The host’s immune status may affect virus evolution. Little is known about how developing fetal and placental immune milieus affect virus heterogeneity. This knowledge will help us better understand intra-host virus evolution and how new virus variants emerge. The goal of our study was to find out whether the isolated in utero environment—an environment with specialized placental immunity and developing fetal immunity—supports the emergence of RNA and DNA virus variants. We used well-established porcine models for isolated Zika virus (RNA virus) and porcine circovirus 2 (DNA virus) fetal infections. We found that the isolated in utero environment was conducive to the emergence of RNA and DNA virus variants. Next-generation sequencing of nearly whole virus genomes and validated bioinformatics pipelines identified both unique and convergent single nucleotide variations in virus genomes isolated from different fetuses. Zika virus and PCV2 in utero evolution also resulted in single nucleotide variations previously reported in the human and porcine field samples. These findings should encourage further studies on virus evolution in placenta and fetuses, to better understand how virus variants emerge and how in utero viral evolution affects congenital virus transmission and pathogenicity. The emerging concept is that pregnancy-modulated maternal immunity is favorable for virus evolution towards a more pathogenic infection phenotype, i.e., a failure to mount an efficient antiviral response in pregnant mice is associated with the emergence of influenza variants with increased pathogenicity . Along wLimitations imposed by the sampling of human tissues restrict comprehensive studies on virus evolution in fetuses. Thus, viruses that cause in utero infections in animals may serve as valuable models to study the complexity of viral evolution in fetuses. However, only a few research efforts on in utero viral evolution in the natural animal host are reported ,3,4. TheViral evolution studies in fetuses may increase our fundamental understanding of intra-host virus heterogeneity and virus variant emergence. Towards this goal, we designed a focused study aiming to understand whether an isolated in utero environment—the environment with the developing fetal and placental immunity—is conducive to the emergence of RNA and DNA virus variants. We used well-established porcine models for isolated in utero ZIKV and porcine circovirus 2 (PCV2) infections. Afterward, using the NGS of nearly whole-virus genomes and validated bioinformatics pipelines, we profiled the in utero heterogeneity of ZIKV and PCV2 genomes.® 3000 (Thermo Fisher Scientific), and incubated for 7 days at +28 °C. After two passages in C6/36 cells, cell culture media containing ZIKV was centrifuged  and frozen (−80 °C). Viral titers were quantified in triplicates in VERO cells  with an endpoint dilution assay, as described below.To reduce virus genomic heterogeneity and reproduce a hypothetical scenario wherein founder viruses are transmitted via the placental barrier , we gene® 3000 . To generate sufficient titers for the in vivo infection study, supernatants were passaged three times in VR1BL and three times in Dulac cells [g, 20 min, +4 °C) and frozen (−80 °C). Viral titers were quantified in Dulac cells with the endpoint dilution assay as described below.For the DNA virus, the whole genome of the PCV2 strain BaPCV2b (GenBank: FJ233905.1) was de novo synthesized (GenScript). The circular BaPCV2b genome has a single SacII restriction site at 491–496 nt; the DNA was synthesized starting from 495 nt with CGGC and GG added to the 5’ and 3´ fragment ends to generate two SacII flanking restriction sites (File S1). The extended DNA fragment was inserted into the pUC57 plasmid at the multiple cloning site (EcoRV). The plasmid was amplified with the isothermal rolling-circle method using the TempliPhi 100 Amplification kit  and digested with SacII restriction enzyme ; the 1767 bp band was selected with preparative gel electrophoresis, purified with an Invitrogen PureLink Quick Gel Extraction Kit , circularized with T4 DNA Ligase , and transfected into VR1BL cells [ac cells ; cell cuThe absence of mycoplasma contamination was confirmed in all virus stocks and cell cultures with the LookOut Mycoplasma PCR Detection Kit .5 TCID50 of ZIKV or intraperitoneally + intra-amniotic (200 μL + 500 μL) with 103.9 TCID50 of PCV2. Conceptuses were labeled with non-absorbable surgical sutures on the adjacent uterine walls to directly identify inoculated fetuses during sampling. Pigs were sampled 28 days after ZIKV and PCV2 in utero inoculation [g, 20 min, +4 °C), plasma was aliquoted and frozen (−80 °C). Fetuses were visually examined, and gross pathology was recorded. Fetal organs were sampled based on known ZIKV [We followed the Canadian Council on Animal Care guidelines . All efforts were made to minimize animal suffering. Pigs were humanely euthanized with Euthanyl overdose followed by exsanguination. We used well-established porcine models for isolated ZIKV ,12,13,14own ZIKV ,18 tissu50) per ml. Media from mock-inoculated cells were used as negative controls.We used the endpoint dilution assay to quantify infectious titers in the ZIKV stock ,12,13,14Despite the presence of infectious virus in amniotic fluids, as well as persistent infection and molecular pathology in fetal organs, the isolation of infectious ZIKV from tissues of immunocompetent animals is challenging, and the reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay is a generally used approach to determine in vivo viral loads . To conf50 per ml or gram.The endpoint dilution assay is a standard method for quantifying infectious PCV2 titers in stock and confirming in utero infection ,18. TissWe used qPCR to quantify the PCV2 genomic DNA copies in the virus stock, body fluids, and tissues for NGS library construction. We extracted DNA with the PureLink DNA/RNA Mini Kit  from fetal blood plasma, heart, spleen, placenta, and brain, according to the manufacturer’s instructions. Before DNA extraction, the tissue samples were weighed on an analytical balance. Tissues were homogenized using RNase-free stainless steel beads and TissueLyser II operated for 5 min at 25 Hz. We used PCV2-specific SYBR green-based one-step qPCR 13 with a reaction mixture (50 μL) consisting of 25 μL 2× SensiFAST SYBR Lo-ROX Kit Mix , 2 μL 10 µM) of each primer , 19 μL nuclease-free water and 2 μL DNA template. The amplification of PCV2 DNA was performed via 35 cycles of denaturing at +95 °C for 15 s, annealing at +65 °C for 15 s, and extension at +72 °C for 10 s. DNA from a stock of BaPCV2b was used to generate a standard curve  with a wide dynamic range, with the high linear correlation (R2 = 0.9984) between the cycle threshold (Ct) value and template concentration. The slope of the standard curve (−3.358) corresponded to the 98.52% reaction efficiency level. The PCR values were corrected for fluid volumes or tissue weights and after log transformation expressed as PCV2 DNA genome copies per ml or gram. Kit reagent controls were included in every DNA isolation and PCR run. In all endpoint dilution assays and PCR tests, we included samples from mock-inoculated and non-manipulated animals from our previous studies  µM of ea,12,13,14® II Reverse Transcriptase  and Random Hexamers . As previously validated for accurate iSNV calling, we used more than 1000 viral RNA copies for cDNA synthesis [® Hot Start High-Fidelity 2× Master Mix . An enzyme activation step of 30 s at +95 °C was followed by 35 amplification cycles for 15 s at +95 °C and 5 min at +65 °C. The correct size of amplicons in each reaction was verified by 1% agarose gel electrophoresis.RNA and DNA from virus stocks and placental and fetal samples were used to construct ZIKV and PCV2 whole-genome NGS libraries. We used the PrimalSeq protocol, an approach wherein a nearly whole virus genome is amplified in the ~400–480 bp overlapping fragments with multiplexed PCR reactions and two technical replicates for each multiplex reaction . The ampLibrary construction and Illumina sequencing were performed as previously described with some modifications . Virus a® Hot Start High-Fidelity 2× Master Mix (12.5 μL reactions). Each individual reaction was performed in two technical duplicates. An enzyme activation step of 30 s at +98 °C was followed by 35 amplification cycles for 7 s at +98 °C, 25 s at +68 °C, and 30 s at +72 °C. The correct sizes of the amplicons in each reaction were verified by 1% agarose gel electrophoresis. Amplicons from the six individual PCR reactions  were combined, bead-purified, and used for library construction and sequencing, as described above for ZIKV.The multiplex primer design tool Primal Scheme has previously been used to sequence RNA viruses—ZIKV, yellow fever virus and West Nile virus . Here, wFor NGS data processing and variant calling, we used an open-source software package iVar  to process virus sequencing data and call single nucleotide variations (SNVs) from technical replicates . To idenAs per the previously described and validated best practices , we analThe percentage of SNVs in virus sequences from each sample was calculated by dividing the number of SNV sites by the total number of sequenced nucleotides; the mean percentage from two NGS technical replicates was represented. Mean SNV frequencies were also calculated from NGS technical replicates. To quantify the degree of convergent evolution in ZIKV or PCV2 genomes, any iSNV that emerged in more than one fetus/fetal organ was defined as convergent, and the total convergence was quantified as the percentage of convergent iSNVs from the total number of unique iSNVs.We used GraphPad PRISM 8. Data are expressed as individual and arithmetic mean values.We tested whether the in utero environment is conducive to the emergence of new RNA and DNA virus variants. As a model for RNA viruses, we used ZIKV; as a model for DNA viruses, we used PCV2. The experimental design is shown in Fetuses were injected with ZIKV to induce isolated in utero infection. The ultrasound-guided protocol ,12,13,14We extracted RNA from the initial ZIKV stock that was used for fetal injection, placental tissues, and fetal lymph node tissues . The extThe initial ZIKV stock and placental samples had correct DNA bands after amplification with multiplex PCR primers, 86.6–98.2% of ZIKV genome coverage, and high  average sequencing depth . Out of We compared sequences from experimental samples with the parental H/PF/2013 strain sequence (GenBank: KJ776791.2) to identify ZIKV SNVs. We used iVar for NGS processing and variant calling with a sequence depth of at least 400×, a frequency of at least 3%, and variants that were detected in both technical replicates [All seven SNVs in the ZIKV stock were most probably introduced during virus rescue with ISA and in vitro virus adaptation to the cell line. While all seven stock-specific SNVs had high frequencies from 17% to 40%, the in utero environment selected against stock-specific SNVs because they were not identified in placental or lymph node samples . To idenPlacental and lymph node samples showed unique patterns of ZIKV iSNVs A; howeveWe also compared iSNVs to ZIKV sequences available in public databases. Fifteen iSNVs were recently (2014–2020) reported in Asian ZIKV strain sequences from Brazil, Colombia, Dominican Republic, French Polynesia, Honduras, India, Mexico, New Caledonia, Nicaragua, Puerto Rico, Singapore, Suriname, Thailand, and the USA . Twelve To study whether the in utero environment is conducive to the emergence of new DNA virus variants, we injected five fetuses with PCV2—the virus with the smallest known DNA genome that causes natural infection in pig fetuses. We confirmed high levels of PCV2-specific antibodies in maternal blood before breeding and before in utero inoculation; antibodies prevent maternal infection, thus providing a model for isolated in utero infection. Fetal and placental tissues were sampled 28 days after injection. In utero PCV2 infection caused typical clinical outcomes ,16—a mixWe used DNA extracted from virus stock and from blood plasma, heart, spleen, brain, and placenta tissues from four injected fetuses . Porcine circovirus 2 showed high stability in vitro: SNVs were not identified in the initial virus stock, even after six passages in cell cultures. However, even with high in vitro stability and conservative iVar parameters, de novo-emerging PCV2 iSNVs were identified in six tissues derived from four fetuses . The perTo further confirm that tissue-specific iSNVs are not virus stock-derived artifacts, we called variants in the PCV2 stock sequences with a low (0.01%) frequency threshold and compared low-frequency stock-specific SNVs to iSNVs. One iSNV out of six was not identified in the PCV2 stock sequences, even with the very low 0.01% threshold . Three tWe also compared tissue-specific iSNVs to the PCV2 sequences available in public databases. Four PCV2 iSNVs were previously reported in sequences from pig farms in China, Europe, India, Indonesia, and the USA . AltogetWe designed this study to understand whether the isolated in utero environment—a fetus with fetal placental mesenchyme—affects the evolution of RNA (ZIKV) and DNA (PCV2) viruses. The key finding is that the isolated in utero environment is conducive to the emergence of RNA and DNA virus variants. The emergence of these variants can result from the de novo development of mutations, and from the selection of and increase in initial low-frequency variants.To induce isolated infection in the fetus with fetal placental membranes, we used ISA-derived ZIKV and precise ultrasound-guided in utero inoculation. In utero inoculation did not cause maternal infection because maternal tissues were negative for ZIKV, and the blood did not have ZIKV-specific antibodies. Thus, in our fetal pig model, isolated in utero virus evolution can be analyzed in the context of specific fetal immune responses. Additionally, the fetal pig model reproduces in utero ZIKV infection in humans well; e.g., the virus persists in the fetal placental mesenchyme for at least 90 days . The fetEach placental sample had a unique pattern of ZIKV iSNVs A, suggesInterestingly, a recent study showed ZIKV circulation in mosquitoes from pig farms in Mexico, as determined by PCR, and natural ZIKV infection in farm pigs, as determined by serological tests . Thus, iZika virus iSNVs identified in our study were previously reported in human and mosquito samples collected between 2014 and 2020 . Thus, iPorcine circovirus 2 is a ubiquitous pig pathogen that naturally causes infection in adults and fetuses. In our PCV2 model, the virus is injected directly into fetuses and does not cause maternal infection, because most adult pigs have immunity against PCV2 that is acquired naturally during the life span or after vaccination ,16. In tWe used PCV2 with a low mutant spectrum complexity and precise ultrasound-guided in utero inoculation to induce isolated infection in fetuses. For replication, PCV2 uses the host’s polymerase with proofreading activity, but despite proofreading the polymerase-guided replication, we identified unique PCV2 iSNVs as well as convergent iSNVs in different fetuses . In contPorcine and bovine offspring exposed during the fetal period to congenital viruses—classical swine fever virus, Japanese encephalitis virus, and bovine viral diarrhea virus—may shed virus into the environment, and develop disease ,37,38,39Collectively, we have shown that the isolated in utero environment is conducive to the emergence of RNA and DNA virus variants. These findings should encourage further studies on virus evolution in fetuses and placenta, in order to better understand how congenital virus variants emerge and spread. Additional studies are also urged to identify whether in utero-emerging virus variants may pose more severe transmission and disease development risks. Understanding how in utero evolution affects the transmission and pathogenicity of new virus variants may promote scientific and public health efforts to develop monitoring strategies and preventive interventions, including efficient vaccines that will be safe for pregnant individuals.


Rhabdoviruses infect a large number of plant species and cause significant crop diseases. They have a negative-sense, single-stranded unsegmented or bisegmented RNA genome. The number of plant-associated rhabdovirid sequences has grown in the last few years in concert with the extensive use of high-throughput sequencing platforms. Here, we report the discovery of 27 novel rhabdovirus genomes associated with 25 different host plant species and one insect, which were hidden in public databases. These viral sequences were identified through homology searches in more than 3000 plant and insect transcriptomes from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) using known plant rhabdovirus sequences as the query. The identification, assembly and curation of raw SRA reads resulted in sixteen viral genome sequences with full-length coding regions and ten partial genomes. Highlights of the obtained sequences include viruses with unique and novel genome organizations among known plant rhabdoviruses. Phylogenetic analysis showed that thirteen of the novel viruses were related to cytorhabdoviruses, one to alphanucleorhabdoviruses, five to betanucleorhabdoviruses, one to dichorhaviruses and seven to varicosaviruses. These findings resulted in the most complete phylogeny of plant rhabdoviruses to date and shed new light on the phylogenetic relationships and evolutionary landscape of this group of plant viruses. Furthermore, this study provided additional evidence for the complexity and diversity of plant rhabdovirus genomes and demonstrated that analyzing SRA public data provides an invaluable tool to accelerate virus discovery, gain evolutionary insights and refine virus taxonomy. Over 16,000 petabases  database hosted at NCBI and identified 27 novel plant rhabdoviruses from 25 plant and one insect species, showing structural, functional and evolutionary cues to be classified in the family 5), shortlisted as likely corresponding to novel virus transcripts, which were then further analyzed. Given the redundant nature of many retrieved hits, a step of contig clustering was implemented using the CD-hit suite with the standard parameters available at http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit (accessed on 10 March 2021). In addition, the raw sequence data corresponding to several SRA experiments associated with different NCBI Bioprojects (The detection of plant rhabdovirus sequences was done as described by Longdon and colleagues . Brieflyprojects  were rethttp://www.usadellab.org/cms/?page=trimmomatic (accessed on 15 March 2021), and the resulting reads were assembled de novo with Trinity v2.6.6 using the standard parameters. The transcripts obtained from de novo transcriptome assembly were subjected to bulk local BLASTX searches  against a Refseq virus protein database available at ftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral/viral.1.protein.faa.gz (accessed on 3 March 2021). The resulting viral sequence hits of each bioproject were visually explored. Tentative virus contigs were extended by iterative mapping of each SRA library’s raw reads. This strategy employed BLAST/nhmmer to extract a subset of reads related to the query contig, and these retrieved reads were used to extend the contig, and then, the process was repeated iteratively using as the query the extended sequence. The extended and polished transcripts, now presenting overlapping regions, were reassembled using the Geneious v8.1.9  alignment tool.The nucleotide (nt) raw sequence reads from each analyzed SRA experiment linked to the TSA projects returning rhabdovirus-like hits were downloaded and preprocessed by trimming and filtering with the Trimmomatic tool, as implemented in https://www.ncbi.nlm.nih.gov/orffinder/) (accessed on 19 March 2021), functional domains, and the architecture of the translated gene products was determined using InterPro (https://www.ebi.ac.uk/interpro/search/sequence-search) (accessed on 19 March 2021) and the NCBI Conserved domain database v3.16 (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (accessed on 19 March 2021). Further, HHPred and HHBlits as implemented in https://toolkit.tuebingen.mpg.de/#/tools/ (accessed on 19 March 2021) were used to complement the annotation of divergent predicted proteins by hidden Markov models. Importin-α-dependent nuclear localization signals were predicted using cNLS Mapper available at http://nls-mapper.iab.keio.ac.jp/ (accessed on 23 March 2021), nuclear export signals were predicted using NetNES 1.1 available at www.cbs.dtu.dk/services/NetNES/ (accessed on 19 March 2021) and transmembrane domains were predicted using the TMHMM version 2.0 tool (http://www.cbs.dtu.dk/services/TMHMM/) (accessed on 23 March 2021).ORFs were predicted with ORFfinder (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ (accessed on 23 March 2021).Percentage amino acid (aa) sequence identities of the predicted ORFs of each plant-associated rhabdovirid identified in this study based on the available plant-associated rhabdovirus genome sequences were calculated using https://mafft.cbrc.jp/alignment/software (accessed on 29 March 2021), with multiple aa sequence alignments using FFT-NS-i as the best-fit model. The aligned aa sequences were used as the input in MegaX software [Phylogenetic analysis based on the predicted nucleocapsid proteins of all plant rhabdovirids, listed in software  to generAlphanucleorhabdovirus, Betanucleorhabdovirus, Cytorhabdovirus, Dichoravirus and Varicosavirus. Most of the tentative plant hosts of the novel viruses are herbaceous dicots (16/25), seven are herbaceous monocots, one a woody dicot and one a gymnosperm , with the genome organization 3′-N-P–P3-M-G-L-5′ , Plectranthus aromaticus virus 1 (PleArV1) and Rhododendron delavayi virus 1 (RhoDeV1), as well as the partial genomes of Cuscuta reflexa virus 1 (CusReV1) and Persicaria minor virus 1 (PerMiV1), were assembled in this study , bird’s-foot trefoil associated virus 1 (BFTaV1), black currant-associated rhabdovirus (BCaRV), cardamom vein clearing virus 1 (CdVCV1), datura yellow vein virus (DYVV), green Sichuan pepper nucleorhabdovirus (GSPNuV), sonchus yellow net virus (SYNV) and sowthistle yellow vein virus (SYVV) , apple rootstock virus A (ApRVA), s (SYVV) .Pairwise aa sequence identity values between each encoded protein of the five novel viruses and those from other betanucleorhabdoviruses vary significantly . The higThe phylogenetic analysis based on the deduced N protein aa sequences showed that CusReV1, RhoDeV1, PerMiV1 and PleArV1 clustered with the betanucleorhabdoviruses BFTaV1, BCaRV, BmV2, CdVCV1, DYVV, GSPNuV, SYNV and SYVV , which aAnthurium amnicola virus 1 (AntAmV1), Asclepias syriaca virus 1 (AscSyV1), Bemisia tabaci-associated virus 1 (BeTaV1), Glehnia littoralis virus 1 (GlLV1), Gymnadenia densiflora virus 1 (GymDenV1), Nymphaea alba virus 1 (NymAV1), Tagetes erecta virus 1 (TaEV1) and Trachyspermum ammi virus 1 (TrAV1), as well as the partial genomes of five other cytorhabdoviruses, named Allium chinense virus 1 (AChV1), Dioscorea composite virus 1 (DiCoV1), Lotus corniculatus virus 1 (LotCorV1), Pelargonium radula virus 1 (PelRaV1) and Suaeda salsa virus 1 (SuSV1), were assembled in this study , strawberry virus 1 (StrV1) and tomato yellow mottle-associated virus (TYMaV), with whom they share a similar genomic organization. AChV1 and SuSV1 formed a clade with alfalfa dwarf virus (ADV), chrysanthemum yellow dwarf-associated virus (ChYDaV), raspberry vein chlorosis virus (RVCV) and strawberry crinkle virus (SCV). AscSyV1, LotCorV1 and PelRaV1 clustered with Actinidia cytorhabdovirus (AcCV), Bacopa monnieri virus 1 (BmV1) and Wuhan insect virus 4 (WhIV4), and all these viruses have a similar genomic organization. BeTaV1 clustered with cucurbit cytorhabdovirus 1 (CuCV1), and these viruses share a similar genomic organization. AntAmV1 clustered with paper mulberry mosaic-associated virus (PMuMaV), with which it shares a similar genomic organization. TaEV1 clustered with the cluster formed by AntAmV1 and PMuMaV, but these viruses have a dissimilar genomic organization. DiCoV1 clustered with Colocasia bobone disease-associated virus (CBDaV), but these viruses have a dissimilar genomic organization, while GymDenV1 and TrAV1 formed a monophyletic sister clade with the aphid-transmitted cytorhabdoviruses , oviruses .Viola verecunda virus 1 (VVeV1) were identified in this study. We assembled three nonoverlapping fragments corresponding to RNA 1 containing the complete N, P3 and G genes and the near-complete RNA 2, which encodes the L protein , Brassica rapa virus 1 (BrRV1), Lolium perenne virus 1 (LoPV1), Melampyrum roseum virus 1 (MelRoV1) and Pinus flexilis virus 1 (PiFleV1), as well as the partial genomes of two others, named Phlox pilosa virus 1 (PhPiV1) and spinach virus 1 (SpV1), were assembled in this study , lettuce big-vein associated virus  and vitis varicosavirus (VVV). The gene junction sequences of BrRV1 and PiFleV1 differ slightly: the BrRV1 5′-end sequence is CUCA instead of CUCU, while the PiFleV1 3′-end sequence is GU(N)5UUUUU instead of AU(N)5UUUUU (The consensus gene junction sequences 3′ AU(N)N)5UUUUU .The pairwise aa sequence identities between the cognate-encoded proteins of AAnV1, BrRV1, LoPV1, MelRoV1, PhPiV1, PiFleV1 and SpV1 and those from other varicosaviruses vary significantly . The phyIn the last few years, several novel plant rhabdoviruses, which may be asymptomatic, have been reported in HTS studies ,53,54,55The coding-complete or complete genomic sequences of 66 plant rhabdoviruses were reported by early 2021. Thirty-three of them are cytorhabdoviruses, thirteen alphanucleorhabdoviruses, ten betanucleorhabdoviruses, five dichorhaviruses, four varicosaviruses and one gammanucleorhabdovirus. Similarly, half of the novel sequences reported in this study are those of putative cytorhabdoviruses, indicating the extensive diversification of this group of viruses. The novel 27 viruses discovered in this study all appear to be members of new species and account for nearly half of the plant rhabdovirus species reported so far. In addition, five new putative betanucleorhabdoviruses shed more light on the diversity and evolution of this recently created genus. One newly identified putative alphanucleorhabdovirus and one novel putative dichorhavirus expand the diversity of their respective genera. Finally, we detected seven novel varicosa-like viruses, which doubles the number of known varicosaviruses, providing new information about the genomic diversity and evolution of these little-studied fungi-transmitted viruses. Thus, our study provides the most complete insight to date about the genomic diversity and evolution of plant rhabdoviruses, complementing the status quo of these viruses with additional data on genomic organization and highlighting their apparent genetic flexibility. Overall, the sequence identity between the newly discovered viruses and those plant rhabdoviruses already described was low, a common feature among these taxa characterized by a high level of diversity in both genome sequence and organization [Alphanucleorhabdovirus, Betanucleorhabdovirus and Cytorhabdovirus have a nucleotide sequence identity lower than 75% in the complete genome sequence and occupy different ecological niches as evidenced by differences in the host species and/or arthropod vectors  (accessed 11 April 2021). Cytorhabdoviruses may also have an aa sequence identity of less than 80% in all cognate ORFs. Viruses assigned to different species in the genus Dichorhavirus have less than 80% nucleotide sequence identity in the L gene. Based on these species demarcation criteria, ATV1 should be classified in a new Alphanucleorhabdovirus species and AscSyV2, PleArV1 and RhoDeV1 in a new species in the genus Betanucleorhabdovirus; due to incomplete sequences, PerMiV1 and CusReV1 cannot be classified based on these criteria. AntAmnV1, AscSyV1, BeTaV1, GlLV1, GymDenV1, LotCorV1, NymAV1, TaEV and TrAV1 should be classified in a new species in the genus Cytorhabdovirus, whereas AChV1, DiCoV1, PelRaV1 and SuSV1, due to incomplete genome sequence data, cannot be classified. The dichorha-like VVeV1 also awaits at least coding-complete sequence data before it can be formally classified in this genus. The species demarcation criteria recently proposed for the genus Varicosavirus by the ICTV Rhabdoviridae Study Group included a minimum nucleotide sequence divergence of 50% in the L protein. Based on this genetic distance, the varicosa-like viruses AAnV1, BrRV1, LoPV1, MelRoV1, PiFleV1 and SpV1 would not be considered different species, and PhPiV1 cannot be classified due to incomplete sequencing data. Considering the diverse host plant species these viruses were identified in and their phylogenetic relationships, these viruses should likely be classified in different species. Thus, we propose a nucleotide sequence identity of 75% across the genome and in the N gene as thresholds for species demarcation in the Varicosavirus genus, in line with the species demarcation criteria for the other genera of plant rhabdoviruses.Viruses assigned to different species within the genera Pinus flexilis virus 1, was associated with a gymnosperm host. Few viruses were identified previously from gymnosperms [Sciadopitys verticillata and proposed to belong to a varicosavirus [Most of the plant hosts in which the novel viruses of this study were identified are dicots; nevertheless, AChV1, which was detected in Chinese onions, is likely the first monocot-infecting cytorhabdovirus that belongs to a clade of aphid-transmitted viruses. Furthermore, most of the source hosts in which the novel viruses were identified are herbaceous plants, which, overall, are the most common hosts of plant rhabdoviruses . Howevernosperms ,57,58,59osavirus . TherefoInterestingly, the genome sequence of four novel viruses discovered in this study, GymDenV1 (only four predicted genes), TaEV and TrAV1 (both with only five predicted genes) and PiFleV1 (an unsegmented varicosa-like virus), have a unique genome architecture among plant rhabdoviruses that differs from the consensus genome organization reported for previously known cytorhabdoviruses and varicosaviruses . The genFive canonical structural protein genes 3′-N-P-M-G-L-5′ are thought to be conserved among all rhabdoviruses , while ptrans-kingdom horizontal transfer between fungi and plants [The discovery of diverse novel rhabdo-like viruses in metagenomics studies of arthropods  supportsd plants . A G prod plants . NeverthBrevipalpus mites, and the varicosa-like viruses may be transmitted by chytrid fungi. Based on its phylogenetic clustering, the alphanucleorhabdovirus ATV1 is likely transmitted by a planthopper. Among the novel cytorhabdoviruses identified in this study, AChV1, AscSyV1, GlLV1, LotCorV1, NymAV1, PelRaV1 and SuSV1 are likely aphid-transmitted, while the vectors for AntAmV1, DiCoV1, GymDenV1, TaEV1 and TrAV1 cannot be predicted. The BeTaV1 genome was assembled from whitefly transcriptome data and, therefore, is likely a whitefly-transmitted cytorhabdovirus. The L protein-like TSA sequences included in the assembly of the BeTaV1 genome were previously reported to be 95–98% identical to soybean blotchy mosaic virus partial L gene sequences [Among all plant rhabdoviruses studied so far, there is a strong correlation between phylogenetic relationships and vector types ,20. We tequences ; thus, iequences , thus suAt least one transmembrane domain was identified in each P′ protein predicted in the genomes of a group of cytorhabdoviruses discovered in this study, consistent with a previous analysis of other cytorhabdoviruses, which also identified at least one transmembrane domain in every P′ protein . InteresTwo of the cytorhabdoviruses, AChV1 and SuSV1, whose genomes were only partially assembled, clustered phylogenetically in a monophyletic clade with ADV, ChYDaV, RVCV and SCV, all the viruses that contain a P6 accessory ORF between the G and L genes ,67,68,69Cytorhabdovirus to establish three genera that represent different evolutionary lineages: (I) Alphacytohabdovirus would include species for all aphid-transmitted cytorhabdoviruses; (II) Betacytorhabdovirus would include species for all those cytorhabdoviruses likely transmitted by leafhoppers, planthoppers and whiteflies, as well as AntAmnV1, DiCoV1 and TaEV1, and (III) Gammacytorhabdovirus would include species for GymDenV1 and TrAV1 (The phylogenetic relationships of the now expanded number of known cytorhabdoviruses provide some support for splitting the genus nd TrAV1 .The ATV1 genome that was assembled from the transcriptome data of the monocot agave does not encode any accessory ORFs, like most of the alphanucleorhabdoviruses, except the group of PYDV-like viruses that encode an X ORF of unknown function . ATV1, wAll the betanucleorhabdoviruses identified in this study, AscSyV2, CusReV1, PerMiV1, PleArV1 and RhoDeV1, appear to be associated with dicot hosts, which is consistent with every betanucleorhabdovirus reported so far . The dicNucleorhabdovirus into the three genera Alphanucleorhabdovirus, Betanucleorhabdovirus and Gammanucleorhabdovirus [The phylogenetic relationships of nucleorhabdoviruses, including the novel alpha- and betanucleorhabdoviruses discovered in our analysis, clearly support the recent split of the previous genus bdovirus .Currently, there are only three varicosaviruses recognized by the ICTV, LBVaV, AMVV1 and RCaVV, and a novel varicosavirus, VVV, was recently identified . NeverthThe N protein encoded by the unsegmented PiFLeV1 is basic; on the other hand, the N proteins encoded by the bisegmented varicosaviruses AAnV1, AMVV1, BrRV1, LBVaV, LoPV1, MelRoV1, PhPiV1, RCaVV, SpV1 and VVV are acidic. This difference in the IEP may be associated with a different replication mechanism of the unsegmented PiFLeV1 compared to the segmented varicosaviruses. Moreover, AAnV1 and BrRV1 P2s are basic, while the P2s of AMVV1, LBVaV, LoPV1, MelRoV1, PiFleV1, RCaVV, SpV1 and VVV are neutral or acidic. The biological significance of this difference is unknown, because the functional role of P2 still needs to be unraveled.Poaceae. However, LoPV1 RNA1 has one ORF, while AMVV1 RNA1 has two [LoPV1 and AMVV1 are phylogenetically closely related; their N and P3 aa sequences are >50% identical, and both are associated with grasses in the family  has two . These m has two . Interes has two ,76. On t has two ,77. The  has two ,52.Sciadopitys verticillata previously reported by Mushegian and colleagues [Among the varicosa-like viruses identified in this study, PiFleV1 is unique in terms of genome organization, since its genome is unsegmented, a characteristic that differs from the currently known bisegmented genome architecture of varicosaviruses . This malleagues  to suppoIn summary, this study illustrates the complexity and diversity of plant rhabdoviruses genomes and demonstrates that analyzing SRA public data is a valuable tool not only to accelerate the discovery of novel viruses but, also, to gain insight into their evolution and to refine virus taxonomy. However, the inability to go back to the biological material to confirm viral genome sequences and to link the presence of the viruses to a specific host is the main drawback of the data mining approach for virus discovery. This limitation could lead to the potential misidentification of host species linked to viruses. Thus, researchers need to be cautious when analyzing SRA public data for virus discoveries.


To evaluate the intra and interobserver reproducibility of a new goniometric method for evaluating the isolated passive range of motion of the glenohumeral joint in an outpatient setting.This is a prospective observational study on healthy subjects. The Glenohumeral ROM Assessment with Scapular Pinch (GRASP) method is a new method for assessing the isolated range of motion (ROM) of the glenohumeral joint (GH) by a single examiner with a clinical goniometer. It measures the isolated glenohumeral passive abduction (GH-AB), passive external rotation (GH-ER) and internal rotation (GH-IR) with the arm at 45º of abduction.These three GH ROM parameters were measured in both shoulders of 30 healthy volunteers . The full shoulder passive abduction, passive external rotation and internal rotation 45º of abduction were measured by the same examiners with a goniometer for comparison. One examiner made two evaluations and a second examiner made a third one. The primary outcome was the intra- and interobserver reproducibility of the measurements assessed with intraclass correlation coefficients (ICC) and the Bland–Altman plot.The intra-observer ICC for isolated glenohumeral ROM were: 0.84 ± 0.07 for GH-ABD, 0.63 ± 0.09 for GH-ER, and 0.61 ± 0.14 for GH-IR. The inter-observer ICC for isolated glenohumeral ROM were: 0.86 ± 0.06 for GH-ABD, 0.68 ± 0.12 for GH-ER, and 0.62 ± 0.14 for GH-IR. These results were similar to those obtained for full shoulder ROM assessment with a goniometer.The GRASP method is reproducible for quick assessment of isolated glenohumeral ROM.IIIThe online version contains supplementary material available at 10.1186/s40634-021-00352-z. The shoulder joint is a complex system in which four different joints  work synchronically. All these different joints have a role in normal function, during sporting activities, and in shoulder pathology . CertainThe importance of understanding the degree of involvement of the glenohumeral joint in the total shoulder ROM has been shown in the general population and in athletes for problems such as adhesive capsulitis, 16 or gSome authors have suggested different methods for measuring the isolated passive glenohumeral ROM (GH-ROM) with a goniometer in clinic , 8. ThesThe main objective of this study was to evaluate the reproducibility in healthy adults of a new simple goniometric method, the Glenohumeral ROM Assessment with Scapular Pinch (GRASP), that measures selectively the PABD, PER and PIR of the glenohumeral joint and can be used by a single examiner using a simple clinical goniometer. The null hypothesis was that the presented method was not reproducible.The Glenohumeral ROM Assessment with Scapular Pinch (GRASP) method was developed to allow for quick assessment of the passive abduction, external and internal rotation ROM of the glenohumeral joint with a goniometer by a single individual in a busy clinical setting.The subject to be measured is asked to undress from the thigh up and to sit in a stool Fig. a. Then sAdditional file 1: A video in mp4 format is included as SDC 1Based on the sample size formulas for estimating intraclass correlation coefficients with precision of Zou et al.  a minimuThirty healthy subjects  with no history of shoulder pathology agreed to participate in this research study. Both shoulders´ GH-ROM and FS-ROM was measured in all subjects. Since agreement between measurements does not depend on the side in which measurements are made, right and left shoulder of the subjects were considered independently for a total of 60 shoulders.Written informed consent to participate in the study was obtained for each subject. The study was approved by the Local Institutional Review Board .Two examiners took part in data collection, both had extensive experience using the method. Three measurement sessions were completed for each 60 shoulders: two by examiner A (named sessions A1 and A2) and one by examiner B (named session B), following an A1–B–A2 sequence. The actual examiner assigned to the role of A and B was randomized for each patient with a flip of a coin. All sessions were planned in the same day. When one of the examiners was making the measurements, the other examiner left the room to be blinded to the results obtained by his/her fellow investigator.Each measurement session included sequential measurements of the isolated glenohumeral passive abduction (GH-AB) ROM, passive external rotation (GH-ER) and internal rotation (GH-IR) with the arm at 45º of abduction, followed by measurement of the full passive shoulder ROM in abduction (FS-PABD), external rotation (FS-PER) and internal rotation (FS-PIR) with the arm at 45º of abduction in the sitting position. This second set of measurements was performed as a control for reproducibility. All measurements were performed with a set of long-arm (12″) goniometers .The ROM values were tested for normality using the Kolmogorow-Smirnoff test. Intra and interobserver reproducibility was assessed for all six variables using Two Model intraclass correlation coeficients (ICC) . ICC belAn arbitrary cut-off point of 10º was stablished to further assess reproducibility: the percentage of measures that varied more than 10º between repeated measurements was calculated. The minimal clinically important difference (MCID) in ROM for shoulder movements has not been clearly established, but a range of 2º to 10º has been proposed .The mean [standard deviation] values of the six angular parameters measured were: FS-ABD.176º[6.57º]; FS-ER:79.6º[8.94º]; FS-IR:77.8º[7.59º]; GH-ABD: 81.4º[8.94º]; GH-ER: 61.0º[11.8º]: and GH-IR: 49.9º[10.7º].The ICC obtained for intra and interobserver reproducibility can be seen in Table The most important finding of this study is that the Glenohumeral ROM Assessment with Scapular Pinch (GRASP) method for the measurement of isolated passive glenohumeral ROM has reproducibility in line with other standard techniques used in clinical practice to assess full-shoulder passive range of motion. It provides very good reproducibility in abduction measurements and good acceptable reproducibility in external and internal rotation.A precise assessment of the role that each individual joint has in the full shoulder ROM is something that is rarely done in the typical busy clinical setting . The maiUnfortunately, the available tools to assess reliably the GH-ROM have significant limitations that make them unusable in a typical clinical setting. Systems that use cumbersome motion tracking systems , 2, fluoThe new method presented here was developed with the following requirement in mind: it should use only a manual goniometer , it should require only one examiner, it should be quick to use and painless to the patient and should be as reproducible as the usual methods to assess ROM in a busy clinical setting. The GRASP method has clear advantages for the clinician: it is simple to use, requires only one examiner, uses a simple goniometer, does not require the patient to lie or place the shoulder in 90º of abduction , the measurements are easy to interpret and comparisons to the contralateral side or previous exams can be done seamlessly.The intra and interobserver reproducibility of the GRASP method to assess GH-ROM described are in par with the measurements we took of FS-ROM. This suggest that it might be as adequate as the typical methods that we use in our practice to assess ROM. The ICC values for GH-ABD are especially relevant, with 95% confidence intervals over 0.75, the reproducibility should be considered good and there is no other system, to our knowledge, that allows for GH passive abduction assessment in the clinical setting. The values for GH-ER 0.68) and for GH-IR (0.62) are also acceptable but lower than for abduction, but precise assessment of rotation movements is more difficult . The met8 and forThe ICC values obtained are not optimal, this being a limitation of this method, as this tool might be of limited use when very precise assessment is needed. Despite of this, using the MCID threshold of 10º showed that abduction and probably external rotation can be measured relatively safely with this new method and it seems as reproducible if not more reproducible as other available methods. A clear limitation of the measurement method is that it requires the subject to keep the shoulder in 45º of active adduction during rotational measurements, some patients, with significant rotator cuff problems might struggle to keep the arm in that position for long. Another limitation of this study is sample size: although a sample size calculation for the reproducibility analysis was indeed performed, the results obtained are limited in scope regarding the adquisition of normative data for healthy individuals, as the sample size is too small and includes a relatively young population. Ideally further reproducibility assessment could have been performed making repeated measurements in different days, this was not done and is a limitation of this study. To finish, a clinical tool should not be only accurate, but it should also be useful; this study only evaluates reproducibility, it lacks a clear clinical indication for use; this is a limitation that our research team is working to solve.The Glenohumeral ROM Assessment with Scapular Pinch (GRASP) method is an easy to use, reproducible method for quick and assessment of the isolated glenohumeral range of motion in healthy adults. The quality of the data obtained is similar to that obtained when assessing full shoulder passive ROM with a goniometer.


In the 1980s, virus inactivation steps were implemented into the manufacturing of biopharmaceuticals in response to earlier unforeseen virus transmissions. The most effective inactivation process for lipid‐enveloped viruses is the treatment by a combination of detergents, often including Triton X‐100 (TX‐100). Based on recent environmental concerns, the use of TX‐100 in Europe will be ultimately banned, which forces the pharmaceutical industry, among others, to switch to an environmentally friendly alternative detergent with fully equivalent virus inactivation performance such as TX‐100. In this study, a structure–activity relationship study was conducted that ultimately led to the synthesis of several new detergents. One of them, named “Nereid,” displayed inactivation activity fully equivalent to TX‐100. The synthesis of this replacement candidate has been optimized to allow for the production of several kg of detergent at lab scale, to enable the required feasibility and comparison virus inactivation studies needed to support a potential future transition. The 3‐step, chromatography‐free synthesis process described herein uses inexpensive starting materials, has a robust and simple work‐up, and allows production in a standard organic laboratory to deliver batches of several hundred grams with >99% purity.  In particular, the phenol moiety of TX‐100 ‐tetramethylbutylphenol) is resistant to biodegradation and as such toxic to the environment, for example, it may impair the reproductive cycles of aquatic organisms.I)I), and indeed, some alternative detergents have recently been suggested.More recently it has been realized that TX‐100 (I) with a detergent of analogous activity and behavior, thus minimizing any impact on the virus inactivation capacity, physical behavior, and removability during subsequent manufacturing steps, to ultimately support a smooth transition away from the phenol‐containing detergent TX‐100 (I). As detergents with a close structural similarity to TX‐100 (I) were not available, an attempt was made to develop non‐phenolic TX‐100‐like detergents and to evaluate their ability to inactivate lipid‐enveloped viruses.In order to not to adversely affect pharmaceutical bioprocesses, the best solution would be to replace TX‐100  was diluted to 0.6 mg/ml using a buffer that contained 396 mM NaCl, 20 mM MES acid monohydrate, 10 mM CaCl2 dihydrate, and 0.099% (v/v) PS80. This matrix is representative for an intermediate of the production process of recombinant factor VIII . The starting material had a pH of 6.4 and was filtered through a 0.2 µm filter immediately before virus inactivation runs were performed.As a model matrix for plasma‐derived products, Fraction II was used, that is, the process intermediate obtained during immunoglobulin production at the stage before virus inactivation/reduction steps.2.2l‐glutamine, 1 mM sodium pyruvate, 0.75% sodium bicarbonate, 1x non‐essential amino acids, and 100 µg/ml gentamicin sulfate . Mus dunni cells  and PG4 cells  were propagated in Mc Coy's 5A Medium that was supplemented with 10% FCS, 2 mM l‐glutamine, and 100 µg/ml gentamicin sulfate.Vero cells  were propagated in an in‐house manufactured basal medium that was supplemented with 5% fetal calf serum , 2 mM Pseudorabies virus  was obtained from Eberhard Karls University, Tübingen, Germany. The virus was propagated on Vero cells, which were also used as an indicator cell line for virus titrations. Xenotropic Murine Leukemia Virus  was obtained from the ATCC (VR‐1447). The virus was propagated on Mus dunni cells and titrated on PG4 cells.50) assays were employed using eightfold replicates of serial half‐log sample dilutions of virus‐containing samples that were incubated on the respective indicator cell lines. After incubation, cytopathic effects were evaluated by microscopic visual inspection. TCID50 titers were calculated according to the Poisson distribution and expressed as log10[TCID50/ml]. Virus reduction factors were calculated in accordance with the EU Committee for Proprietary Medicinal Products guidance.The choice of viruses reflects requirements as defined in the relevant guidelines2.310[TCID50/ml], indicating that neither matrix constituents nor the chosen physicochemical parameters led to virus inactivation. As for manufacturing, the final target concentrations of PS80, TNBP, and TX‐100 are 0.3%, 0.3%, and 1% (w/w), respectively. Taking the ratio of SD components into account, different S/D mixes were prepared by combining PS80  and TNBP  with either TX‐100  or the potential surrogate compounds. Among these substances, TX‐100 reduced , Ecosurf EH‐9, and Tergitol TNM‐100X  were obtained commercially, while the other detergents were synthesized in‐house (see ESI). The spiked process material was weighed to calculate the amount of S/D mix to be added to reach 5% (plasma‐derived model matrix) or 10% (recombinant model matrix) of the concentration as specified for manufacturing. These considerably reduced final concentrations enable to demonstrate virus inactivation kinetics, a regulatory requirement,50 virus titration were drawn at 1–2, 10 ± 1, 30 ± 1, and 59 ± 1 min after detergent addition.Each combination of S/D mix, virus, and matrix was investigated in duplicate runs using 30–50 ml of filtered process material per run. Throughout the entire experimental runs, the process material was kept at 17 ± 1°C (plasma‐derived model matrix) or 1 ± 1°C (recombinant model matrix) and continuously mixed by a magnetic stirrer. Spiking with the respective virus stock solution was performed at a ratio of 1:31 (v/v). Subsequently, two samples termed spike control (SC) and hold control (HC) were drawn. SC was titrated immediately while HC was incubated in the same cooling unit as the SD‐treated process material and titrated at the end of the respective run. For the vast majority of experiments, the difference in virus titer between SC and HC pairs was ≤0.5 logFor all used process materials, the cytotoxicity of the respective S/D mix, as well as any possible matrix effects on cell lines used for virus detection were tested and taken into consideration for the calculation of reduction factors.33.1I) while retaining its full inactivation activity against viruses as well as its well‐established process compatibility.Bioisosteres are surrogate compounds designed to exhibit similar physical properties with broadly similar biological activity to another chemical compound. The purpose of exchanging one bioisostere for another can be multiple such as improved potency, enhanced selectivity, altered physical properties, reduced or redirected metabolism, acquisition of novel intellectual property or circumvention of undesired metabolites.I) replacement compounds.Bioisosteres possess near‐equal molecular shapes and volumes and have approximately the same electron distribution.3.2I) are responsible for its outstanding virus inactivation activity,It is appealing to understand which structural elements of TX‐100 (I) features structural elements such as the 1,1,3,3‐tetramethylbutyl alkyl chain, the aromatic ring, and the linkage to the polyethylene glycol chain. All those elements can be chemically modified one by one by designed chemical synthesis, to reveal their contribution to the activity in subsequent virus inactivation studies.In addition to its polyethylene glycol chain, TX‐100 (3.3I), we obtained the structure of TX‐100 reduced (II), a commercially available detergent  failed to effectively inactivate a model retrovirus in a recombinant model matrix at such cold temperature  has a diminished penetrating activity compared to TX‐100 (I), which is both efficient at low and elevated temperature in plasmatic and recombinant matrices. A possible cause for the more moderate inactivation at low temperature might be the decrease of the virus membrane fluidity and its porosity as described in the fluid mosaic model.It is tempting to speculate that TX‐100 reduced  directly linked to the hydrophilic polyethylene glycol chain.In a further SAR step, the whole ring was removed to yield model compound III, the corresponding highly substituted alcohol was mesylated followed by nucleophilic attack of a monoprotonated PEG polymer, which delivered the candidate III in a satisfactory 64% overall yield after a chromatographic purification in multigram scale  surrogates Ecosurf EH‐9 and Tergitol TNM‐100X  which equally failed to show significant inactivation, especially at a cold temperature (data not shown).Interestingly, the inactivation properties of compound s Figure . These rA hypothesis for the low activity of structures without the aromatic ring might be that the planar geometry of the aromatic ring enables to create a denser interaction with the virus membrane, by π–π stacking and/or hydrophobic interaction with membrane proteins, for instance, thereby favoring the formation of mixed micelles and disruption of the lipid envelope.3.5IV containing two double bonds, based on the Geraniol structure as well as its fully hydrogenated derivative V were synthesized in a similar manner in multigram scale and in acceptable overall yield  with the sole exception of having an extra methylene group inserted between the aromatic ring and the polyethylene glycol chain, VII features, in addition to the latter change, a shortened alky chain (tert‐butyl instead of tert‐octyl para‐substituted to the aromatic ring).At this point of the investigation, we had elaborated the crucial presence of the aromatic ring in the detergent structure to reach maximum virus inactivation and more information was required to evaluate the role of other elements, such as the VI, a 6‐step synthesis was envisaged taking advantage of the already established octyl alky chain in the para position of the aromatic ring in the starting material octyl‐phenol. First, the corresponding triflate intermediate VIII was obtained quantitatively using triflate anhydride. Next, a palladium catalyzed reaction in combination with a stoichiometric amount of zinc(II) cyanide delivered, after a chromatographic step, the para‐substituted benzonitrile IX. Hydrolysis followed by reduction of the carboxylic acid X yielded the benzyl alcohol XI as a solid. The last 2 steps, following similar protocols as described above, finalized the synthesis of over 120 g of VI with an overall yield of almost 23% over 6 steps  from the commercially available tert‐butyl benzyl alcohol following standard mesylation/nucleophilic substitution, described previously in gram scale   Figure . In starI) can be replaced by another linkage such as a benzyl ether while fully preserving virus inactivation activity, corresponded to our aim of removing the phenol group. This undesired group is in fact the root cause for the estrogen receptor‐binding  activity of TX‐100 (I) degradation products The crucial finding that the phenol polyethylene glycol connection in TX‐100  Suzuki coupling step. Following standard protocol, both detergents XIII and XIV were obtained in gram quantities in respectively 34% and 24% overall yield  compared to VI and TX‐100 (I) . However, virus inactivation studies at standard temperature revealed unsatisfactory potency of both compounds . Firstly, VI showed effective and reliable virus inactivation for the process matrices and viruses tested. Secondly, its very similar structure to TX‐100 (I) suggests that also the chemo‐physical properties of the detergent , as well as interaction with the protein(s) of interest will be highly similar. This should ensure that changes in the affected processes, such as analytical method or removal process, will be only minimal. Finally, from an ecological point of view, after release of compound VI into the environment, the polyethylene glycol chain is broken down in a similar manner as for TX‐100; however, the revealed benzyl alcohol will be easily oxidized to the corresponding benzoic acid,I) degradation. In addition, further differences between phenols et benzyl alcohols in their abilities to be metabolized either through oxidative or bacterial processes might favor benzylic compounds in regard to the impact on their biodegradations.Detergent 3.82O, Pd catalyst), toxic 2, DMF, MsCl), and hazardous (LiAlH4) chemicals. Moreover, as the demand for material grew rapidly to accommodate feasibility studies, the synthetic route was upgraded to a convenient 3‐step synthesis using cheap, nontoxic and safe starting materials available in bulk , no ortho product was detected. The work‐up included a simple quench of the acid followed by extractive aqueous washes. Other isomers originating from likely cation rearrangements of the alkyl chain were also formed and could be removed entirely during the purification of the following Step 2. Exhaustive evaporation of the solvent is needed to remove the totality of the toluene, as it might react competitively with the NBS reagent during the next step.Taking advantage of the inducing effect of the methyl group of toluene to achieve the para‐substitution of the target molecule, an acid‐catalyzed Friedel‐Craft alkylation using diisobutylene was envisaged to access the hydrocarbon 3.8.2XV was then engaged at 70°C with AIBN and NBS to form benzyl bromide XVI in a radical bromination in cyclohexane, during which after radical propagation, succinimide precipitates as a white solid. After filtration and concentration, the crude product containing some starting material as well as dibrominated product XVII was precipitated in IPA to yield a product with an approximate 90% purity. Dibrominated product XVII was synthesized separately to confirm the identity of this main side‐product. Further two recrystallizations in IPA delivered the benzyl bromide intermediate XVI in moderate yield over two steps (25%–30%) but at >99% purity (200 nm). Thus, all side products from the first and second step could be removed and a crystalline colorless solid (large plates) was obtained after drying.The crude oily hydrocarbon 3.8.3XVI was added in one portion before the reaction mixture was cooled 30 min later by addition of ice/water followed by pH adjustment, removal of the discoloration, and addition of EtOAc. Several aqueous washes assured that the excess of PEG400 was entirely removed. After concentration of the EtOAc, the residue containing some bi‐functional product XVIII was re‐dissolved in EtOH/water and non‐polar washes were performed with cyclohexane to remove a large portion of the bi‐functional product XVIII. An independent synthesis of bifunctional side‐product XVIII confirmed the structure and served as reference material. A final concentration delivered several hundred grams of product VI as a yellowish clear oil with a purity of >99% (LC‐ELSD). It can be noted that the route utilizes only five solvents , all of them being nonhazardous and recoverable after concentration.Without solvent, a 5‐molar excess of PEG400 (polyethylene glycol with an average MW of 380–420 g/mol) was deprotonated at 60°C by addition of tBuOK. To the mono‐deprotonated PEG400, the benzyl bromide intermediate This material is currently in use for feasibility studies, engineering and confirmation runs. Further chemical development studies and improvements are in progress to continuously optimize the process and prepare for large‐scale industrial production of this detergent, which was named “Nereid” after Amphitrite, the Greek sea goddess and wife of Poseidon but also the mother of Triton, from whom comes the prefix “amphi‐“ (ἀμφί) meaning “both kinds, on both sides” used in modern words such as amphibious, amphitheater or amphiphile (detergent).4I) dramatically narrowed the screening possibilities described in this study. In this regard, the plan arose to develop a tailor‐made detergent that fulfills the prerequisites of the virus inactivation potency of TX‐100 (I) without being harmful to the environment. Structure‐function analyses indicated that the alkyl chain substitution must follow the 1,1,3,3‐tetramethylbutyl pattern of TX‐100 (I) and must be connected to an aromatic phenyl ring to retain full virus inactivation properties. The installation of a –CH2– between the aromatic ring and the polyethylene glycol chain resulted in conservation of the inactivation potential, thus the phenol‐free detergent Nereid was discovered. A convenient 3‐step synthesis was developed, which allows the production of hundreds of grams of pure detergent using reliable protocols and standard equipment. Further work is in progress to adapt the synthesis route to the multi‐kg, and ultimately ton scales. Investigations are underway to analyze and dismantle the exact molecular mechanism of the S/D treatment. Although protein compatibility as well as process compatibility have been verified on a considerable range of products ,The limited number of commercially available detergents that meet the pre‐defined structural requirements of similarity with TX‐100 (Jean‐Baptiste Farcet, Richard Scheinecker, and Otto Kostner are employees of Baxalta Innovations GmbH, Vienna, Austria, now part of the Takeda group of companies. Johanna Kindermann, Michael Karbiener, and Thomas R. Kreil are employees of Baxter AG, Vienna, Austria, now part of the Takeda group of companies. Jean‐Baptiste Farcet, Michael Karbiener, and Thomas R. Kreil are Takeda stock owners. Jean‐Baptiste Farcet, Johanna Kindermann, and Thomas R. Kreil are inventors of a submitted patent application related to Brij C10, Triton X‐100 reduced, and Nereid.Jean‐Baptiste Farcet conceived the Sar study, designed and synthesized the new compounds. Jean‐Baptiste Farcet, Richard Scheinecker, and Otto Kostner analyzed the new chemicals. Jean‐Baptiste Farcet and Otto Kostner optimized the synthesis of Nereid. Johanna Kindermann planned and coordinated the virus inactivation experiments, Michael Karbiener gave conceptional input and structured the manuscript. Thomas R. Kreil initiated and coordinated the study. Jean‐Baptiste Farcet, Michael Karbiener, and Thomas R. Kreil contributed to the writing of the manuscript.Supplementary information.Click here for additional data file.


Orthotospovirus species have been reported in China, including eight newly identified species from this genus. The number of new highly pathogenic Orthotospovirus strains or species has increased, likely because of the virus species diversity, the wide range of available hosts, adaptation of the viruses to different climates, and multiple transmission routes. This review describes the distribution of Orthotospovirus species, host plants, typical symptoms of infection under natural conditions, the systemic infection of host plants, spatial clustering characteristics of virus particles in host cells, and the orthotospoviral infection cycle in the field. The evolutionary relationships of orthotospoviruses isolated from China and epidemiology are also discussed. In order to effectively manage orthotospoviral disease, future research needs to focus on deciphering the underlying mechanisms of systemic infection, studying complex/mixed infections involving the same or different Orthotospovirus species or other viruses, elucidating orthotospovirus adaptative mechanisms to multiple climate types, breeding virus-resistant plants, identifying new strains and species, developing early monitoring and early warning systems for plant infection, and studying infection transmission routes.Orthotospoviruses are responsible for serious crop losses worldwide. Orthotospoviral diseases have spread rapidly in China over the past 10 years and are now found in 19 provinces. Currently, 17  Tospovirus in the family Bunyaviridae and were divided into seven or nine serogroups based on serology , which are responsible for the virus acquisition and transmission by the thrips vectors. The core of the virion contains three viral RNA fragments, named L RNA, M RNA, and S RNAs according to their lengths, encapsulated by the nucleocapsid protein (N). The orthotospovirus are single-negative-stranded, ambisense RNA viruses. The L RNA (∼8.9 kb) encodes the RNA-dependent RNA polymerase (RdRp) in the complementary (vcRNA) strand; M RNA (∼4.8 kb) encodes the movement protein (NSm) in the viral (vRNA) strand and the Gn and Gc proteins in the vcRNA strand; and RNA S (∼2.9 kb) encodes the N protein in the vcRNA and silencing suppressor (NSs) in the vRNA strand .To date, many articles have illustrated details of the global occurrence, epidemiology, and molecular interactions between orthotospoviruses and their thrips vectors . In thisSymptoms of orthotospoviral disease in host plants are very similar, with only minor differences between species of virus. The major symptoms are ringspots , bud necrosis, silver mottle, and vein banding. Zonate spots are characteristic of orthotospovirus infection. Although symptoms vary between disease stages, chlorotic, yellow, and necrotic ringspots can occur at all stages . Herbaceous plants with severe disease die in the late stage. The symptoms occur in the leaves and fruit, with a few cases of stem necrosis .Bidens bipinnata (B. bipinnata L.), and woody plants   .Orthotospovirus species known worldwide, comprising 11 definitive species  and 19 tentative species (written in upright letters) (tomato spotted wilt orthotospovirus (TSWV) clade, watermelon silver mottle orthotospovirus (WSMoV) clade, soybean vein necrosis-associated orthotospovirus (SVNV) clade, iris yellow spot orthotospovirus (IYSV) clade, and groundnut yellow spot orthotospovirus (GYSV) clade  . Orthotoletters) , namely V) clade . Based oV) clade . VirusesV) clade , 2020. VV) clade . VirusesV) clade . The SVNV) clade . SeveralV) clade . Howeverglobally .Orthotospovirus species has been reported is China. A total of 17 Orthotospovirus species are known in China to date  was reported in tomatoes from Yunnan province, based on whole-genome sequencing and viral particle clustering characteristics in host cells    and moremperate) . The ortan: TZSV , hippeasan: TZSV , and chian: TZSV . TSWV anan: TZSV . The ortThysanoptera. More than 14 species of thrips are known to act as vectors for orthotospoviruses.Orthotospoviruses are becoming more and more harmful due to their multiple transmission routes and wide host ranges. The main transmission routes under natural conditions are via the vector thrips, which are insects in the order Ceratothrip oidesclaratris, Dictyothrips betae, Frankliniella occidentalis, Frankliniella schultzei, Frankliniella gemina, Frankliniella intonosa, Frankliniella cephalica, Frankliniella bispinosa, Frankliniella fusca, Frankliniella zucchini, Scirtothrips dorsalis, Neohydatothrips variabilis, Thrips palmi, and Thrips tabaci . A single species of thrips can transmit one or many Orthotospovirus species  (The thrips that are known to transmit orthotospovirus include s tabaci . A singlspecies) .Besides transmission by thrips, other routes of transmission facilitate spread of orthotospoviruses. Orthotospoviruses such as SVNV spread through seeds . In addiOrthotospovirus species, we have recently found WSMoV, TZSV, and HCRV in Tibet, Hainan, and other Chinese provinces. Orthotospoviruses tend to quickly replace other viral pathogens (such as tobacco mosaic virus and cucumber mosaic virus) or form complex/mixed infections with other viruses (such as potato virus Y and whitefly transmitted geminivirus) in solanaceous crops, based on our research results over the past 20 years in Yunnan province.Orthotospoviruses have spread rapidly through China in recent years. Before 2015, orthotospoviral diseases mainly occurred in Southwest and Southeast China. However, over the last 5 years, orthotospoviral diseases have also occurred in Central, Northwest, and Northeast China. Although TSWV is the main epidemical Orthotospovirus species, especially TSWV, in China are as follows: (1) Over the last 20 years, the rapid popularization and development of greenhouses in China has provided perfect conditions for the propagation of the vector insects, thrips. Particularly in the north of China, greenhouses provide suitable temperatures for the overwintering of the virus-transmitting thrips, and indeed the orthotospoviral diseases in most northern provinces occur mainly in greenhouses (The main reasons for the rapid expansion of enhouses . In soutenhouses . (2) Chaenhouses . This isenhouses .Orthotospoviruses in host cells have distinct histopathological characteristics that mainly relate to the virus species rather than to the host plant species, and these characteristics have important diagnostic value. The histopathological characteristics differ between the different stages of infection. In general, at 3 days post inoculation (dpi), there are a large number of vesicles in the host cells. At 7 dpi, virus particles form in the vesicles. At this stage (in the early stages of infection), double-enveloped virions (DEV) are also observed in host cells. At 9 dpi, virus particle aggregates appear in the host cells. By 12 dpi (in the late stages of infection), the cells are filled with virus particles .Orthotospovirus species. TSWV virus particles always cluster in the endoplasmic reticulum (ER) or in special vesicles (based on transmission electron microscopy of ultrathin sections or negative staining). TZSV virus particles usually cluster in a moniliform structure in the ER membrane  or as double-enveloped virions (cross-section) (Impatiens necrotic spot orthotospovirus (INSV) virus particles usually cluster in the lumen of the ER (Orthotospovirus species.The virus particle clustering characteristics differ among section) . WSMoV vf the ER . AlthougTo establish a systemic infection in a host plant, the TSWV ribonucleoprotein (RNP) complex migrates along the ER membrane. The N protein wraps with TSWV RNAs to form the RNP complex, and these RNPs are subsequently driven along the ER membrane and actin by the action of myosin, after which the viral RNPs enter into the Golgi body, where they form mature virus particles . To infeBiological control is an effective method to control thrips-borne orthotospovirus disease, which includes diversified prevention and control measures. These include a reasonable rotation or continuous replanting mode ; releaseSw-5 and Tsw resistance genes, respectively. After selection trials, virus-resistant plants were grown in areas of high TSWV occurrence in China. However, the resistance of these plants was poor, due to single-gene resistance combined with the existence of complex/mixed infections. To date, there are no varieties with high levels of comprehensive resistance.Growing highly virus-resistant plant varieties can be an effective way to prevent and control viral diseases. TSWV-resistant tomato and pepper varieties have been bred by Bayer, Syngenta, and other companies. These varieties possess the Orthotospovirus TSWV, GYSV, INSV, and GCFCV has been developed by transforming the conserved motifs of the RdRp gene of WSMoV through hagrobacterium-mediated transformation  pathway, as well as up-regulate the levels of miRNAs  that contribute to inhibiting TSWV infection  signaling pathway and inhibition of NSs, NSm, and RdRp gene expression , isolated from nfection , 2017. Anfection . The terpression . Tagitinc leaves , 2020. IViral infection at the seedling stage (a highly susceptible stage) is the main reason for disease outbreaks in the middle and late plant growth periods. To obtain virus-free seeds and effectively reduce the occurrence and loss caused by orthotospovirus diseases, seedlings should be grown in greenhouses with insect-proof netting or maximal barrier precautions, yellow or blue sticky plates should be used to attract and trap the vector insects (thrips), and the virus carriage rate of the seedlings should be regularly monitored. Comprehensive measures to reduce the vector insects (thrips) should also be applied.Orthotospovirus species) have increased the frequency of disease. Orthotospoviruses are therefore a threat to crop production because of their high pathogenicity and the complex/mixed infections involving different Orthotospovirus species or other viruses.Orthotospoviruses are expected to further expand worldwide because of their wide host range, multiple transmission routes, and ability to adapt to diverse climates. Additionally, increases in agricultural trade have accelerated global transmission. In China, new species or strains with high pathogenicity .It is necessary to enhance the monitoring of orthotospovirus infections and set up early warning systems in high-incidence areas; to allow the rapid diagnosis of symptoms caused by ZZ conceived and designed the review and wrote most of the manuscript. KZ wrote the text on orthotospoviruses evolutionary relationships, conducted the statistical analyses shown in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Early childhood development (ECD) programs can help address disadvantages for the 43% of children under 5 in low- and middle-income countries (LMICs) experiencing compromised development. However, very few studies from LMIC settings include information on their program’s cost-effectiveness or potential returns to investment. We estimated the cost-effectiveness, benefit–cost ratios (BCRs), and returns on investment (ROIs) for 2 effective group-based delivery models of an ECD parenting intervention that utilized Kenya’s network of local community health volunteers (CHVs).Between October 1 and November 12, 2018, 1,152 mothers with children aged 6 to 24 months were surveyed from 60 villages in rural western Kenya. After baseline, villages were randomly assigned to one of 3 intervention arms: a group-only delivery model with 16 fortnightly sessions, a mixed-delivery model combining 12 group sessions with 4 home visits, and a control group. At endline , 1,070 children were retained and assessed for primary outcomes including cognitive and receptive language development  and socioemotional development . Children in the 2 intervention arms showed better developmental outcomes than children in the control arm, although the group-only delivery model generally had larger effects on children. Total program costs included provider’s implementation costs collected during the intervention period using financial reports from the local nongovernmental organization (NGO) implementer, as well as societal costs such as opportunity costs to mothers and delivery agents. We combined program impacts with these total costs to estimate incremental cost-effectiveness ratios (ICERs), as well as BCRs and the program’s ROI for the government based on predictions of future lifetime wages and societal costs. Total costs per child were US$140 in the group-only arm and US$145 in the mixed-delivery arm. Because of higher intention-to-treat (ITT) impacts at marginally lower costs, the group-only model was the most cost-effective across all child outcomes. Focusing on child cognition in this arm, we estimated an ICER of a 0.37 standard deviation (SD) improvement in cognition per US$100 invested, a BCR of 15.5, and an ROI of 127%. A limitation of our study is that our estimated BCR and ROI necessarily make assumptions about the discount rate, income tax rates, and predictions of intervention impacts on future wages and schooling. We examine the sensitivity of our results to these assumptions.To the best of our knowledge, this study is the first economic evaluation of an effective ECD parenting intervention targeted to young children in sub-Saharan Africa (SSA) and the first to adopt a societal perspective in calculating cost-effectiveness that accounts for opportunity costs to delivery agents and program participants. Our cost-effectiveness and benefit–cost estimates are higher than most of the limited number of prior studies from LMIC settings providing information about costs. Our results represent a strong case for scaling similar interventions in impoverished rural settings, and, under reasonable assumptions about the future, demonstrate that the private and social returns of such investments are likely to largely outweigh their costs.NCT03548558, June 7, 2018. American Economic Association RCT Registry trial AEARCTR-0002913.This trial is registered at ClinicalTrials.gov,  Italo Lopez Garcia and colleagues conduct an economic evaluation of an effective parenting intervention to promote cognitive, receptive language, and socioemotional development among children under 3 years in Kenya. It is estimated that 43% of children under 5 years of age from low- and middle-income countries (LMICs) are at risk of not reaching their full developmental potential as adults due to poverty, lack of stimulation, and nutritional deficiencies.Numerous early childhood development (ECD) parenting interventions that teach parents strategies to improve responsive stimulation and nutritional practices with children have demonstrated that they can help improve children’s outcomes.Yet very few ECD parenting studies have shared information about their program’s costs to enable an analysis of its cost-effectiveness or potential economic returns, which is crucial to inform policy decisions on whether to invest in ECD parenting programs.Among the very few studies that report a program’s cost-effectiveness, none has accounted for the opportunity costs that program participants must incur to attend a program and enact any recommended behavioral changes.We calculate the total costs, cost-effectiveness, and compare the benefits versus costs for an ECD parenting intervention recently tested across 60 villages in rural western Kenya involving nearly 1,200 mothers and their young children.The program compared 2 different delivery models, one featuring only group visits within villages and a second featuring a combination of group and home visits. Both were delivered by local community health volunteers (CHVs) over 8 months.We estimate the opportunity costs for the CHVs and participants in our program under both delivery models and predict the future benefits and costs stemming from participation, such as increases in lifetime wages and schooling costs.We find that children experienced large improvements in their cognitive development at very reasonable costs under both delivery models, but the group-only model was the most cost-effective.Under reasonable assumptions about the future, we find that long-term program benefits could outweigh its associated costs by a factor of 15, implying a large potential return on investment (ROI) for a government willing to scale this program.Our findings are very encouraging for the investment into ECD parenting programs for resource-poor and rural settings.As national and international policymakers increasingly encourage investment into ECD parenting programs, our results represent a strong case study for a promising means to do so. Scientific evidence confirms that the first 3 years of life are when risk factors such as inadequate parental stimulation exert the greatest harm on child development and when effective interventions can have the greatest benefit ,2. NumerData on program costs can enable different types of economic evaluations of ECD interventions. Cost-effectiveness analysis (CEA) compares the relative costs of a program to its generated impacts on outcomes and is one of the most important indicators used to inform scalability . HoweverA more comprehensive economic evaluation may also include a benefit–cost analysis (BCA), which compares the monetized expected program benefits relative to its costs, and can better inform policy on the potential benefits to society of scaling a program. Yet BCAs are even more rare than CEAs in the evaluation of ECD parenting programs in LMICs , likely In addition to a lack of economic evaluations of ECD parenting programs, there has been growing interest in understanding the delivery format that is most scalable to reach young children before age 3 in LMICs ,15. ECD The aims of this study are to perform an economic evaluation of an effective ECD parenting intervention targeted to families with children under age 3 in SSA that can contribute new insights about the relative cost-effectiveness of 2 different delivery models and to estimate their potential BCRs and returns to investment.ClinicalTrials.govNCT03548558, and the study protocol has been published . Al. Alp = 0This study is reported as per the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) guideline  20]. We. We20]. All costs and benefits in local currency included in our analyses were converted at 101.8 Kenyan Shillings = US$1 and reported in 2020 prices, adjusted for inflation.For the calculation of cost-effectiveness, we adopt a societal perspective that combines the implementation costs a health service provider would incur if the program were to be scaled using local CHVs with present period societal costs to the household and the community. These societal costs include the opportunity costs of mothers and CHVs to travel to and attend sessions, as well as rental costs for venues to host the sessions if operating at scale . We use accounting cost methods for the provider costs and economic costing methods to incorporate the opportunity costs of participation for mothers and CHVs. For the calculation of BCRs and ROIs, we additionally incorporate the predicted future costs and benefits stemming from gains in cognitive skills following participation in the program. Future costs include the costs of increased schooling attendance among participants in a scaled version of the program , and futProvider costs relevant to implementing the Msingi Bora program were collected during the 8-month intervention period using quantitative financial data reported by SWAP. For these costs, we used a step-down accounting costing methodology using actual program costs rather than budgeted costs ,25 and wUnit costs by intervention arm are presented in We present the costs that were required to implement the intervention “as it happened,” which include additional costs incurred due to unexpected delays in fieldwork, as well as cost inefficiencies in practice . The only exception is that we account for the higher time use costs for CHVs delivering home visits for the 4 review sessions in the mixed-delivery arm using estimates of the opportunity cost of their time. For the sake of comparison, in Direct costs included salaries and stipends for personnel , training, travel and accommodation for training, and any food and supplements necessary for the implementation of the program.PersonnelWe include the flat monthly stipends paid to CHVs and mentor CHVs, as well as salary costs for 3 subcounty supervisors and 4 SWAP staff members involved in day-to-day activities of implementation, supervision, and training, prorated to their percentage time commitment to the project. These base salary costs were equally shared among the 2 intervention arms. For the 4 review sessions that were delivered through home visits in the mixed-delivery arm, we add to these salary costs the time use costs that CHVs, mentor CHVs, and subcounty supervisors spent delivering or supervising these sessions using an estimated hourly wage rate and the time taken to conduct these activities . These tTravel and accommodationThese costs include transportation, meals, and lodging for CHVs, mentor CHVs, and subcounty supervisors to attend the 2 centralized 5-day training sessions in Kisumu, transportation costs for supervisors to supervise sessions, and transportation and meal costs for mentor CHVs and CHVs to attend the monthly refresher trainings hosted in the respective subcounties. These costs again differ by delivery arm due to the travel time necessary to conduct home visit review sessions in the mixed-delivery arm, again priced at local wage rates.Supplements and incentivesWe include milk, soap, or eggs given to participants during sessions, as well as a locally published picture book given to all children in session 14.These costs included start-up costs such as the cost of tablets used for session monitoring and an online data transfer platform, administrative costs such as the design and printing of manuals for CHVs used during training, mobile phone credit for CHVs, mentor CHVs, and supervisors, and the printing of T-shirts for CHVs and certificates for participants who completed the program.In addition to the provider costs above, we include the following societal costs.Mothers’ opportunity cost of timeMothers who participate in Msingi Bora pay opportunity costs of their time stemming from attending sessions, as well as enacting the new recommended behaviors with their children at home. To price this time, we again use the 2015 to 2016 KIHBS to estimate an implied hourly wage for all mothers based on the average wage rate for females in the 2 study counties, regardless of their employment status, and updated to 2020 prices . We combVenuesWhile our program enjoyed the free use of community centers or churches for the group-based sessions, under a scaled version of Msingi Bora, we estimate that venues would no longer be free and thus include a daily rental fee per venue per group session, for which there are 12 in the mixed-delivery arm and 16 in the group-only arm .We calculate incremental cost-effectiveness separately for each delivery model as the ratio of that intervention model’s ITT impact on each child outcome divided by that model’s total present period costs, where we use a societal perspective and present results in 2020 US$. This allows us to interpret our results in terms of SD improvement in a given outcome per US$100 investment by intervention arm. Following standard recommendations for reporting of cost-effectiveness , we alsoTo estimate BCRs and ROIs of our program in the long term, we additionally consider future costs and benefits that would result from increased cognitive abilities among participants in a scaled version of our program. Future costs include additional schooling costs due to increased attendance, and future benefits include net gains in lifetime wages.p < 0.0001) years of schooling after adjusting for covariates including parental education, survey wave and month of interview, a female indicator variable, and baseline 1998 school grade fixed effects  rounds 1 to 4  ,28, whicp < 0.0001) increase in annual wages after controlling for the same covariates as the schooling regressions  data on wage profiles by age of children as adults for our setting; and (b) the predicted intervention impacts on wages. We obtain potential wage profiles by age of children as adults from a representative sample of individuals aged 16 to 64 again using the 2015 to 2016 KIHBS. We restrict the sample to at least halftime workers (working at least 20 hours per week in any paid occupation) and calculate the life stream of average earnings by age expressed in 2020 prices, as well as the sum of discounted earnings to present values adjusting for expected survival probabilities using age life tables from Kenya . To predFor our BCA, we follow standard practice to translate the long-term costs and benefits of our program into present discounted value (PDV) terms using an annual discount rate to reflect their delayed nature. We assume an annual discount rate of 5%, which is in line with previous benefit–cost analyses of ECD programs from LMIC settings –33. HoweWe use our estimates of the full societal benefits and costs that includes these future schooling costs and wage increases to estimate both a BCR and the expected ROI. The BCR compares the child’s expected increase in discounted lifetime earnings resulting from early cognitive gains following participation in Msingi Bora versus its average costs per child, also expressed in PDV terms. The ROI is the difference in additional expected tax revenues due to the increased PDV of lifetime earnings from the intervention relative to the average costs per child, expressed in terms of a percentage change. For calculating ROI, we assume a 14.6% average tax rate, again based on KIHBS earnings data updated to 2020 prices and matched to Kenya’s marginal tax rate schedule from 2020 . In a seIn order to derive SEs for our estimated BCRs and ROIs, we perform 1,000 Monte Carlo simulations that allow for uncertainty in our estimates of wage and schooling returns to cognitive abilities, assuming that all these parameters are normally distributed random variables.We conduct 2 types of sensitivity analyses. We first test for the robustness of our BCRs and ROIs by systematically varying one parameter at a time while holding all others constant. This allows us to identify what “extreme values” are needed for our BCR to be less than 1 and our ROI less than 0, which, in turn, would make scaling Msingi Bora a poor societal investment. We vary the discount rates, total costs per child, average tax rate, cognitive wage returns, and the public cost of an additional grade per child per year to find these extreme values. In addition to these parameters, we test for the degree of fade-out in impacts in the longer-term that our program could sustain while still maintaining positive net benefits and ROIs, which is motivated by a recent systematic review of ECD parenting trials from LMICs that finds that early intervention impacts on children’s developmental outcomes tend to fade over time .In addition, in Table B in The total costs for the program were US$56,171 for the group-only arm and US$58,103 for the mixed-delivery arm , which tThe present period societal costs were US$8,748 in the group-only arm (US$22 per child) and US$9,433 in the mixed-delivery arm (US$24 per child) . This smWe estimate gains in lifetime wages from participation in the program of US$2,729 per child in the group-only arm and US$1,784 in the mixed-delivery arm , a diffeIn Table B in We conducted an economic evaluation of 2 potentially scalable and effective group-based ECD parenting interventions targeted to mothers with young children in rural Kenya. We found that the total costs per child were slightly smaller for the group-only model (US$140) than for the mixed-delivery model (US$145) that substituted periodic home visits for group sessions. Coupled with the fact that effect sizes in child outcomes were consistently higher for the group-only delivery model , these cost estimates imply that the group-only model was also the most cost-effective, with effect sizes per US$100 additional investment of 0.37 SD in cognition, 0.30 SD in receptive language, and 0.16 SD in socioemotional outcomes.We also found that the long-term economic returns of scaling Msingi Bora could be extremely high if our predicted long-term program impacts hold. Focusing on cognitive outcomes, we find a BCR of 15.5 for the group-only delivery model and 10.6 for the mixed-delivery model, while the average ROIs for a government scaling the program are 127% and 55%, respectively. We would also require very extreme values of our input parameters to have negative ROIs and BCRs less than 1. For example, program costs would outweigh benefits under the group-only model with a discount rate of 17% or if our early impacts in cognition almost entirely fade out in the longer term from 0.52 SD to 0.03 SD.The small difference in costs across delivery models of US$5 per child is perhaps surprising. However, it is worth remembering that in 12 of 16 sessions (75%), the delivery model was identical across intervention arms, so the only difference in costs comes from the 4 review sessions. In addition, even though CHVs in the mixed-delivery arm spent an average of 27 additional hours traveling to and delivering each review session as home visits, when priced at current local wage rates, the additional hours spent did not translate into large differences in monetary costs.Our group-only delivery model remains highly cost-effective when we compare our ICERs based on a health provider’s perspective to those we calculate for 2 recent effectiveness studies from Pakistan and India reporting unit costs for similar parenting interventions targeted to children aged 0 to 3 from LMICs and focused on similar outcomes of children’s cognitive and language development ,14. We cAnother recent effectiveness study conducted in Odisha, India compared group meetings versus individual home visits over 2 years and similarly found group visits to be much more cost-effective for a relatively similar sample to ours . This stMsingi Bora’s estimated BCRs are also comparatively high with the very limited number of related interventions reporting similar estimates  includinOur study has numerous strengths related to methods and the economic evaluation itself. To the best of our knowledge, our study is the first economic evaluation of an ECD parenting intervention targeted to children under 3 years of age in SSA. Moreover, our study is unique in comparing the cost-effectiveness of different models of delivery contributing to the debate of potentially scalable models for ECD parenting interventions in low-resource settings. Strengths of our study include adoption of a societal perspective for our CEA that accounts for private opportunity costs to participants and delivery agents, the combination of cost-effectiveness and BCAs to assess outcomes, and the inclusion of sensitivity analyses showing the values at which the program generates negative returns. All of these complement each other to provide policymakers a more comprehensive portrait of private and social costs as well as expected economic returns of our program. Limitations of our analysis include that our BCA necessarily relies on different assumptions of how program impacts translate into long-term benefits and costs using external data sources and that we assume that our early impacts can be sustained in the long term. Future work could address these limitations, but obviously necessitate the passage of time. We also do not account for varying levels of schooling quality across public or private schools that may present different economic opportunities for children enrolled in Msingi Bora, and we do not account for patterns of formal and informal employment or internal migration that may influence the profiles of wages by age we used to estimate benefits. We also examine the economic benefits of enhanced human capital only in terms of increased wages, but we do not include other outcomes that could potentially improve such as labor market participation, share of workers in the informal sector, health, life expectancy, or prosocial behaviors. We may similarly be missing some societal costs in addition to those included. As discussed above, we believe the BCR outcomes can be interpreted as the lower bound of the true societal returns as long as our early impacts are sustained, but if there are large costs incurred to society as a result of intervention-related improvements, leaving these out may modify our BCR and ROI estimates.Our findings highlight the need for careful cost analyses to accompany evaluations of ECD parenting programs in LMICs to help inform governments and policymakers on the potential returns to society and to the government of investing in such programs. The growing recognition worldwide about the importance of a child’s early years and the potential promise of responsive parenting interventions to improve children’s outcomes are not likely to affect policy decision-making without accompanying information about the cost-effectiveness of such investments. We also believe that cost-effectiveness analyses of ECD parenting programs should account for the private opportunity costs to participants and delivery agents for both ethical and practical reasons: Excluding these costs could create perverse incentives for governments to offload program costs onto users and delivery agents, who are often vulnerable and poor citizens whose burden of time should not be assumed to be free. We hope our results can motivate similar analyses from other ECD parenting programs from LMIC settings.Our results show that an integrated child development intervention featuring exclusively group sessions delivered by paraprofessional community health workers can be very cost-effective. We find that a mixed-delivery model that substitutes periodic home visits for group sessions comes at a slightly higher cost, albeit with a lower benefit, and thus is less cost-effective for our setting. Our results represent a strong case for exploring pathways to scale similar interventions in low-resource rural settings where many children and families could potentially benefit from ECD parenting programming, and investments could pay significant dividends over time.S1 ChecklistCHEERS, Consolidated Health Economic Evaluation Reporting Standards.(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S1 DataBCA, benefit–cost analysis; CEA, cost-effectiveness analysis.(ZIP)Click here for additional data file.