological lesions and no detectable viral antigen in the gastro-intestinal tract of the examined birds.The high number of birds and the increasing number of wild bird species infected during recent HPAI outbreaks suggest that HPAI H5 viruses are adapting to wild birds. During past epizootics, domestic birds had played an important role for the Gs/Gd lineage by acting as a main reservoir where virus evolution could take place ,2. GraduSecond, cloacal swabs were consistently positive for viral RNA in naturally infected Eurasian wigeons and barnacle geese, and were more reliable in detecting the infection in barnacle geese. In addition, the jejunum of the five infected Eurasian wigeons was consistently positive for virus RNA by RT-PCR. These findings contrast with previous studies, where the cloacal excretion of HPAI virus infected birds was usually uncommon and, if present, lower than pharyngeal excretion ,15,16. THowever, a finding against our hypothesis was the lack of virus antigen expression and associated histological lesions in the intestinal epithelium of naturally infected Eurasian wigeons and barnacle geese. Virus antigen expression had been previously reported in the gastro-intestinal epithelium of Eurasian wigeon and graylag goose naturally infected with 2016 H4N8 [Eurasian wigeons and geese have similar biological traits that may be relevant for the epidemiology of HPAI. Eurasian wigeons have already been described as long-distant vector; HPAI virus infection in barnacle and graylag geese is instead a more recent and less-studied event. Although migratory goose species were not previously found to disperse LPAIVs , this quAnother common trait between Eurasian wigeons and geese is their feeding biology. Apart from dabbling, Eurasian wigeons use a diverse feeding strategy that includes foraging by grazing on land ,21. DuriRespirotropism is retained for the 2020 H5N8 virus, based on patterns of virus attachment and virus antigen expression observed in this study. The combination of these two mechanisms  could give HPAI viruses an advantage over LPAIVs that are mainly excreted via the orofecal route.Aythya ferina) infected with HPAI 2005 H5N1 virus [Anas platyrhynchos domesticus) infected with HPAI 2016 H5N8 virus [This study also described the virology and pathology of HPAI virus infection in barnacle geese, a new host species for the virus. Infection with HPAI H5 viruses in both Eurasian wigeons and barnacle geese was characterized by a high level of neurotropism. Both species presented with brain lesions associated with virus antigen expression. Most likely, this also represented the cause of death for the birds. Brain lesions were also compatible with neurological signs  shown by many birds and described by field operators during the 2020/2021 HPAI surveillance . The higN1 virus ; polioenN8 virus . SimilarStercorarius skua) and golden eagle (Aquila chrysaetos) [Vulpes vulpes), gray seals  and harbor seals (Phoca vitulina) were found dead, probably from contact or ingestion of infected wild birds [The mortality of waterbirds from the neurotropism of the 2020 HPAIV H5N8 could serve in reaching yet more new host species. Infected waterbirds with neurological signs are more visible and trigger a hunt response of their predators; they are easier prey to catch and more likely to be eaten ,29. In 2ld birds ,31, and In conclusion, HPAI H5 viruses from the 2020/2021 epidemic expressed a diverse repertoire of tissue tropism, including respirotropism and neurotropism, as well as a high level of in vitro enterotropism, in infecting wild ducks and geese. Because of the constant evolution shown by HPAI viruses, continued monitoring of the future outbreaks is vital to better understand the new dynamics between virus and its hosts.


Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae in sand fly samples collected from southern Thailand.The distribution of phlebotomine sand flies is changing rapidly due to climate change. This issue has implications for the epidemiology of sand fly-borne diseases, especially sand fly-associated viruses. Few studies concerning sand fly-associated viruses have been conducted in Thailand. Therefore, this study aimed to perform a molecular survey of groups of pathogenic RNA viruses belonging to the Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae molecular determination was performed by RT-PCR, and positive samples were identified by cloning and sequencing, cell culture inoculation, and phylogenetic analysis.Sand flies were collected at two locations in Trang and Songkhla provinces of southern Thailand, and individual sand fly samples were processed for species identification and virus detection. The Rhabdoviridae family virus presence in Phlebotomus papatasi . The findings demonstrated a new cluster of rhabdovirus that was closely related to Bactrocera dorsalis sigmavirus strain BDSV.abc5 and the lineages of insect-specific Rhabdoviridae. In addition, the Bayesian tree suggested that the common ancestor of this group was the dimarhabdovirus clade. It was assumed that the virus may have switched hosts during its evolution. However, the detection of Orbivirus, Phlebovirus, and Flavivirus genera using specific primers for RT-PCR was negative in the collected sand flies.The results presented in this study were based on the analysis of a total of 331 female sand flies. This molecular study revealed evidence of Rhabdoviridae in Thailand. This is the first data regarding the circulation of Rhabdoviridae in Ph. papatasi from Thailand. We found a new cluster of rhabdoviruses that was close to the new B. dorsalis sigmavirus. It is possible that there is a great deal of diversity in this family yet to be discovered, and a more extensive survey for new rhabdoviruses may uncover viruses from a wide diversity of host taxa and broaden our understanding of the relationships among the Rhabdoviridae.There is limited knowledge on the genetic diversity and ecology of  Leishmania parasites in Sergentomyia (Neophlebotomus) gammae, Se. khawi, and Se. (Parrotomyia) barraudi sand flies, and Trypanosoma parasites in Phlebotomus stantoni, Se. khawi, Se. indica, Se. anodontis, Ph. asperulus, and Ph. betisi species [Bunyaviridae, genus Phlebovirus) are sand fly fever Sicilian virus (SFSV), sand fly fever Naples virus (SFNV) and Toscana virus (TOSV), which are transmitted to humans by different species of sand flies, especially Phlebotomus species [Rhabdoviridae [Reoviridae [Flaviviridae [http://www.ictvonline.org/virusTaxonomy.asp, comprising Cytorhabdovirus, Dichorhavirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Sprivivirus, Tibrovirus, Tupavirus, Varicosavirus, and Vesiculovirus [Sigmavirus, Vesiculovirus, and Ephemerovirus), cytorhabdoviruses, nucleorhabdoviruses, and rhabdoviruses are carried by arthropods [Orbivirus genus in Idiophlebotomus spp., Phlebotomus papatasi, and Se. khawi from southern Thailand was closely related to Changuinola virus (CGLV) [Orbivirus, Phlebovirus, and Flavivirus and family Rhabdoviridae, in field-caught sand flies collected from the southern region of Thailand. The detection method used was reverse transcription polymerase chain reaction (RT-PCR) with the use of viral-specific primers to precisely target each virus and sequence analysis. This technique provides a rapid means for obtaining expansive results that facilitate the investigation of the phylogenetic relationships of the virus and its host. Moreover, identification of the host and virus associations remains a key challenge for virus research. The impacts of virus screening in sand flies from Thailand can offer an important contribution to the establishment of vector control programs for sand fly-associated viruses in the country and the region.Phlebotomine sand flies (Diptera: Psychodidae) are a medically important group of insects widely distributed throughout the world. They are responsible for causing several diseases in humans and other animals, including leishmaniasis, sand fly fever, and human bartonellosis –3. In so species , 5. Sand species , 7. The  species , 9. Moreoviridae , family oviridae , and famiviridae . Interesiviridae . In 2015ulovirus , 15. Simthropods . Despitethropods . The moss (CGLV) . HoweverCytB)-PCR [A total of 331 female sand flies were collected in Huai Yot District, Trang Province , and in Na Thawi District, Songkhla Province , Thailand, during the period 14–20 December 2020. The sand flies were collected using five to seven miniature Centers for Disease Control and Prevention (CDC) light traps, which were installed and hung from tree branches or hooks at a height of 1 m measuring from the trap hood to the ground. All traps were activated for 12 h from 6.00 p.m. to 6.00 a.m. Insects were collected from the light traps the following day. These sand flies were stored in 2-ml cryovials in liquid nitrogen and then transported to the laboratory for sorting and taxonomic identification. Individual female sand flies were placed on a sterilized slide and dissected under a stereomicroscope equipped with an ice block, using 26G  ×  1/2″ sterile needles. Head and genitalia were mounted with Hoyer’s medium on a slide, which was later used for morphological species identification based on keys proposed by Lewis , Phumee ytB)-PCR . The abdg for 5 min. Subsequently, 100 μl of supernatant with 300 μl of minimum essential medium (MEM) was used for virus culture. The pellet and some of the supernatant with lysis buffer were processed for viral RNA extraction using the Invisorb Spin Virus RNA Mini Kit  following the manufacturer’s instructions. The extracted RNA samples were immediately used for detection of virus families, and the remaining samples were stored at −80 °C. The RNA samples were amplified for the detection of the Orbivirus [Phlebovirus [Flavivirus [Rhabdoviridae [Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae were 188, 244, 270, and 460 base pairs (bp), respectively. A synthesized plasmid was used as a positive control to prevent sample contamination.All sand fly samples were ground in 200 μl of 1× PBS and then centrifuged at 11,000×rbivirus , Phlebovebovirus , and Flaavivirus  genera aoviridae . All theoviridae . PCR ampAedes albopictus C6/36 (ATCC CRL-1660) or rhesus monkey kidney epithelial (LLC-MK2) (ATCC CCL-7) cells for 1 h in 24-well plates grown in MEM  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 100 U/ml penicillin, and 100 μg/ml streptomycin . The cultures were incubated at 28 °C (C6/36) and 37 °C (LLC-MK2) in 5% CO2. Cytopathic effects (CPEs) were monitored and observed daily for the following 6–10 days. The positive isolates were continually propagated in C6/36 cells and LLC-MK2 cells grown in MEM supplemented with 10% heat-inactivated FBS  at 28 °C (C6/36) and 37 °C (LLC-MK2) in a 5% CO2 environment for additional passages. The culture supernatants were harvested for identification and sequencing. Uninfected C6/36 or LLC-MK2 cells were used as negative controls.One hundred microliters of each virus-positive suspension was inoculated onto confluent monolayers of PCR products were either directly sequenced or cloned into the pGEM-T Easy Vector , transformed into DH5α competent cells, and screened by a blue–white colony selection system. The suspected positive colonies were cultured and used for further plasmid DNA extraction using the Invisorb Spin Plasmid Mini kit  following the manufacturer’s instructions. Purified plasmids were sent for sequencing by 1st BASE DNA sequencing services  using universal forward T7 primers. Several independent PCR clones (3–5 clones) were analyzed to produce a consensus nucleotide sequence for each virus. Because of genetic variation within the degenerate primer sequences, they were excluded from the analysis. Nucleotide sequences were analyzed by comparing them with the reference viral sequence in the GenBank database using the BLASTn tool in the BLAST program. This tool searches nucleotide databases using percentage nucleotide queries and identity.https://mafft.cbrc.jp/alignment/software/) [http://iqtree.cibiv.univie.ac.at/) with 1000 ultrafast bootstrap replicates. The best-fit model of substitution was identified using the auto function on the IQ-TREE web server [http://tree.bio.ed.ac.uk/software/figtree/).The nucleotide sequences were aligned using the BioEdit version 7.2.5 sequence alignment editor  and MAFFftware/) . Phylogeb server . FinallyThis study is a descriptive analysis. The sample size was calculated following a simple formula  by estimPhlebotomus stantoni and Se. indica were the most abundant sand fly species found in Trang and Songkhla provinces, respectively. Idiophlebotomus spp., Ph. mascomai, and Ph. papatasi were found only in Trang province. All samples were screened for viruses: Orbivirus, Phlebovirus, and Flavivirus genera and family Rhabdoviridae. The Orbivirus, Phlebovirus, and Flavivirus genera RT-PCRs were negative in all sand fly samples (Table Rhabdoviridae RNA was detected in three (0.9%) Ph. papatasi samples. Viral isolation was not successful in cell culture inoculation of the positive samples. No cytopathic effect (CPE) was observed in three positive sand fly samples, and culture supernatants remained negative for the family Rhabdoviridae. Rhabdoviridae family RNA was detected in three samples of Ph. papatasi. Sequencing of the L-region amplicon revealed a sequence length of approximately 450 bp. Sequences of all three samples were deposited in GenBank (accession nos. OK205076–OK205078). We observed that these three sequences had similarity of 74.16% compared to Bactrocera dorsalis sigmavirus strain BDSV.abc5 (accession no. MN745080), which has been reported as a new sigmavirus in the family Rhabdoviridae from fruit flies. The intramural diversity of three sequences and other sigmaviruses showed 19.2–41.5% nucleotide similarity. The ML analysis using the sequences from the three positives of family Rhabdoviridae in this study revealed a separate clustering from B. dorsalis sigmavirus strain BDSV.abc5 (accession no. MN745080), B. tryoni rhabdovirus 1 strain d (accession no. MW208811), rhabdoviruses or dimarhabdoviruses supergroup; vesiculovirus, Le Dantec and Kern Canyon group, Almpiwar group, sigmavirus, Hart Park group, and ephemerovirus  sand flies collected in Trang and 169 (51.1%) sand flies collected in Songkhla. Rhabdoviridae RNA in three (0.9%) samples of Ph. papatasi sand flies from Trang Province. The Ph. papatasi was not found in Songkhla Province in this study. However, the results obtained did not reveal the circulation of the Orbivirus, Phlebovirus, or Flavivirus genera in field-caught sand flies. To the best of our knowledge, this is the first study undertaken to identify a new cluster of rhabdoviruses from Ph. papatasi in Thailand that was genetically closely related to B. dorsalis  sigmavirus strain BDSV.abc5. Rhabdoviruses, a family of negative-sense RNA viruses , are a large and ecologically diverse group of viruses [B. dorsalis sigmavirus strain BDSV.abc5 of the oriental fruit fly (accession no. MN745080) [B. tryoni rhabdovirus 1 strain d of a tephritid fruit fly (accession no. MW208811) [Rhabdoviridae [Sigmavirus genera infects a variety of fruit flies, including Drosophila melanogaster, D. immigrans, and D. ananassae [D. melanogaster (DMelSV) became paralyzed and died upon exposure to CO2, whereas uninfected flies recovered after exposure to CO2 [Culex mosquitoes [Drosophila [2 exposure. From the aforementioned discussion, it is postulated that rhabdoviruses may exist in common groups of insects and may serve as common pathogens in those insect populations. Our findings demonstrated the first use of molecular techniques to detect the family Rhabdoviridae in sand flies from southern Thailand. The results revealed the presence of a new cluster of rhabdoviruses, which was closely related to the new sigmavirus in Ph. papatasi. The phylogeny of these sequences was reconstructed using Bayesian analysis with amino acid sequences and models of protein evolution. Our phylogeny showed that new sigmaviruses in Ph. papatasi are in the same lineage of dimarhabdoviruses, which suggested that infecting arthropod viruses emerged from this clade and might have undergone host switching during its evolution rather than cospeciation with them. Previous studies have revealed that for a pathogen to infect a new host species, it must adapt and adjust to the new environment in which it settles [Ae. albopictus C6/36 (ATCC CRL-1660) and rhesus monkey kidney epithelial (LLC-MK2) (ATCC CCL-7) cells was not successful, and the RT-PCR performed with the infected cells was negative. This likely resulted from a combination of factors associated with the C6/36 and LLC-MK2. Indeed, C6/36 cells have been used extensively for the isolation of rhabdoviruses [Pipistrellus kuhlii) in Vero cells [Lutzomyia longipalpis) with vesicular stomatitis virus (VSV) from a sand fly could establish productive, long-lasting persistent infections. However, sustained persistence was difficult in C6/36 cells in the study [The study of pathogens in arthropods is an important part of surveillance programs. It can provide data about circulating pathogens and their vectors, assess potential public health threats, predict probable disease emergence in susceptible humans or animals, and establish optimal diagnostics and interventions to prevent disease transmission , 31. In  viruses . For theN745080)  and B. tW208811) . The viroviridae , 34. Signanassae . It was e to CO2 . In addie to CO2 . Severale to CO2 , Culex msquitoes , and Droosophila  reduced  settles –42. Addi settles , 44. Her settles  implied oviruses , but thero cells . Anotherhe study –50. ExteThis study reported a new cluster of rhabdoviruses closely related to the new sigmavirus in sand flies from southern Thailand. Additionally, the results also provide information on virus association with insect host adaptation. Although rhabdoviruses are already considered common pathogens in terms of insect-specific viruses, extensive surveys and comprehensive studies of new rhabdoviruses might be able to clarify and demonstrate the wide diversity of their host taxa as well as epidemiological data of this virus in sand flies. Moreover, further studies will be able to provide a better understanding of the relationships between rhabdoviruses and sand flies in Thailand.


Pembrolizumab monotherapy or in combination with chemotherapy are two new treatment options for patients with metastatic non-squamous non-small cell lung cancer (NSCLC) and high (≥ 50%) programmed death ligand 1 (PD-L1) expression. We conducted a cost-effectiveness analysis for Switzerland comparing these two options but also pembrolizumab to chemotherapy.We constructed a 3-state Markov model with a time horizon of 10 years. Parametric functions were fitted to Kaplan–Meier overall survival (OS) and progression-free survival (PFS) using 2-year follow-up data from the KN-024 and KN-189 registration trials. We included estimated costs for further treatment lines and costs for best supportive care. Costs were assessed from the Swiss healthcare payer perspective. We used published utility values.Combination therapy resulted in an expected gain of 0.17 quality-adjusted life years (QALYs) per patient and incremental costs of Swiss Francs (CHF) 81,085 as compared to pembrolizumab. These estimates led to an incremental cost-effectiveness ratio (ICER) of CHF 475,299/QALY. Pembrolizumab in comparison to chemotherapy was estimated to generate mean incremental QALYs of 0.83 and incremental costs of CHF 56,585, resulting in an ICER of CHF 68,580/QALY. Results were most sensitive to changes in costs of 1L pembrolizumab and combination therapy, together with changes in PFS. In the probabilistic sensitivity analysis, we estimated combination therapy was cost-effective in 4.9% of the simulations and pembrolizumab monotherapy in 82.9%, assuming a willingness-to-pay threshold of CHF 100,000 per QALY gained.Pembrolizumab is likely to be cost-effective from the Swiss healthcare payer perspective, whereas pembrolizumab plus chemotherapy is not.The online version contains supplementary material available at 10.1007/s10198-021-01282-4. Lung cancer is the second most common cancer and the most frequent cause of death among all cancers worldwide . Non-smaTreatment options for patients with metastatic NSCLC have evolved significantly over the last years with the implementation of molecular testing, targeted therapy, and immune-checkpoint inhibitors blocking the programmed cell death (ligand 1) (PD-L1/PD-1) pathway . Among tWe developed a 3-state Markov cohort simulation model with mutually exclusive health states of progression-free survival (PFS), progressive disease (PD) and death. The model was programmed in TreeAge software (Version 2019 R1.1) . In TreeAge, second-line (2L) treatment was implemented with the help of tunnels.We chose a model cycle length of 1 month applying half-cycle correction and a time horizon of 10 years based on the poor prognosis of stage IV NSCLC patients and the lack of more extended follow-up data for immunotherapy. An annual discount rate of 3% for costs and quality-adjusted life-years (QALYs) was applied.Incremental cost-effectiveness ratio (ICER) of the pembrolizumab combination strategy versus pembrolizumab monotherapy and of pembrolizumab monotherapy versus chemotherapy were assessed in Switzerland, expressed as costs [in Swiss Franc (CHF)] per QALY gained.We modelled a patient population with the characteristics of the KN-024 and KN-189 registration trials comprising of adult patients with previously untreated stage IV, mainly non-squamous NSCLC , without EGFR or ALK alterations. In KN-024, only patients with PD-L1 expression ≥ 50% had been recruited [In line with the interventions of the aforementioned trials, we compared three treatment strategies: combination strategy  or BSC directly), pembrolizumab strategy , and chemotherapy strategy  .Following discontinuation of 1L treatment, the percentage of patients as reported in each trial were assumed 2L treatment or BSC directly. Patients receiving 2L treatment were simulated to receive docetaxel (after 1L combination) for 4 cycles (= 2.76 months), platinum-based chemotherapy  for 4 cycles (after 1L pembrolizumab), followed by pemetrexed maintenance therapy for up to 35 cycles or until disease progression (only for patients on 2L pemetrexed), or 2L pembrolizumab until progression (after 1L chemotherapy). Patients were simulated to progress after either 6 cycles (4.2 months for 2L chemotherapy) or after 5.2 months (for 2L pembrolizumab) and to receive BSC, based on PFS results from 2L treatment studies  (supplemThe Kaplan–Meier (KM) curves from 2-year follow-up data of the KN-024  trials wThe model considered the occurrence of all reported grade 3 to 4 adverse events (AEs) from KN-024  and KN-1We used published utilities (QALYs) for PFS under 1L treatment as provided by Huang , and utiWe assessed costs from a Swiss healthcare payer perspective, where all direct medical costs were considered. Costs included regimen costs (drug costs), application costs (e.g. intravenous administration), diagnostic costs (e.g. computer tomography (CT) scans), costs for AEs of 1L treatment, and costs for other consumables and clinical visits. We detailed treatment costs during PFS and PD in supplementary Methods section S4 and supplementary Table S3.To investigate parameter and structural uncertainty, we performed one-way and probabilistic sensitivity analyses, as well as several scenario analyses. For the sensitivity analyses, we varied 33 individual parameters  and presented the 15 most influential parameters in tornado diagrams. More details about the sensitivity analyses are given in supplementary section S5.In a first scenario analysis, we assumed lower pemetrexed costs  based on the price of the cheapest available generic product in the United Kingdom (UK) but not available in Switzerland. We used the price specified by the British National Formulary and converted it into Swiss Francs . In a fuScenario analysis six investigated an unlimited pemetrexed maintenance until progression or death, lifting the restriction of maximum 35 cycles. Rather than assuming treatment termination with disease progression, scenario analysis seven used the time-on-treatment (ToT) curves of Insinga et al.  for the Over 10 years, combination therapy relative to pembrolizumab monotherapy was projected to result, after discounting, in an expected gain of 0.17 QALYs per patient and incremental costs of CHF 81,085, resulting in an ICER of CHF 475,299 per QALY gained. Discounted incremental life years (LYs) of 0.29 resulted in incremental costs per LYs gained of CHF 277,600. A comprehensive list of all base-case results is provided in Table Pembrolizumab monotherapy compared to chemotherapy was projected to generate mean incremental QALYs of 0.83 per patient and mean incremental costs of CHF 56,585, resulting in an ICER of CHF 68,580 per QALY gained. Mean total costs of CHF 155,379 for the pembrolizumab monotherapy strategy were largely comprised of 1L pembrolizumab treatment costs . For the chemotherapy strategy, the mean total costs of CHF 98,794 were mostly driven by BSC costs , followed by 1L chemotherapy costs .Table When patients under 1L chemotherapy treatment were assumed not to be able to switch to 2L pembrolizumab, an ICER of CHF 76,126 per QALY gained was estimated for the comparison of pembrolizumab monotherapy versus chemotherapy. Using a shorter time horizon of 5 years resulted in higher estimated ICERs.As expected, unlimited pemetrexed maintenance increased costs particularly for chemotherapy and combination strategies leading to an ICER of CHF 813,101 for the comparison of the combination and pembrolizumab groups. The final conclusions of the comparisons were however not affected. Use of ToT curves to approximate drug and administration costs only slightly increased the ICER for the comparison pemetrexed to chemotherapy. However, the ICER of the comparison of the combination strategy to pembrolizumab is approximately halved. Using a lognormal distribution to approximate and extrapolate the OS KM curves, resulted in an increased ICER of CHF 709,301 per QALY gained for the combination versus pembrolizumab strategy, and a similar ICER of CHF 58,899 per QALY gained for the pembrolizumab versus chemotherapy strategy. Again, the final conclusions of the comparisons were not affected.The tornado graph in Fig. For the comparison of the pembrolizumab strategy relative to the chemotherapy strategy Fig. , variatiIn probabilistic sensitivity analyses, we estimated pembrolizumab to be cost-effective in 82.9% of the simulations and the combination therapy in 4.9% of the simulations, assuming a willingness-to-pay threshold (WTP) threshold of CHF 100,000 per QALY gained .Over a 10-year time horizon, we estimated mean improvements of 0.34 LYs (undiscounted) and 0.17 QALYs (discounted) per patient, together with an increase in mean costs of CHF 81,085, when comparing the combination strategy from KN-189  to the pembrolizumab monotherapy strategy from KN-024. The resulting ICER was CHF 475,299 per QALY gained. This suggests that combination therapy is not cost-effective in Switzerland if a threshold of CHF 100,000 per QALY gained is assumed. Results were robust to changes in the majority of the input parameters but were sensitive to changes in parameter estimates of the modelled PFS curves as well as in costs of the combination and the pembrolizumab therapies. High costs of 1L and 2L chemotherapy were partially driven by high Swiss costs of commercial pemetrexed. Currently, there is no pemetrexed generic approved in Switzerland. In the US, Insinga et al. [The cost-effectiveness of 1L pembrolizumab strategy relative to the chemotherapy strategy was CHF 68,580 per QALY gained, suggesting that pembrolizumab monotherapy is cost-effective in Switzerland. Over a 10-year time horizon, we estimated mean improvements of 1.14 LYs (undiscounted) and 0.83 QALYs (discounted) per patient, together with an increase in mean costs of CHF 56,585. Variation in the cost of 1L pembrolizumab had a substantial influence on the ICER. Assuming it was not possible for patients previously receiving 1L chemotherapy to switch to 2L pembrolizumab after disease progression, the estimated ICER increased to CHF 76,126 per QALY gained, but would still be cost-effective. The findings for the pembrolizumab versus chemotherapy comparison are in line with previous CEA results from the partitioned survival model by Huang et al. for the US  and its One major strength of our analysis is the use of recently published 2-year survival data of the KN-024 , and theA further strength is that we also used published utilities for progression-free disease under 1L treatment , directlThere are some limitations to our analysis. Due to the independent modelling of survival curves, we could not directly vary the treatment effect in sensitivity analyses. Survival curves with the best fit were selected based on the AIC and BIC criteria in relation to the 2-year KM-curves and also based on visual inspection of the longer-term fit (10 years), assuming the majority of the patients being dead after 10 years. Extrapolations beyond the 2-year clinical effectiveness data may need adaptation when longer-term follow-up data become available. Recent evidence , 23 indiA further limitation is that a proportion of KN-024 patients had squamous NSCLC (18.4%). We were unable to exclude these patients from our analysis, as published PFS and OS results were not stratified by squamous versus non-squamous histology. Assumptions on 2L treatments and their duration were sourced from information provided in the KN-024 and KN-189 trials. In case of missing or unclear information these had to be complemented with own clinical assumptions. We only included cost of AEs due to 1L treatment and assumed that costs of AEs due to 2L treatment could be neglected.In conclusion, Pembrolizumab monotherapy is likely to be cost-effective from the Swiss healthcare payer perspective, whereas pembrolizumab plus chemotherapy is not. INSIGNA (NCT03793179), and further trials comparing immunotherapy with combination immuno-chemotherapy, will allow validation of these findings, and in-depth analysis of NSCLC subsets characterized by PD-L1 in the near future.Supplementary file1 (DOCX 522 KB)Below is the link to the electronic supplementary material.