Baculoviridae) that infect lepidopteran pests have an established record as safe and effective biological insecticides. Here, we describe a new approach for the development of NPV-based insecticides. This technology takes advantage of the unique way in which these viruses are transmitted as collective infectious units, and the genotypic diversity present in natural virus populations. A ten-step procedure is described involving genotypic variant selection, mixing, coinfection and intraspecific coocclusion of variants within viral occlusion bodies. Using two examples, we demonstrate how this approach can be used to produce highly pathogenic virus preparations for pest control. As restricted host range limits the uptake of NPV-based insecticides, this technology has recently been adapted to produce custom-designed interspecific mixtures of viruses that can be applied to control complexes of lepidopteran pests on particular crops, as long as a shared host species is available for virus production. This approach to the development of NPV-based insecticides has the potential to be applied across a broad range of NPV-pest pathosystems.Nucleopolyhedroviruses (NPV,  Biological insecticides based on pathogenic microorganisms form part of the larger group of biorational insecticides that are selective, low-risk products and technologies that conserve natural enemy populations and present a reduced risk to non-target organisms and the environment . The neeBaculoviridae, genus Alphabaculovirus) are virulent pathogens that lethally infect the larval stages of these insects, including many species of pests. Natural isolates of these viruses have been used as the active ingredients of biological insecticides in developed and developing countries worldwide (Lepidopteran nucleopolyhedroviruses (NPVs)  that form a protein complex that is essential to gain access to the host cell during the primary infection process  gene  spread across the genome and insertions, deletions, inversions and duplications that are frequently located around hotspots such as ro genes . Signifiol) gene , and in ol) gene , and alssmission .In other cases, structural variants with large deletions, sometimes representing over 10% of the genome, are present in up to one third of all variants in some NPV populations . For exa–7 substitutions per nucleotide per round of copying, typical of the very low mutation rates observed in DNA viruses (–3) in NPV populations  are enveloped together in groups of 1–29 nucleocapsids per ODV . ConsequCooperative social interactions among viruses can have clear benefits for their transmission, replication, and ability to overcome host immune defenses . For exa(i)The structure of the OB itself allows the virus to persist and disperse in the environment, in soil or plant surfaces, for extended periods until consumed by a susceptible larva. This allows virus survival during periods of low host density, including overwintering .(ii)Multi-nucleocapsid ODVs deliver various nucleocapsids to the host cell, a fraction of which may uncoat in the cell nucleus and initiate replication, whereas others can travel to the basal membrane, acquire the GP64 protein (group I NPVs) that is produced early in infection. These nucleocapsids can then leave the cell as budded virus to establish a rapid systemic infection that does not depend on the destiny of the primary infection of the midgut cell . In effe(iii)Simultaneous infection by multiple virus genomes could provide a mechanism of cooperative positive feedback between virus genome templates and the virally encoded products that promote replication , althoug(iv)A group of virus genomes may be better able to overcome the innate immune response of the host cell . As NPVs(v)Spodoptera frugiperda was demonstrated to result in the transmission of between one and five genotypic variants, including rare variants that comprised less than 1% of the virus inoculum , ac78 and sf32 have been demonstrated to influence the number of nucleocapsids (genomes) enveloped in each ODV  affects the occlusion of ODVs in cell culture conditions  are the main structural components of the viral OB . These peach ODV . In contthin OBs , whereasnditions . Of theselection .Here, we propose the coocclusion of laboratory-controlled selected combinations of genotypic variants as a novel strategy for the development of NPV preparations with improved insecticidal activity. This approach originated from the observation that a natural Nicaraguan isolate of Spodoptera frugiperda multiple NPV (SfMNPV) comprised nine different genotypic variants. Of these, three defective variants were not infectious when administered perorally to larvae because they lack the genes for certain PIF factors that are essential for infection in the midgut of the larval host. These defective variants were able to take advantage of the cellular pool of virally encoded proteins produced by the other variants present within the cell, including the PIF factors . OBs comThese findings indicated that interactions among cooccluded variants (that by definition must have replicated in the same host cell) could alter the phenotype of progeny OBs. Additional studies with this pathosystem revealed that the number and identity of variants in cooccluded mixtures and their ratio resulted in mixtures with altered OB pathogenicity, speed-of-kill and OB production characteristics , 2008.These findings led us to design the following ten-step pathway for the development of the active material for NPV-based insecticides .(i)Briefly, this involves access to a collection of natural NPV isolates from the insect pest of interest (step 1).(ii)Dose or concentration-mortality bioassays are then performed in the target host to identify the most pathogenic, fast-killing and productive natural isolates available (step 2).(iii)in vivo cloning using very low concentrations of BV inoculum injected into larvae over several rounds, although this is a laborious procedure compared to plaque purification (step 3). Importantly, cell culture is likely to select for variants that are particularly amenable to replication in an in vitro system, whereas in vivo cloning of BVs replicates natural conditions, except for the critical gut infection process.The selected isolates are then subjected to purification in a suitable cell line (plaque purification) or by (iv)Sampling of a large number of isolated plaques in combination with a genotype characterization procedure, such as the use of restriction endonuclease profiles, allows the classification of genotypic variants according to their prevalence in the natural isolate population. It is usually necessary to amplify plaque picks by injection into larvae to produce sufficient genomic DNA for restriction enzyme analysis (step 4).(v)Each identified variant  is then subjected to insect bioassay to determine OB pathogenicity, speed of kill and OB production characteristics (step 5).(vi)Variants with particularly interesting insecticidal traits can then be selected for coocclusion studies. At this stage it is useful to acquire genome sequence information for the variable regions so that qPCR primers can be designed for subsequent quantification of laboratory-controlled mixtures of the variants. For coocclusion, pure variant OBs can be mixed and used for peroral inoculation of larvae in different ratios for coocclusion of the selected combinations of variants (step 6). The use of high inoculum concentrations or doses ensures the infection of larvae by variants that differ in OB pathogenicity. Alternatively, if variants have no or very low peroral infectivity, the injection of known mixtures of variant BVs into host larvae allows variant replication in coinfected cells and, as a result, the production of cooccluded mixtures of variants.(vii)Once cooccluded mixtures of variants have been produced , their i(viii)Following this, the most promising combinations are subjected to serial passage in larvae at an intermediate inoculum concentration, such as a 50% lethal concentration of OBs (step 8).(ix)Serial passage involves inoculating larvae and collecting the OBs from those that die from lethal infection. These OBs are then used as the inoculum for the following round of inoculation. In essence, this is a process of artificial selection for variants in the most transmissible proportions. Between four and six steps of serial passage can result in marked changes in the proportions of the variants present in the cooccluded mixture. These changes can be readily quantified by qPCR. Following this procedure, the resulting OBs are subjected to insect bioassay to determine the improvement, if any, in pathogenicity, speed-of-kill and OB production characteristics of the passaged OBs (step 9).(x)Bacillus thuringiensis (step 10).Finally, the selected combination of variants can be subjected to greenhouse and field testing to determine the efficacy of the preparation compared to reference biorational products, such as insect growth regulators, spinosyns or insecticides based on cis and trans acting factors. An example of a trans effect is that of viral enhancins, located in the OBs, which are released in the midgut as OBs dissolve   . As a reIn reality, for multiple type NPVs, variants that replicate together will most likely be enveloped together in ODVs and cooccluded in mixed variant OBs. In contrast, for single type NPVs in which nucleocapsids are enveloped individually, the OB, containing numerous such ODVs, defines the collective infectious unit of transmission. The situation is different for granuloviruses, as each granulovirus OB usually occludes only a single ODV comprising a single nucleocapsid , althougThe application of the intraspecific coocclusion procedure can best be exemplified by two examples.The Chysodeixis chalcites nucleopolyhedrovirus (ChchNPV) is a single type NPV in which ODVs comprise a single nucleocapsid with a single genome. Coocclusion of selected variants of ChchNPV resulted in a faster killing and more pathogenic preparation than a wild-type isolate from Tenerife, Spain . An inithoar and bro-d genes  had an OB potency 6.3-fold higher than that of the SP1 wild isolate, although with a slightly (6%) slower speed of kill . Serial e tested . Subsequngiensis . PotentiS. frugiperda larvae, and (iv) SeMNPV + SfMNPV (95:5%) which replicated in S. exigua larvae. As host susceptibility to each virus and the replication efficiency of each virus differed, the composition of the cooccluded mixture was adjusted by altering the doses of OBs of each virus used to inoculate larvae, or by inoculating larvae with the heterologous virus followed 24 h later by the homologous virus (S. exigua larvae, albeit at decreasing prevalence, despite lacking the pif-1 and pif-2 genes. The defective variant was cotransmitted with SeMNPV due to the presence of PIF factors produced by SeMNPV in cells infected by both viruses. A subsequent study demonstrated that the asynchronous inoculation of larvae at intervals of 12–72 h resulted in marked changes in the composition of the cooccluded mixture of viruses (As mentioned previously, the narrow host range of NPVs limits their use in crops attacked simultaneously by several species of pests . Followius virus . In the  viruses . This wa viruses .in vitro due to the presence of AcMNPV proteins in the ODV envelope (Plaque purification and end-point dilution of ODVs released from mixed-virus OBs revealed that the viruses had been coenveloped to form mixed-virus ODVs prior to coocclusion . For exaenvelope . The exaenvelope . The mixenvelope .An additional laboratory demonstration of the value of the technology came from a study in which the single nucleocapsid HearNPV was cooccluded with the multinucleocapsid nucleopolyhedrovirus, HearMNPV. The cooccluded mixture was able to infect, replicate and persist during insect serial passage in two host species that are not susceptible to HearNPV alone, again as a result of the sharing of pooled proteins in coinfected cells . Such fiThe advances described in the present review open the way to a diversity of future studies, both as a means to improving the efficacy of NPV-based insecticides and as a research tool to better understand novel aspects of host-virus and virus-virus interactions in NPV-based pathosystems. Potential improvements to NPV-based insecticides include the possible potentiation of the insecticidal properties of mixtures of viruses, as observed when mixtures of variants are cooccluded. The use of virus mixtures and the findings that mixtures of HearNPV and HearMNPV resulted in altered host range suggest that this technology could provide a means to control complexes of lepidopteran pests in certain crops. It may also allow a broader range of host species to be used for the production of mixed-virus preparations, although the impact of the use of alternative hosts on the insecticidal characteristics of the resulting preparation would require careful evaluation.The industrial production of mixed-variant OBs could be achieved following established procedures , althougThe need for full testing of the efficacy of virus mixtures to control complexes of pests in the field is also a priority for establishing which pests and pest complexes are most amenable to control by mixed-virus technology. This approach has begun to be studied by others, for example for the control of lepidopteran pests in soybean , 2021. SFrom a research perspective, proteomic studies on the composition of pseudotyped viruses produced in coinfected hosts could contribute to defining the influence of ODV or BV composition on both virion infectivity and the virion-associated factors that determine host range.cis interactions. Nonetheless, other possible interactions could occur between viruses infecting the same host, but different cells, in a trans interaction mode. For NPVs, the success of primary infections appears to rely on the former, but granuloviruses probably use the latter, possibly mediated through cells infected by helper genotypes, which release a diffusible factor that circulates within the infected larva (Finally, this review explored the interactions between viruses infecting the same cell, i.e., ed larva . Future ed larva , and aidTW wrote the manuscript. PC and ML-F edited and improved the manuscript. All authors conceptualized the review, contributed to the article, and approved the submitted version.Bioinsectis SL is a spinoff company of the Universidad Pública de Navarra that is currently evaluating the commercial applications of the coocclusion technology described in this review. 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.


High throughput sequencing (HTS) has revolutionised virus detection and discovery, allowing for the untargeted characterisation of whole viromes. Viral metagenomics studies have demonstrated the ubiquity of virus infection – often in the absence of disease symptoms – and tend to discover many novel viruses, highlighting the small fraction of virus biodiversity described to date. The majority of the studies using high-throughput sequencing to characterise plant viromes have focused on economically important crops, and only a small number of studies have considered weeds and wild plants. Characterising the viromes of wild plants is highly relevant, as these plants can affect disease dynamics in crops, often by acting as viral reservoirs. Moreover, the viruses in unmanaged systems may also have important effects on wild plant populations and communities. Here, we review metagenomic studies on weeds and wild plants to show the benefits and limitations of this approach and identify knowledge gaps. We consider key genomics developments that are likely to benefit the field in the near future. Although only a small number of HTS studies have been performed on weeds and wild plants, these studies have already discovered many novel viruses, demonstrated unexpected trends in virus distributions, and highlighted the potential of metagenomics as an approach. Metagenomics is the analysis of environmental samples using high-throughput sequencing (HTS), with the intent of characterising microbial communities. HTS has the advantage of the identification and genomic characterisation of viruses in a target-independent manner, and it has proved to be a valuable tool for virus detection, discovery, or diversity studies. Classic virus detection methods, such as the enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR), usually target one or a few viral species and require prior knowledge of the virus to be detected, while HTS methods have been developed to look for virus-like sequences without the bias of only detecting known viruses. HTS technologies allow a generic approach to virus identification and can deliver species, or even strain-specific results . There aIn this review, we focus on the role of weeds and wild plants in virus emergence, and discuss recent metagenomic studies of weeds which show the potential of this approach for untargeted virus detection in wild hosts. We review recent discoveries of plant viruses inhabiting various ecological niches and discuss “pros and cons” of using metagenomics for this purpose. Moreover, we show the potential of using HTS to explore the diversity and richness of the viruses associated with plant populations. Finally, we discuss the future directions of plant virus metagenomics in weeds and wild plants.In agroecosystems, crop plants often grow side by side with weeds and wild plants. These wild plants constitute potential reservoirs for viruses that may spread into cultivated plants, thereby leading to epidemics or to the emergence of novel viruses ,4,5,6. MCavariella aegopodii. PYFV can be transmitted to carrot (Daucus carota) by aphids only when they have first acquired AYV. Carrot is not susceptible to infection by AYV, but introduction of the virus complex into carrot crops leads to scattered infections because individual aphids retain the transmission-dependent combination for only a few days  comprising 400 specimens of the target hosts collected from twenty sites in the Tallgrass Prairie Preserve in northeastern Oklahoma over four years. Samples were tested for the presence of plant viruses applying VANA and double-stranded RNA enrichment methods, allowing for comparisons of community composition based on the host species, location, or time of collection. This large-scale metagenomic study classified viral sequences into OTUs for a better assessment of virus diversity. Results indicated that host species had a significant effect on virome composition compared to the location and sampling time. The majority of plant viruses identified during the study were novel viral species that had not previously been reported.All of the above-mentioned studies explore the spatial variation between viral communities in weeds and wild plants; however, information on the temporal variation in wild plant virus communities is still very limited. Thapa et al.  analysedHTS techniques can also be used to study different host–pathogen interactions . By combArabidopsis halleri populations shows seasonal variation in accumulation, whilst the prevalence and genome of the virus populations are surprisingly consistent [We have provided an overview of different studies that use HTS methods on wild plants and weeds in nsistent .Are there any general features of plant-associated viromes that can be tentatively identified, based on the studies reviewed here? First, there is the observation that diverse dsRNA viruses, a very large proportion of which appear to be novel, usually dominate the plant-associated viromes ,31. A seLuteovirus genus (Tombusviridae family) and infect a wide range of grass species [In natural ecosystems, host populations typically have greater genetic diversity and more complex age structures than populations in agroecosystems, particularly in regions where crops are grown as monocultures with low genetic diversity. In wild plant communities, by contrast, plant genomes are under natural selection. This may favour the persistence of multiple resistance alleles or result in selection for novel resistance alleles. Understanding the role of plant genetic diversity in controlling patterns of virus emergence is a long-standing goal of biological research, and it is central for the management of current infectious diseases and developing long-term strategies to prevent the emergence of new ones . Increas species . CYDV an species . The int species ,12,58,59P. lancelota populations across the Aland Islands in Finland. They investigated whether the structure of virus communities differed between natural and agriculture edge settings. The study revealed that virus species richness declined with increasing plant diversity in a natural ecosystem, while in cultivated areas virus species richness was moderately higher, and not associated with plant richness. This difference was not explained by changes in host richness between these two habitats, suggesting potential virus spillover and increased transmission of viruses across the agroecological interface [The opposite scenario has been shown in the study of Susi and Laine , who invnterface . The effPolerovirus genus, Luteoviridae family) can induce changes in the alighting, settling, and probing behaviour of its vector, the cotton aphid Aphis gossypii [Some viruses capable of devastating crops and harvested products have become more active and damaging because their geographical ranges have expanded due to climate change. A changing climate can contribute to the successful spread of newly introduced viruses or their vectors, and the establishment of these organisms in new habitats that were previously unfavourable. Non-native plants and insects may also expand their geographic ranges and hereby invade communities where they were previously not present. These species might constitute reservoir hosts or vectors of plant viruses capable of causing epidemics in nearby crops . In terrgossypii . CABYV igossypii .The efficacy of viral metagenomics to detect variable virus communities in weeds and wild plants relies on sampling, nucleic acid extraction, high-throughput sequencing, and bioinformatic analysis strategies. Each of these steps will pose its own challenges and may affect estimates of virus community composition.The choice between protocols using viral particle enrichment or total nucleic acid extraction protocol will affect the relative abundance of viral reads, as reviewed by Maclot et al. . It is iSecondly, another critical step in plant virus metagenomics is the preparation of high-quality nucleic acid extracts. Polysaccharides, high levels of RNases, polyphenols, and fibrous tissues are often encountered in plant samples, and fibrous tissues such as lignin (wood) are difficult to break up and remove, which makes extracting high-quality nucleic acids challenging for wild plant species. Numerous adaptations have been made to nucleic acid isolation methods—including modifications to tissue storage, maceration, preparation and extraction—to obtain sufficient, high-purity nucleic acids for non-model plant species, depending on the tissue type and the presence of secondary metabolites and inhibitors . These etombusviridae [Medicago arborea, a shrub in the Fabaceae family [Analysing virus metagenomics data in weeds and wild plants is challenging, both conceptually and practically. Currently, the HTS method commonly used for viral metagenomics is Illumina sequencing. Illumina HiSeq and NextSeq instruments generate short, paired end reads of 2 × 75−150 bp in length, read from the short fragments produced during the library preparation process. Illumina MiSeq offers a considerably lower volume of reads, but read length can be doubled up to 2 × 300 bp paired end reads. From these short sequencing reads, longer virus genome sequences can be assembled using a variety of pipelines and software packages. After the assembly process, viral genomes can, in some cases, be incomplete , or assembly can result in chimeric sequences (where reads from different genetic origins within a sample are similar and therefore erroneously assembled into one sequence). Chimeric assemblies are more likely more likely to happen when metagenomics are performed on pooled samples, which is common in plant virus metagenomics studies. In the future work, the problem of chimeras may be solved by using HTS methods which generate longer reads, such as Oxford Nanopore sequencing, or PacBio SMRT, or HiFi sequencing. Due to the small genome size of plant viruses, ranging from 4 to 30 kb , tombusvsviridae , and sevsviridae , Plum posviridae  and Yam sviridae . Howevere family . In thesAlthough long read sequencing provides opportunities for plant virus metagenomics, there are a few drawbacks. Firstly, all library preparation protocols for RNA sequencing require poly-A tailed RNA as input, as sequencing adapters are designed to bind to this region. However, many plant virus species do not have a poly-A tail. This limitation can be circumvented by adding a poly-A tailing step prior to sequencing, but this reaction is not specific to viral RNA and will also affect any host RNA present in the sample . SecondlWhile other microbial communities, such as bacteria or fungi, may be routinely characterised through a conserved gene , viruses have no such conserved genomic regions due to their varied evolutionary history. Since there is no universal marker for viruses, viral genome segments are recognised through sequence similarity to other viruses. This results in several problems in identifying viral metagenomes from weeds. Firstly, virus genome databases are incomplete, and there is currently a bias towards plant viruses found in crops. This means that viruses that occur exclusively in wild plants may not be recognised due to a lack of sequence similarity with known viruses. The significant proportion of viral metagenomics data that has no detectable sequence similarity with any known biological entity has been referred to as “dark matter”, and its viral nature remains an open question ,51. The Lastly, the analysis of virus metagenomics data is a practical challenge for non-specialised teams. Working with HTS-data requires computing equipment capable of storing and processing large datasets, as well as expertise on genome assembly analysis pipelines and packages. However, the decrease in its basic operational costs, further development of simple, reliable, and user-friendly bioinformatics tools for data analysis and sequence mapping will make the utilisation of metagenomics affordable to a larger group of researchers and phytosanitary services. Although virus detection using HTS is a powerful tool, in case of limited coverage across a viral genome and low sequencing depth, confirmatory tests may be required.In summary, metagenomics approaches have a huge potential for detecting viruses, characterising viromes and exploring virus emergence and epidemiology. Plant virus metagenomic studies have shown that viruses are common in weeds and wild plants, even in the absence of symptoms, and these studies have uncovered many new, uncharacterised viruses. These results highlight how powerful HTS approaches are for virus discovery, whilst also showing the spectacular amount of viral biodiversity harboured by wild plants. Nonetheless, HTS methods have also some pitfalls and several challenges remain in this field of study: choices regarding protocols for enrichment and sequencing of plant viromes will affect the virus species and viral genetic variation that can be uncovered. This can be achieved by the wider implementation of plant virus metagenomics studies, which have the capacity to detect viruses alone or in mixed infection and the potential to reveal the presence of novel or unsuspected viruses ,86. Key


Cryptococcus gattii is a primary fungal pathogen, which causes pulmonary and brain infections in healthy as well as immunocompromised individuals. Genetic manipulations in this pathogen are widely employed to study its biology and pathogenesis, and require integration of foreign DNA into the genome. Thus, identification of gene free regions where integrated foreign DNA can be expressed without influencing, or being influenced by, nearby genes would be extremely valuable. To achieve this goal, we examined publicly available genomes and transcriptomes of C. gattii, and identified two intergenic regions in the reference strain R265 as potential “safe haven” regions, named as CgSH1 and CgSH2. We found that insertion of a fluorescent reporter gene and a selection marker at these two intergenic regions did not affect the expression of their neighboring genes and were also expressed efficiently, as expected. Furthermore, DNA integration at CgSH1 or CgSH2 had no apparent effect on the growth of C. gattii, its response to various stresses, or phagocytosis by macrophages. Thus, the identified safe haven regions in C. gattii provide an effective tool for researchers to reduce variation and increase reproducibility in genetic experiments. Cryptococcus species complexes is responsible for 181,100 deaths annually [Cryptococcus neoformans species complex [C. gattii species complex is a primary pathogen and can cause fatal infections in both immunocompromised individuals and individuals with no apparent immunodeficiencies [C. neoformans and C. gattii cause fatal meningitis, C. gattii is more likely to cause severe lung disease and death, without CNS dissemination [C. gattii is responsible for the Vancouver Island outbreak in 1999, and it has spread to the north–western US [C. gattii diverged from C. neoformans roughly 37 million years ago [C. gattii species complex comprises four distinct molecular types, VGI-VGIV [Cryptococcosis caused by the pathogenic  complex ,3,4. By ciencies ,6. Althomination . C. gattCryptococcus species complexes [C. neoformans species complex (H99 and XL280), and Af293/CEA10 strains of A. fumigatus [C. gattii.In the past two decades, major advances in molecular technology have facilitated our understanding of the pathogenic omplexes ,12,13,14omplexes ,16. Thisomplexes ,18. Thusumigatus ,20,21. Tumigatus ,23,24,25C. gattii to facilitate the identification of potential gene free regions that are unlikely to be in a constitutive heterochromatin state. We identified two such gene free regions as potential “safe haven” sites: CgSH1 and CgSH2. We found that integration of a fluorescent and a selection marker gene at CgSH1 and CgSH2 did not affect the expression of their neighboring genes. Moreover, DNA integration at these regions per se had no apparent effect on all the phenotypes examined, including growth, thermotolerance, melanization, capsulation, tolerance of cell wall stress, response to osmotic stresses, and phagocytosis. Therefore, we believe that CgSH1 and CgSH2 provide safe haven sites to facilitate genetic manipulations in C. gattii strains. Together with the previous work for the C. neoformans species complex, this study completes the important task of identifying safe haven regions for the entire pathogenic cryptococcal species complexes.In this study, we took advantage of the genomic data and transcriptome data that are publicly available for C. gattii R265 were downloaded from NCBI (GenBank assembly accession: GCA_002954075.1). According to the orientation and positions of two neighboring genes, we classified the intergenic regions as convergent, divergent, or tandem and calculated their sizes. BAM  files and FPKM values were generated from published RNA-seq data [The genomic sequence and annotation files of C. gattii strains, a BLAST search was conducted using the sequence of CNBG_3433 as a query to identify homologues in other C. gattii strains. Then we performed subsequent BLAST analyses using the sequences of neighboring genes of the homologues as queries, and we then identified the best match for CNBG_3434 in these strains. Although CNBG_3433-CNBG_3434 and their homologue pairs remain neighboring genes in other C. gattii strains examined, the orientation of these two neighboring genes is not conserved among the 6 strains. The same strategy was used for identifying potential CgSH2 in other strains of C. gattii.To identify the potential CgSH1 in other Cryptococcus gattii strains and plasmids used in this study are listed in the 2PO4, 4% agar, pH 7), and L-DOPA medium  were used to test the phenotypes as described previously [eviously ,28. Strahttp://grna.ctegd.uga.edu/, accessed on 21 January 2022) embedded in FungiDB. Cryptococcus native U6 promoter was amplified from JEC21 while the scaffold of sgRNA was amplified from the plasmid pDD162 [TEF1 promoter. To that end, the plasmid pXL-Cas9 with the GPD1 promoter driven Cas9 was digested with NotI and FseI and then purified as the backbone vector. The TEF1 promoter was amplified from pYZ79 and purified [TEF1-Cas9 was generated and used as the template to amplify the whole Cas9 expression cassette with primers M13F/M13R [TEF1-Cas9—were introduced into R265 by electroporation and transformants were selected on YPD plates with hygromycin (200 µg/mL) and passaged on non-selective YPD media for stability testing. Transformants with correct integration into the CgSH1 were expected to yield a band of ~1.2 kb for the forward orientation and ~1 kb for the reverse orientation with the primer set Linlab3779/Lianlab8147/Linlab8148  system, as described previously . Firstlyd pDD162 . Then thpurified . After Tnlab8148 . Transfonlab8148 .ACT1 are included in t test.The indicated strains were cultured in liquid YPD medium at 30 °C with shaking at 220 rpm for 16 h. Cells were collected and flash frozen in liquid nitrogen, and then lyophilized for 24 h. Total RNA samples were extracted using the PureLink RNA Mini Kit following the instruction from the manufacture. The integrity of RNA was confirmed by gel electrophoresis. Each RNA sample (10 µg) was treated with DNase (TURBO DNA-free) according to the manufacture’s instruction. DNase-treated RNA samples were used as templates to generate the first strand cDNA by the GoScript Reverse Transcription System. The cDNA products were diluted to 8 ng/µL and 2 µL was used as template for quantitative real-time PCR (qRT-PCR) using Power SYBR Green as described previously . Primers4, pH 7.5) as described previously [Pulsed field gel electrophoresis (PFGE) was performed according to the procedures described previously ,35. Brieeviously . The agaeviously . After s2O. Images were acquired with an AxioCam MRm camera and processed with Zen pro software. The fluorescence intensity of 30 individual cells from each strain imaged at 63× magnification was quantified using ZEN “Histo definition” quantification software application. Each cell and background were selected using the circular selection tool and the average fluorescence intensity within that circle was recorded. The fluorescence intensity of each cell and its background was measured. The relative fluorescence intensity of each cell was normalized by subtracting the fluorescence intensity of the cell’s background.Fluorescent microscopy was performed using a Zeiss Imager M2 microscope . The indicated strains were cultured in liquid YPD medium overnight at 30 °C and washed with sterile ddH2O and adjusted to the same cell density , and then serially diluted. The serial dilutions of each strain (3 µL) were spotted onto various agar media for phenotypical analyses. To test thermotolerance of the indicated strains, cells were spotted onto YPD medium and incubated at 22 °C, 30 °C and 37 °C for 2 days. To analyze melanization, cells were spotted onto agar medium containing L-dihydroxyphenylalanine (L-DOPA) and incubated at 37 °C in the dark. To observe capsule production, cells were spotted onto RPMI agar medium and grown at 37 °C with 10% CO2. To test the ability of fungal cells to grow in cell wall stress and osmotic stresses, cells were grown on YPD medium supplemented with Congo Red (0.05%), NaCI (1.5 M), or KCI (1.5 M).The indicated strains were cultured overnight in liquid YPD medium at 30 °C with 220 rpm. The cells were washed with ddH5 cells per well) and cultured at 37 °C with 5% CO2 for 24 h. Before the coculture, C. gattii cells (2 × 106 cells per well) were opsonized with mouse serum for 30 min. The serum was collected from mice infected with H99 strain from a previous study [C. gattii cells. After mixing, the cocultures were incubated at 37 °C with 5% CO2 for 2 h. Then cocultures were washed three times with warm Dulbecco’s phosphate-buffered saline (DPBS) to remove non-adherent cells. The macrophages were checked under an inverted microscope  and then lysed by chilled PBS with 0.1% Triton X-100. The cell suspension was collected and diluted, plated on the YNB medium, and incubated at 30 °C for 2 days for colony forming unit (CFU) enumeration.Murine macrophage cells were used to evaluate phagocytosis as we previously described . Mouse mus study .Then theC. gattii reference strain R265 was used to identify potential safe haven regions. R265 belongs to the VGII molecular type, which is predominant among clinical and environmental isolates from Vancouver Island and is responsible for the majority of cases of cryptococcosis caused by C. gattii [a and AIR265α), which are genetically similar and differ primarily in the mating type locus, are only available in an R265 background [C. gattii reference strain would likely generate most impact in this field.. gattii ,8,38. Fu. gattii  and tran. gattii . Lastly,ckground ,42. TherC. gattii reference strain R265 were identified and classified into three categories based on the relative transcriptional orientations of the two neighboring genes: convergent (terminator–terminator or tail–tail), divergent (promoter–promoter or head–head), and tandem (promoter–terminator or head–tail). A total of 40 convergent intergenic regions (out of 2153) larger than 2 kb were selected to minimize potential disruption of any nearby promoters and terminators. We examined transcript profiles at these 40 intergenic regions and their neighboring genes based on the RNA-seq data of R265 cultured in YNB medium with or without the addition of the zinc chelator TPEN [C. gattii genes in these data sets generally lie somewhere between 10 and 20 FPKM (fragments per kilobase of transcript per million). Examining the transcript profiles of these regions allowed us to exclude 18 intergenic regions where transcripts were detected. To ensure safe haven regions are accessible for the expression of the inserted genes, 10 out of remaining 22 intergenic regions where both neighboring genes showed no transcripts were excluded because these regions could potentially be in a heterochromatin state, repressed for transcription. Eventually, we picked 3 out of remaining 12 intergenic regions for further characterization  system that we established previously [C. gattii R265 differs from that of C. neoformans H99 . To test the impact of DNA insertion in the candidate safe haven sites, we examined various phenotypes of the transformants of candidate 1 and candidate 2, and compared them to the wild-type strain R265. As shown in a-α bisexual mating by mixing these α strains with the JEC20a tester strain and co-culturing them on V8 juice agar medium. During mating, α and a cells fuse and form dikaryotic zygotes. The zygotes then transition to hyphal growth and aerial hyphal tips eventually form club-shaped basidial heads where meiosis and sporulation occur, forming four chains of basidiospores [a , it is not surprising that there are considerable genome differences among them, including chromosomal rearrangements and nucleotide sequence divergence [C. gattii species complex, we analyzed the genome sequences of six C. gattii strains that encompass all four molecular types: VGI , VGII (R265), VGIII (CA1873), and VGIV (IND107). Based on homology analyses of the neighboring genes of CgSH1 and CgSH2 in R265 and these other strains , we found that both gene pairs remain as neighbors in all these strains (C. gattii species complex.Considering the evolutionary separation of the four molecular types within the vergence ,33. To d strains . However strains B, implyi strains A. Thus, C. gattii serotype B reference strain R265 and identified two gene free regions CgSH1 and CgSH2 that can serve as safe haven sites for DNA integration. In addition to the information of genome sequence and gene annotations, we capitalized the publicly available RNA-seq data to minimize the risk of selecting unrecognized transcribed regions or potentially repressed heterochromatin regions [C. gattii species complex contains at least five recognized species or four molecular types [C. gattii in this study. According to the alignment of CgSH1 and CgSH2 based on neighboring genes in multiple strains representing all four molecular types of the C. gattii species complex, similar gene free regions exist in other C. gattii strains. In particular, the intergenic region represented by CgSH1 in all strains examined are of a reasonable size, suggesting that CgSH1 might be useful for integrating foreign DNA fragments in most, if not all, strains of the C. gattii species complex. Taken together, these newly identified CgSH1 and CgSH2 regions provide a good tool for genetic manipulation in C. gattii.In this work, we screened the genomes of the  regions . Our resar types . Howeverar types ,44. For


Identifying disease-related costs is a crucial step to plan for proper allocation of resources and future healthcare services for persons with inflammatory bowel disease (IBD). Data on pediatric inflammatory bowel disease-associated costs are limited.We aimed to estimate indirect and out of pocket (OOP) pediatric IBD-associated costs in Canada.In a nation-wide cross-sectional analysis, caregivers of children and young adults (<17 years) with IBD were invited to complete a questionnaire on lost work hours and OOP costs related to IBD in the 4 weeks prior to the survey. Participants were re-invited to periodically answer the same questionnaire every 3–9 months for 2 years. Lost productivity was calculated using the Human Capital method. Costs were reported in 2018 inflation-adjusted Canadian dollars. Predictors of high cost users (top 25%) were examined using negative binomial regression.Consecutive 243 (82 incident cases) of 262 (92.7%) approached participants completed the first survey with a total of 450 surveys longitudinally completed over 2 years. The annual median indirect costs per patient were $5,951 , with $5,776  for Crohn’s disease (CD) and $6,084  for ulcerative colitis (UC) (p=0.77). The annual median per patient OOP costs were $2,925  with $3,021  for CD and $2,600  for UC (p=0.55). Older age (10-17y) at diagnosis (p=0.04) and parents in part-time employment (p=0.01) were predictors of higher indirect costs, while female sex (p<0.001), parents with a lower education level (p<0.001) and lower annual family income (p<0.01) were associated with higher OOP costs.Indirect and OOP IBD-associated costs are substantial and more likely to affect families with unstable employment and lower annual income. Examining different strategies and interventions to reduce these costs such as virtual platforms, telephone and outreach clinics especially in poor communities and families with low annual income is warranted.CIHRThe Children’s Hospital Research Institute of Manitoba


Erythema multiforme in pigs is an acute, self-limiting disease characterized by red skin areas and often associated with anorexia, fever and respiratory problems. The cause of the disease remains unknown. In a recent study, animals of a commercial breeding herd in Greece were examined, and all animals were found seropositive for porcine reproductive and respiratory syndrome virus (PRRSV). However, neither PRRSV and porcine circovirus type 2 (PCV2) viremia nor antibodies against Aujeszky’s disease virus, African swine fever virus and classical swine fever virus were detected. Here, an extended examination of these pigs was performed on a wide range of porcine viruses using highly sensitive polymerase chain reaction (PCR)-based methods. Affected skin of five animals revealed the presence of porcine lymphotropic herpesvirus-1 (PLHV-1) in all cases, PLHV-2 in one animal and PLHV-3 in four animals. However, neither porcine cytomegalovirus (PCMV) nor porcine circoviruses  were detected. In blood samples, PLHV-1 was present in two animals and PLHV-2, PCV2 and PCV3 in one individual, with PCMV, PCV1 and PCV4 in none of the animals. In one animal, four viruses were found in the blood . A PRRSV viremia was also not detected. All animals carried porcine endogenous retrovirus C (PERV-C) in their genome, but recombinant PERV-A/C was not detected. The results suggest that porcine viruses may be involved in erythema multiforme in these animals and that further studies are needed to assess the role of these pathogens in the disease. Mycoplasma pneumoniae, whereby the target lesions predominantly occur on the trunk. Other triggers of erythema multiforme may be parapoxvirus, varicella-zoster virus, adenoviruses, hepatitis viruses, human immunodeficiency virus and cytomegalovirus. Fungal infections have also been reported to be associated with erythema multiforme [Erythema multiforme is a disease described not only in humans but also in pigs and other animals. In humans, erythema multiforme is characterized by skin eruption with typical target lesions. It is acute and self-limiting and usually resolves without complications. There may be mucous membrane involvement. Erythema multiforme in humans is considered to be a hyperergic mucocutaneous immune-mediated reaction to infections. Herpes simplex virus (HSV) is usually the causative agent in the majority of adults . HSV caultiforme .Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis) seems to have been activated, negatively affecting the health status and performance of the breeding stock. All animals were found seropositive for porcine reproductive and respiratory syndrome virus (PRRSV); however, no PRRSV, porcine circovirus type 2 (PCV2) viremia, antibodies against Aujeszky’s disease virus , African swine fever virus or classical swine fever virus were detected [In pigs, skin lesions or skin abnormalities, such as abnormal color changes, are caused by bacterial pathogens  or viral pathogens  infections) ,4,5. Howdetected . Moreovedetected  and in wdetected ,9. In Gödetected ,10.To analyze whether virus infections may be associated with erythema multiforme as described in humans, diseased pigs in a pig farm in Greece were screened by molecular biological methods, including highly sensitive PCR-based methods. These methods had been previously developed to screen donor pigs and recipients before and after xenotransplantation ,12,13.The animals analyzed in the present case report were part of a commercial breeding stock (Large White × Landrace), suffering from a disease that was described previously and characterized as erythema multiforme associated with respiratory disease . The aniPasteurella multocida; erysipelas, caused by infection with Erysipelothrix rhusiopathiae, Escherichia coli and Clostridium perfringens. Weaners were vaccinated against PCV2 and Mycoplasma hyopneumoniae. Suvaxyn PRRS MLV (ZOETIS) was used against PRRSV. For antiparasitic control, all breeding females were treated with a single ivermectin injection 14 days prior to each farrowing. The feed provided to the animals was self-prepared based on a corn/barley/wheat–soya feed ration, depending on the season.The capacity of the farm, which is located in Central Greece, was 620 sows under production. All sows were moved to dry period after their weaning for the application of artificial insemination, remained for 30–35 days in individual stalls and then were removed to group-housing rooms. All sows of the farm were vaccinated against PRRSV; SuHV-1; porcine parvovirus 1; atrophic rhinitis, caused by infection with toxigenic The selected sows showed clinical signs of previously described erythema multiforme associated with respiratory disease . ParticuDuring an outbreak of these clinical signs, the percentage of diseased animals ranged from 50 to 60%. The mean number of each batch of sows/gilts in dry period is about 220–250 animals.All procedures were conducted according to the ethical standards in the Helsinki Declaration of 1975, as revised in 2000, as well as the national law and after receiving approval  from the Thessaly University Ethics Committee. Blood and skin punches from the affected skin areas of five pregnant sows suffering from red-colored, raised skin lesions were collected , frozen For histological examinations, one punch biopsy (6 mm in diameter) was taken per animal. The biopsy was fixed in 10% buffered formalin, embedded in paraffin, cut and stained with hematoxylin and eosin following standard procedures .g at room temperature. DNA was extracted from the pellet using the DNeasy blood and tissue kit. To quantify DNA, a NanoDrop ND-1000 spectrophotometer  was used, and the samples were stored at −20 °C until further processing.DNA extraction from frozen blood was set up by using a DNeasy blood and tissue kit  according to the manufacturer’s recommendations. The skin samples were treated prior to DNA extraction with collagenase  and hyaluronidase  and incubated at 37 °C at 500 rpm for 30 min on a thermoshaker , followed by 5 min centrifugation at 1000× 3 G  with a final volume of 20 μL.Briefly, to detect PERV-C, a quantitative TaqMan-PCR  was performed as described by Takeuchi et al. . The speThe investigation of PERV-A/C was performed as a conventional PCR based on a primer-probe mix detecting a 1.266 basepair amplicon as described by Wood et al. . For thi3 G .For the molecular biological investigation of several porcine viruses , species-specific real-time PCR methods were applied as previously described  18,19,2,21,22. TPRRSV testing was performed by the private laboratory “VET IN PROGRESS, GREECE”. RNA was extracted using a QIAamp cador pathogen mini kit  according to the manufacturer’s instructions. Carrier RNA was added to each sample, as it enhances the adsorption of viral RNA to the silica membranes. An internal positive control RNA  was added to all samples to ensure recovery of RNA and prove the functionality of the PCR reaction. An RT-PCR kit  was used for the detection of both viral lineages of the PRRSV, PRRSV-1 (European) and PRRSV-2 (North American) strains. The specific gene targets of the commercial assay are proprietary and not publicly available. The reaction conditions involved a reverse-transcription (RT) step at 55 °C for 10 min and RT inactivation and initial denaturation step at 95 °C for 1 min, followed by 45 cycles of a denaturation step at 95 °C for 10 s and a combined annealing-extension step at 60 °C for 1 min. A Roche LightCycler 2.0 was used.The five animals analyzed here were treated twice intramuscularly with amoxicillin  at dose of 15 mg/kg body weight, with an interval of 48 h between treatments. However, the treatment outcomes were not adequate. Typical lesions were detected in all examined sows a,b, and Histopathological examination of skin samples revealed hyperkeratotic and rare parakeratotic hyperkeratosis and moderate-to-severe epidermal hyperplasia. Scattered apoptotic bodies and few lymphocytes were present in all layers of the epidermis and follicular infundibular epithelium. Superficial and middle dermis presented increased vascularization and hyperemia .DNA was isolated and screened for several viruses using PCR or real-time PCR . PCMV, PPERV-A and PERV-B are present in the genome of all pigs, whereas PERV-C is not. PERV-C was found in all five pigs, although with different Ct values, suggesting that the copy number might vary in these pigs. Since PERV-C is present, recombination with PERV-A resulting in a PERV-A/C can occur. However, our analysis revealed that no recombination took place. In our previous publication, the animals suffering from erythema multiforme were all seropositive for PRRSV but PCR negative for this virus . The aniMycoplasma pneumoniae infections, whereas adult erythema multiforme was mainly assigned to HSV infections [Erythema multiforme is a multifactorial disease described for humans ,24,25, pfections . Since tfections ,24,29. Efections ,31,32. In pigs, group housing can result in fighting, fear and anxiety coinciding with the onset of disease . TherefoThe fact that several viruses were detected in the analyzed pigs suggests that they may contribute to the diagnosed erythema multiforme, possibly together with stress or other cofactors. However additional investigations are required to prove this. In the affected skin, PLHV-1 was present in all five animals. Only two of the five animals were positive for PLHV-1 in the blood, suggesting that virus replication occurs mostly in the skin and is not systemic. As a lymphotropic herpesvirus, PLHV-1 certainly replicates in lymphocytes in the skin. Until now, no association between PLHVs and any pig diseases had been described. PLHV-1 causes a post-transplantation lymphoproliferative disorder (PTLD) after experimental allogenic bone marrow transplantations in minipigs ,35,36. TInterestingly, in one animal, pig #4, four viruses were found: PLHV-1, PLHV-2, PCV2 and PCV3. PCV2 was found despite vaccination against the virus. PCV1 and PCV4 were not found. PCMV, a roseolovirus closely related to human herpesviruses HHV-6 and HHV-7, was also not found in any of the analyzed animals. PCMV is widely distributed in pigs, and sensitive detection methods were developed because PCMV poses a serious risk for xenotransplantation. Transmission of PCMV with transplanted pig hearts drastically reduced the lifetime of the xenotransplant in baboons  and of pThe methods used here to detect different viruses are highly sensitive and specific ; howeverPERVs are present in the genome of pigs. PERV-A and PERV-B are present in the genome of all pigs. They can be released as infectious virus particles and infect human cells in vitro and therefore pose a risk for xenotransplantation . PERV-C Five sows with diagnosed erythema multiforme were analyzed using sensitive PCR-based methods. PLHV-1 was found in all animals, and PLHV-2, PCV2 and PCV3 were found in some of the animals, but no PCMV, PCV1, PCV4, PRRSV-1 or PRRSV-2 was detected. This study indicates that viruses may be involved in the pathogenesis of the disease; however, this has to be proven by additional investigations of larger numbers of animals and using additional methods.


Rhabdoviridae is the most diverse family of the negative, single-stranded RNA viruses, which includes 40 ecologically different genera that infect plants, insects, reptiles, fishes, and mammals, including humans, and birds. To date, only a few bird-related rhabdoviruses among the genera Sunrhavirus, Hapavirus, and Tupavirus have been described and analyzed at the molecular level. In this study, we characterized seven additional and previously unclassified rhabdoviruses, which were isolated from various bird species collected in Africa during the 1960s and 1970s. Based on the analysis of their genome sequences obtained by next generation sequencing, we observed a classical genomic structure, with the presence of the five canonical rhabdovirus genes, i.e., nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). In addition, different additional open reading frames which code putative proteins of unknown function were identified, with the common presence of the C and the SH proteins, within the P gene and between the M and G genes, respectively. Genetic comparisons and phylogenetic analysis demonstrated that these seven bird-related rhabdoviruses could be considered as putative new species within the genus Sunrhavirus, where they clustered into a single group (named Clade III), a companion to two other groups that encompass mainly insect-related viruses. The results of this study shed light on the high diversity of the rhabdoviruses circulating in birds, mainly in Africa. Their close relationship with other insect-related sunrhaviruses raise questions about their potential role and impact as arboviruses that affect bird communities. Mononegavirales. They are characterized by a bullet or rod shape and contain a single or segmented molecule of linear negative-strand RNA of a size approximately 10−16 kb, which contains the five canonical genes encoding the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the RNA-dependent RNA polymerase (L) [Rhabdoviruses are enveloped RNA viruses belonging to the order rase (L) . Moreoverase (L) . Each gerase (L) .Rhabdoviridae is the most diverse within the Mononegavirales, with 40 different genera and 246 species according to the latest update by the International Committee on Taxonomy of Viruses  [Vesiculovirus genus [Tupavirus genus [Lyssavirus [The family us genus , but subssavirus . As the ssavirus ,10.Rhabdoviridae, in three different genera, namely Tupavirus, Hapavirus, and Sunrhavirus. Most of them were identified in different bird species in Africa, with the exception of Durham virus (DURV), which was isolated from Fulica americana in North America in 2005 [Tupavirus genus. Similarly, Landjia hapavirus (LJAV), isolated from Riparia paludicola in the Central African Republic in 1970, is the unique representative of bird-related viruses among the Hapavirus genus [Sunrhavirus is associated to a significant degree with birds, and includes Sunguru virus (SUNV), which is described in domestic chicken in Uganda in 2013 [Corythornis cristata in Bangui in the Central African Republic in 1970 [Until now, 12 bird-related viruses were encompassed within the family  in 2005 . This viSunrhavirus [In addition to these characterized viruses, other bird rhabdoviruses are still waiting for taxonomic assignation, and include five viruses collected in the Central African Republic with the Bimbo (BBOV), Kolongo (KOLV), Nasoule (NASV), Ouango (OUAV), and Sandjimba (SAJV) viruses, and two rhabdoviruses collected in Egypt called the Matariya (MTYV) and Burg el Arab (BEAV) viruses. At the time of their collection, initial serological analysis evidenced links to rabies virus groups or bovine ephemeral fever ephemerovirus groups ,14,15. Srhavirus ,17,18,19Sunrhavirus, sharing a common genomic organization and sequence similarities. Our results demonstrated that these seven bird-related rhabdoviruses could represent putative individual new species which are phylogenetically clustered into a single group within the genus Sunrhavirus. These data demonstrate that there is a high level of genetic diversity in the rhabdoviruses that are potentially circulating in African birds. Their close relationship with other insect-related sunrhaviruses, as well as the fact that most of them were isolated from the blood of infected birds, raises questions as to whether they are arthropod-borne and could affect bird communities.In this study, we extended the knowledge on bird-related rhabdoviruses and their associated genetic diversity through the molecular characterization of the nearly complete genome of these seven unclassified virus species, all of them isolated between 1960s to 1970s in wild birds circulating in Africa. Based on molecular analysis conducted on their genome sequences that were obtained by next generation sequencing (NGS), we confirmed that they belonged to the genus Euplectes afer that were collected in 1970 in the suburb of Bangui city. OUAV and SAJV originated from Landjia city in 1970, and were collected from the blood of Sitagra melanocephalus and a pool of crushed organs  from Acrocephalus schoenobaenus, respectively. NASV was isolated from the blood of Eurillas virens (Andropadus virens) that were collected in 1970 in Nasoulé city. Lastly, both Egyptian Matariya virus (09023EGY) and Burg el Arab virus (09027EGY) were obtained from the blood of Sylvia curraca sampled in Port Saïd in 1961 and in Bahig in 1962, respectively. At the time, all these viruses were isolated, amplified after the intra-cerebral  inoculation of suckling newborn mice, and stored long term at −80 °C after freeze-drying. The initial characterization of these viruses was performed serologically, using complement fixation, and immunofluorescent and plaque reduction neutralization assays with reference sera panels, leading to classification by serogroup [All of the viruses characterized in this study were isolated from wild birds in Africa during large field campaigns that were carried out in the 1960s and the 1970s to collect samples of wildlife, with the main aim of evaluating the presence of arboviruses, in particular by virus isolation and serology characterization. Five of them were collected in the Central African Republic, including Bimbo virus , Kolongo virus , Ouango virus , Sandjimba virus , and Nasoule virus , whereas Matariya virus  and Burg el Arab virus  were collected in Egypt [erogroup .Total RNA extraction was performed using Direct-zol™ RNA MiniPrep kit  following the manufacturer’s instructions. Briefly, 600 μL of TRIzol reagent  was added to nearly 200 μL resuspended lyophilized suckling mice brain in nuclease-free water. A DNase I digestion step was performed on-column, according to the manufacturer’s recommendations. Total RNA extraction was eluted in 50 μL nuclease free and stored at −80 °C for further analysis.2, and 1 μL of RNAsin Ribonuclease inhibitor (Promega) for a final volume of 20 μL. The mix was incubated at 25 °C for 10 min then at 50 °C for 90 min. Afterward, double-stranded DNA (dsDNA) was synthesized in a reaction mix containing 20 μL of fresh cDNA, 10 × Second-Strand Synthesis Reaction Buffer , 3 μL of 10 mM dNTP mix (Invitrogen), 1 μL (10 U) of E. coli DNA ligase (New England Biolabs), 4 μL (40 U) of E. coli DNA polymerase I (New England Biolabs), 1 μL (5 U) of E. coli RNase H (New England Biolabs), and 43 μL of nuclease-free water, after incubation at 16 °C for 2 h. The total volume (80 μL) of dsDNA was finally purified for each virus, using a ratio of 1:1.8 of AMPure XP beads (Beckman Coulter) following the manufacturer’s instructions. Finally, dsDNA libraries were constructed using the Nextera XT kit  and sequenced using a 2 × 150 nucleotide paired-end strategy on the NextSeq500 platform as previously described [Genome sequences of the bird-related rhabdovirus were obtained using next generation sequencing (NGS) as previously described ,22,23. Aescribed ,22,23.The NGS data were analyzed using de novo assembly and mapping  with a dedicated workflow built on the Galaxy platform of Institut Pasteur ,22,23,24In case of low covering regions or the presence of gaps, different primer datasets were designed, based either on the sequences obtained by NGS (specific primers) or on the most closely related sequences available in GenBank . These pComplete genome sequences were deposited in GenBank under the accession numbers MW491754-MW491760.Identification of putative additional ORFs (≥180 nt) were performed with Sequencher 5.2.4. The presence of accessory genes was evaluated after comparison with similar genome regions of other rhabdoviruses belonging to the same genus which were available in GenBank.https://www.expasy.org/resources/protparam, accessed on 22 September 2021) [http://www.cbs.dtu.dk/services/TMHMM-2.0/, accessed on 22 September 2021) and TMpred server on the ExPASy server to identify transmembrane helices, and using the normal prediction on Phoebius server  [Phylogenetic analysis of the bird-related viruses and other rhabdoviruses was conducted using datasets of reference sequences downloaded from GenBank . Two dater 2021)  for the er 2021)  to deterThe determination of the genome sequences of the seven bird-related rhabdoviruses was performed using NGS. Between 0.1 to 12 million raw reads were obtained per sample (around 6 million reads on average) . Any remAll of the seven bird-related viruses exhibited a typical rhabdovirus genome organization, with the five canonical genes which encode, in the following order, the N , P , M , G , and the L  proteins . NumerouEach of these viruses presented a putative alternative ORF within the P gene , which corresponds to the C protein already found in other rhabdoviruses, such as Sunguru virus (SUNV), Garba virus (GARV), Durham virus (DURV), Tupaia virus (TUPV), or Klamath virus (KLAV). This protein exhibited a variable spectrum of amino acid conservation among the seven newly described bird-related viruses, including GARV (identity 26.1−80.4%), or after comparison with the three other sunrhaviruses, namely SUNV, Harrison Dam (HDV), and Walkabout Creek (WCB) viruses (10.6−28.7%), and they were highly distinct from the other sunrhaviruses and tupaviruses . For all7, except for the M gene of KOLV and OUAV, which had TGA6 and TGA8, respectively, or for the L gene of SAJV, which exhibited the CGA7 motif. These conserved TI and TTP signal sequences were observed for only one of the putative accessory genes (SH) and for all of the seven bird rhabdoviruses. Surprisingly, the TTP sequence of the SH gene for the virus OUAV was found after the TI sequence of the next gene (G gene) (The transcription initiation (TI) signal was highly conserved among the five canonical genes, with the AACA sequence motif . The con(G gene) . IntriguRhabdoviridae family available in GenBank  for the seven different members already associated with this genus, in addition to the seven new sequences  or after concatenation (concatenated complete nucleotide sequences)  of the seven bird-related rhabdoviruses with the other members within the genus quences) . The clooviruses .Sunrhavirus genus, with amino acid identities ranging from 23.8% to 86.6% and 38.0% to 85.2%, respectively . Seven virus species were associated with this genus, including the two bird-related viruses GARV and SUNV, and five viruses isolated in insects. According to the criteria of the ICTV for species demarcation among the sunrhaviruses, new species need to exhibit a minimum amino acid sequence divergence of 10%, 15%, and 10% for the N, G, and L proteins, respectively. Based on these criteria, all of the seven newly described bird-related rhabdoviruses meet these requirements, with a minimum amino acid sequence divergence of 13.4%, 26.5%, and 16.9% for the N, G, and L proteins, respectively  and SH (between the M and the G genes). Similar C proteins can be observed in rhabdovirus genera other than Sunrhavirus genus, such as Vesiculovirus [Lyssavirus [Ephemerovius (with the P’ protein) [Tupavirus, and Hapavirus genera [Paramyxoviridae family. These C proteins, which expression could occur by leaky ribosomal scanning, are generally considered as highly basic. However, this feature was not observed in the case of the newly described bird-related rhaboviruses. Their precise functions remain unknown, although it has been speculated that they could enhance transcriptional activity or act in viral pathogenesis [Tupavirus genus, including DURV [Sripuvirus genus [In addition to the similar length and genetic relatedness of each viral genome between these bird-related rhabdoviruses and the other members of the genus ulovirus , Lyssavissavirus , Ephemerprotein) , Tupavirs genera , or evenogenesis . The smaogenesis ,17,19,37ing DURV , KLAV [1ing DURV , and morSunrhavirus. This phylogroup encompasses exclusively viruses from wild African birds  [Sunrhavirus genus, which potentially includes now 14 members from three main geographical areas, namely Africa, America, and Australia.Based either on the nucleotide concatenated genome sequences or on the complete amino acid polymerase sequences, all of the seven bird-related rhabdoviruses clustered with GARV in a distinct group, identified here as Clade III, within the genus d Egypt) ,5,15. Twd Egypt) . Lastly,d Egypt) ,37. All Sunrhavirus genus were isolated from insects (mosquitoes with Culex species and biting midges with Culicoides species) or birds is intriguing, and raises the question about a potential arboviral epidemiological cycle in birds, similar to other bird-related arboviruses, such as West Nile virus [Culex species feed on these animals [Tupavius genus was not able to replicate in the insect cell line tested [The fact that the members of the le virus  or Usutule virus . This is animals . However animals . Similare tested . FurtherFulica Americana) that exhibited neurological signs in North Carolina [Conversely, it remains also to be elucidated as to what extent these sunrhaviruses could be specifically associated with birds as vertebrate hosts, and what could be the pathologies induced after infection. Indeed, all of the bird-related sunrhaviruses were isolated from animals during nonspecific epidemiological surveillance of arboviruses, without information related to disease or potential clinical signs associated with the infected animals. Only the tupavirus DURV was suspected to be associated with disease in birds, being isolated from a moribund American coot (Carolina . A firstCarolina . HoweverCarolina ,17,19.Lastly, it is important to underline that the seven bird-related rhabdoviruses described here are part of a large and unique historical collection of arboviruses which were isolated during field campaigns carried out in the 1960s and 1970s on wildlife, particularly in Africa, and of which some of the isolates have yet to be characterized. Thus, complementary studies would make it possible to assess whether these viruses are still circulating and, if so, to investigate their genetic evolution, taking into account changes that may have affected environmental factors or their hosts.


Low- and middle-income countries (LMICs) face disproportionately high mortality rates, yet the causes of death in LMICs are not robustly understood, limiting the effectiveness of interventions to reduce mortality. Minimally invasive tissue sampling (MITS) is a standardized postmortem examination method that holds promise for use in LMICs, where other approaches for determining cause of death are too costly or unacceptable. This study documents the costs associated with implementing the MITS procedure in LMICs from the healthcare provider perspective and aims to inform resource allocation decisions by public health decisionmakers.We surveyed 4 sites in LMICs across Sub-Saharan Africa and South Asia with experience conducting MITS. Using a bottom-up costing approach, we collected direct costs of resources  to conduct MITS and the pre-implementation costs required to initiate MITS.Initial investments range widely yet represent a substantial cost to implement MITS and are determined by the existing infrastructure and needs of a site. The costs to conduct a single case range between $609 and $1028 per case and are driven by labor, sample testing, and MITS supplies costs.Variation in each site’s use of staff roles and testing protocols suggests sites conducting MITS may adapt use of resources based on available expertise, equipment, and surveillance objectives. This study is a first step toward necessary examinations of cost-effectiveness, which may provide insight into cost optimization and economic justification for the expansion of MITS. The vast majority of global childhood deaths are disproportionately concentrated in low- and middle-income countries (LMICs)  and crafEvidence documenting the validity , 11, 12,To date, no comprehensive estimates of the costs to implement MITS in LMIC settings have been published, leading to calls for more evidence . The majSeveral studies reference costs to conduct MITS in LMIC settings but do not conduct a comprehensive accounting of the costs. An examination of barriers to MITS implementation in Mozambique noted that complex microbiology platforms, the need for specialized labor, and the high cost of specific supplies like biopsy needles all present challenges to MITS expansion . Feroz eIn this work, we aim to characterize the costs and cost drivers associated with conducting MITS in LMIC settings, by examining 4 sites where MITS has been implemented at pilot scale. As a result, this case study approach speaks to the potential costs of MITS, rather than generalizable findings. We capture costs from the perspective of the health care provider or health system in order to provide national health ministries and agencies with information on what MITS implementation may cost in order to inform efficient resource allocation decisions.This study adopts a micro-costing approach to capturing costs associated with MITS from the healthcare provider perspective. “Bottom-up” micro-costing estimates the unit resources  at the detailed activity level, and aggregates them to form summary totals . This apWe adopt the healthcare provider perspective because in LMICs, as in many countries, mortality surveillance activities are primarily conducted by healthcare providers. Providers’ activities are directed and funded by national heath ministries, public agencies, or other nonprofit entities interested in efficient resource allocation. For this reason, we acknowledge but do not include costs from the family member or societal perspectives, which often entails unexpected and often large costs to the family of the deceased.In consultation with subject matter experts, we identified key activities required to conduct MITS in a facility  and in the community, and designed a survey to capture the associated unit-level direct costs that were incurred . Direct We surveyed 4 sites conducting MITS at university hospitals in LMICs across Sub-Saharan Africa and South Asia. All sites were grantees of the MITS Surveillance Alliance Secretariat that received incentive grants to expand the use of MITS. Participating sites had experience conducting MITS (>10 MITS cases completed at the time of data collection) and represented varied case populations. We collected data in LMICs between February and August of 2020, before and during the early stages of the coronavirus disease 2019 (COVID-19) pandemic. At each site, the principal investigator and an employee knowledgeable of the site’s finances responded to the computer-based survey. The teams provided additional details and clarifications by follow-up teleconference and email. Each team reviewed and approved the final cost estimates. At sites where data were collected after COVID’s onset, we asked them to report the costs incurred prior to the pandemic, which most readily had affected the price of personal protective equipment.Respondents provided resource information for activities associated with preparing the site to conduct MITS and conducting the MITS procedure, based on actual costs incurred rather than budgets or projected expenses . This inTo capture labor expenses, respondents entered all staff roles that typically contributed to the preparation or performance of MITS. For every staff role, teams indicated the number of persons performing the role, their compensation, and their typical hours worked per week at their site. Respondents provided the number of hours that staff members typically spent on a given activity for an average MITS case. Respondents recorded these hours separately from time spent on administrative tasks related to MITS, like grant management, quarterly reporting, and equipment maintenance, for which we do not account in this study. We compared role-specific wage estimates given by sites to Serje et al’s estimates of health worker earnings as multiples of gross domestic product (GDP) per capita in low- and lower-middle income countries , findingWe developed an Excel-based cost model to aggregate each sites’ activity-specific resource cost estimates and categorized them into 1-time, initial costs  required to be in place for a MITS program to begin and recurring per-case and annual costs to conduct MITS at each site. Costs provided in local currencies were converted to US dollars. We provide a financial accounting of these costs —that is, we did not adjust for depreciation and presented the purchase cost for these items that had been purchased recently or through the MITS Surveillance Alliance grant. We excluded costs to perform additional research activities.The majority of cost categories required simple aggregation. Staff compensation varied by site, including with regular salaries or hourly wages, fixed incentive payments per-case, and per-diem payments for specific activities. We calculated activity-specific labor costs by activity, based on each site’s reported compensation schedule. Where staff were paid hourly wages, we multiplied their wage by the number of hours they spent on an activity, whereas for salaried staff we estimated an hourly wage where appropriate. Start-up costs included some nonconsumable expenditures like equipment purchases and facility renovations that were not used exclusively for MITS. In these cases, we adjusted these costs by the utilization rate of the MITS program. Site-specific estimates were reviewed with respondents before finalizing results.Labor costs constitute roughly 25% of MITS costs per case. Although regional wage differences explain some differences across sites, sites reported a high degree of heterogeneity in staff roles participating in an average MITS procedure . All sitCosts to conduct sample analysis, or testing, make up a significant portion (18%) of the per case cost and are determined by the type and number of tests conducted per case, which varied by the site-specific protocols . All sitMITS kits, which are provided to sites with the needed supplies to conduct the MITS procedure, comprise the largest portion (48%) of the per case MITS cost. This cost includes a standard battery of supplies ($225), a back-up kit sent with each batch of 10 kits ($29 per kit), and assembly and shipping costs to send kits from the United States ($142 labor and shipping). Sites received these kits directly from the MITS Surveillance Alliance program.The 1-time costs required to establish a MITS program, divided into start-up activity and capital costs, far outweigh the cost to complete a single case and differ substantially by site. The wide variation in capital costs by site is driven primarily by the preexisting infrastructure available. Renovation expenses, where necessary, drove capital costs, as some sites adapted existing lab and office space to accommodate MITS. Other capital expenses included equipment and machinery. Site 1 was least well-resourced initially and required a costly autopsy table and embedding machine, whereas site 4 had fewer needs, investing only in a microscope.Start-up costs varied notably by site and reflect each site’s investment in specific pre-implementation activities. For instance, the volume of staff trained in each site differed, which reflects a diversity of available staff roles and/or those selected to receive MITS training. Guidelines shared by the MITS Surveillance Alliance did not stipulate a specific number of hours to complete pre-implementation activities yet did provide standards for their completion.The purpose of this cost analysis was to understand the cost implications of MITS implementation in a range of different LMIC contexts, providing a range of transparent cost estimates that reflect the resources needed for expanding MITS in new locations. These cost estimates are an initial milestone in demonstrating the comprehensive costs of conducting MITS incurred by a provider. Our findings identify important cost drivers and opportunities for reducing cost.The lack of standardization across sites in terms of labor and testing—key drivers of incremental costs—suggests that staff roles and testing protocols may be varied without sacrificing the accuracy of cause of death findings. This variation points to the potential flexibility around labor and testing to reduce per case costs and adapt MITS protocols around resource availability or surveillance goals. First, the notable variation in staff roles applied by each site has the potential to both give greater flexibility to sites that do not have a large number of specialty clinicians available and points to opportunities to reduce labor costs. For instance, with a core team of pathologists for supervision, other staff roles could perform some tasks in place of pathologists. Forthcoming research related to “task-shifting” across roles to conduct MITS explores this in-depth and is an important area of future study.Second, the varied type and number of tests conducted across the 4 sites suggests that improved insight into causes of death from MITS can still be achieved through nonuniform sample testing protocols. Each site’s protocols reflect site-specific surveillance objectives, testing budgets, and the equipment and expertise available. In our study, sites used standard testing procedures across the sites, but no site used more advanced tests, like TAC PCR and traditional PCR tests. This may be due to the limited availability of testing equipment, such as PCR machines, or because more tests may not imply improved cause of death information. Due to the initial investment in infrastructure to conduct tests using molecular techniques like PCR, their inclusion would likely increase the cost to perform MITS, despite the decreasing costs of PCR, in part due to widespread PCR testing for COVID-19. A growing body of evidence suggests that incremental testing may increase data associated with the presence of pathogens yet may not necessarily increase the precision and accuracy of cause of death determination . Ritter As MITS is conducted at relatively few locations globally, expanding its use gives opportunity to reduce per case costs by procuring supplies locally. Although the costs associated with MITS kits are substantial—between 35% and 65% of per case costs—they reflect MITS implementation at pilot-scale and could be reduced in the future by local procurement. The contents of the kits are not highly specialized, suggesting that they may not be limited by supply chains in LMICs.We find that incremental (per case) costs to conduct MITS were relatively minor, once the initial investments to perform MITS were made. This is positive, in that low incremental costs may avoid the challenges of disinvestment over time, as many public health funders may be wary of securing substantial long-term resources to sustain highly costly programs.The initial investments varied by site due to the differing needs and existing infrastructure for capital goods to conduct MITS and highlights the importance of evaluating the infrastructure in-place when considering costs to conduct MITS at a new site. Where necessary, renovation of office and lab space comprised a large portion of these costs, as substantial space is needed to conduct MITS. However, no capital expense was required universally by site, suggesting that potential locations may have the majority of necessary items on hand. Activities to prepare for MITS implementation, including training, reflected each site’s ability to devote resources and/or priority given to these activities.This study represents a small number of sites, and although each site’s costs and context vary, they illustrate the diversity in approaches to implement MITS. As such, obtaining precisely comparable information from each site was difficult. Although we conduct limited cross-site comparisons in this analysis, the individual differences of the economies and healthcare environments limit the utility of these comparisons. Comparing specific resources or staffing roles may be most telling for contextualizing these findings in a new location. We also do not discuss the effectiveness of the MITS program. In the future, it will be important to weigh the body of evidence for MITS to assess the value in replacing existing routine surveillance systems.Our study did not include all possible costs of MITS implementation, such as family counseling, socio-anthropological support , and clinical data abstraction. Importantly, our study also did not include program administration costs , which are a fundamental component of MITS program operational expenses. We were, however, careful to exclude them from our estimates. Administrative costs are notoriously challenging to include and interpret, and often favor a “top-down” costing methodology to account for administrative burden, which is often less precisely tracked and can be under-reported in “bottom-up” costing . FurtherFinally, all activities and costs represented in this study are defined by external grant funding and the technology available to each site. Thus, these results may not represent the activities that would be conducted in the absence of a budget constraint or at expanded scale, but do represent illustrative examples of MITS implementation in LMIC settings where these constraints are common. For instance, sites implementing MITS beyond a pilot-scale may increase community sensitization work in order to improve MITS’ acceptability and overall success. The scale and funding of the costs represented could be both higher and lower than the costs of self-funded MITS implementation.This study is a first step toward additional necessary examinations of cost considerations, including cost-comparison and cost-effectiveness studies, which further provide needed economic justification for the expansion of MITS for improving cause of death data.