Dermolepida albohirtum picorna-like virus (9696 nt) was built from 83,894 assembled reads while only 1350 reads mapped to Lepidiota negatoria beny-like virus (6371 nt). Future studies are essential to determine their natural incidence in different life stages of the host, biodiversity, geographical distributions, and potential as biological control agents for these important pests of sugarcane.Canegrubs (Coleoptera: Scarabaeidae) are major pests of sugarcane crops in Australia, but despite long-term and intensive research, no commercially viable biological control agents have been identified. We used the RNA-Seq approach to explore the viriomes of three different species of canegrubs from central Queensland, Australia to identify potential candidates for biological control. We identified six novel RNA viruses, characterized their genomes, and inferred their evolutionary relationships with other closely related viruses. These novel viruses showed similarity to other known members from picornaviruses, benyviruses, sobemoviruses, totiviruses, and reoviruses. The abundance of viral reads varied in these libraries; for example,  Lepidiota (ten species), Antitrogus (four species), Alepida (three species), Dermolepida (one species), and Rhopaea (one species) [Dermolepida albohirtum), French (Lepidiota frenchi), and negatoria canegrubs (Lepidiota negatoria) are the most damaging and widespread [Sugarcane is grown in tropical and subtropical regions across the globe, and over 1500 insect species have been reported to feed on sugarcane plants . In Austspecies) ,4 are redespread . While tdespread ), the neMetarhiziumanisopliae, no commercially viable biological control strategy is available. One strain of M. anisopliae has been formulated and was commercialized as BioCaneTM [Despite significant research efforts to find effective biological control agents for cane grubs, including the evaluation of the entomopathogenic fungus ioCaneTM ,6. It waioCaneTM ,6, but iCurrently, the Australian sugarcane industry relies solely on the neonicotinoid insecticide imidacloprid for the control of canegrubs. This approach is not sustainable as overreliance on a single compound presents a significant risk that populations will ultimately evolve resistance . FurtherAdelina sp., the fungus M. anisopliae, and the bacterium Paenibacillus popilliae are the major known pathogens of canegrubs in Australia [Previous studies have shown that canegrub larvae are susceptible to a number of pathogens that could, in some cases, be responsible for significant suppression of their local populations. The protozoan ustralia . Sallam ustralia . An unidustralia .Oryctes rhinoceros (Coleoptera: Scarabaeidae), in southeast Asia and the South Pacific islands by a double stranded DNA virus (OrNV: Oryctes rhinoceros nudivirus) [Heteronychus arator [Anomala cuprea (Coleoptera: Scarabaeidae)) [Nudiviridae have great potential for use as biocontrol agents of insect pests, including canegrubs.Several viral pathogens have been reported from scarab beetles across the world , and somdivirus)  is wideldivirus) . OrNV cas arator , which iaeidae)) . Currentaeidae)) , but memD. alborhirtum, L. frenchi, and L. negatoria [Identifying viral pathogens is the first step in investigating their potential as biocontrol agents for canegrubs and other beetle pests. In this study, we have sequenced several RNA viruses from three canegrub species for the first time and conducted phylogenetic analyses to identify novel insect-specific viruses, characterize their genomes, and infer their evolutionary relationships with other closely related viruses. We focused our work on three of the main canegrub species that attack sugarcane in Australia: egatoria ,5. Here,D. albohirtum (n = 15), L.frenchi (n = 10), and L. negatoria (n = 15), were collected from commercial sugarcane farms in Mackay, Central Queensland, Australia in November 2020. Adult beetles were transported to the University of Queensland for viral discovery based on next-generation sequencing.Adult beetles of three different species, w/v) agarose gel. After checking the RNA quality, total RNA samples were submitted to the Genewiz sequencing facility  for library preparation and strand-specific total RNA and mRNA (poly(A)-enriched) sequencing on the NovaSeq platform. For some samples from each species that were used in total RNA sequencing, the eukaryote ribosomal RNA was removed using a VAHTS Total RNA-seq (HMR) Library Prep Kit for Illumina.After 72 h of induced starvation, their gut tissue was dissected and preserved in an RNA protect reagent for RNA extraction and further analysis. The total RNA was extracted from 250 μL of insect gut tissue homogenates using Trizol following the manufacturer’s protocol . The RNA samples were treated with DNase I for 1 h at 37 °C, and then, their concentrations were measured using a spectrophotometer; integrity was ensured through analysis of RNA on a 1% (Oryctes rhinoceros (GCA_020654165.1) and Onthophagus taurus (GCF_000648695.1), as proxy genome references to discard insect-specific reads. We applied a minimum length fraction = 0.5, similarity fraction = 0.8, maximum mismatches = 2, insertion cost = 3, and deletion cost = 3 as mapping criteria in CLC Genomics Workbench. The unmapped reads were retained for downstream analysis after removing rRNA with SortMeRNA [The CLC genomics workbench 21.0.5 and OmicsBox 2.0.36 were used to process the data, and our virus discovery pipeline is summarized in ortMeRNA . We usedortMeRNA  with aut−3. Sequences with positive hits to virus polymerase (RNA-dependent RNA polymerase (RdRP) domain: cd01699) were retained. Any sequences with a positive hit were checked with BLASTn against the nucleotide database (nt) to remove any potential false positive outcomes.BLASTx was used to identify sequence similarity of all assembled contigs with the protein database (nr). To detect highly divergent viruses, we also performed domain-based searches by comparing the assembled contigs against the Conserved Domain Database (CDD) version 3.14 and Pfam v32, with an expected value threshold of 1 × 10Contig sequences with a high degree of similarity to viral proteins were then checked for complete ORFs. ORFs with a minimum length of 150 aa were detected in the CLC Genomic Workbench by using the standard genetic code. We used this tool for viral genome annotation such as the identification of a signal peptide, potential glycosylation sites, and transmembrane domains. We also remapped the RNA-Seq data to putative virus sequences to inspect for sufficient coverage and possible misassembly. The CLC Genomic Workbench’s RNA-Seq function  on a nonstrand-specific option was used.http://www.iqtree.org, accessed on 30 January 2022) to check the best-fit model out of 546 protein models. The maximum likelihood phylogenetic trees were inferred using a JTT substitution matrix and assuming a discretised gamma rate distribution with four rate categories and with 1000 bootstraps. The shape parameter of the gamma distribution was fixed at 1.0 in the CLC Genomics Workbench.The deduced amino acid sequence of predicted ORF regions or RdRP of newly identified viruses were used to calculate their phylogenetic relationship with other members of each respective family. Closely related viruses from the BLASTp analysis of the NCBI nonredundant protein database were downloaded. Multiple amino acid sequence alignments with relevant reference sequences were performed with MUSCLE. We used ModelFinder -enriched and total RNAs extracted from gut tissues of three canegrubs collected from Queensland, Australia. In total, 256,291,462 and 218,995,416 raw reads were produced from three total RNA libraries and three poly(A)-enriched RNA libraries, respectively. The number of unmapped reads to proxy genomes and the assembled contigs and scaffolds are summarized in D. albohirtum) samples, which were collected in Mackay, Central Queensland, Australia. The genome included a single open reading frame encoding a 2909 product , was identified from the greyback canegrub  was detected towards the RNA helicase and RdRp conserved domain regions. This high coverage suggests that this virus might actively replicate in the host. The annotated genomic sequence of this virus has been deposited in GenBank under the accession number OM421671.Picornaviridae and Marnaviridae but indicate that it shares common ancestry with the iflaviruses as well as other picorna-like viruses. Although it shows a high degree of similarity with other putative iflaviruses, the phylogenetic analysis clustered this novel virus with other unclassified picorna-like viruses that have previously been reported from insects (Oryctes rhinoceros (Scarabaeidae) [Aulacophora lewisii (Chrysomelidae) [Harmonia axyridis (Coccinellidae) [Cheilomenes sexmaculata (Coccinellidae) [The maximum likelihood phylogeny separated this novel virus (DaPV1) from other members of the  insects B. In thebaeidae) , Aulacopmelidae) , Harmoniellidae) , and Cheellidae) . Picornavirales and other insect-specific viruses have been reported from birds and bats, and the insectivorous feeding of these vertebrates might be the cause of this spillover and cross-species transmission [Lanius cristatus) in China. Additionally, another virus found in the vesper bat (Yuma myotis) from Washington showed 38.4% similarity with 53% coverage to the greyback canegrub picorna-like virus (DaPV1). Further investigations are required to determine if these insect viruses can replicate and infect birds, bats, and other vertebrates.Recently, several smission . DaPV1 sThere is a growing body of evidence showing that iflaviruses exclusively infect arthropods, but in most cases, their infections remain unnoticed, and the majority of infections are asymptomatic while only small portions of infected hosts produce severe symptoms . HoweverTotiviruses are nonenveloped, double-stranded, positive-sense RNA viruses with icosahedral virions, which consist of a single capsid protein . This isLampyris noctiluca) in Finland [Ligia sp.) in China had the maximum sequence coverage of 97% (sequence similarity of 24.8%) with the first ORF of this newly identified virus [The genome is predicted to encode two large ORFs in which the first ORF is 3984 nt, and encodes a 1328 amino acids (aa) protein that shares 29.4% aa sequence identity  with the hypothetical protein 2 of Hubei toti-like virus 16, which was isolated from spiders in China . This OR Finland . The Beied virus . We coulTotiviridae, and how these viruses adapt to infect higher-order hosts.The second ORF has one nucleotide overlap with ORF 1 and encodes an 852 aa product with a putative RNA-dependent RNA polymerase (RdRP_4) conserved domain (pfam:02123). It showed the maximum sequence identity (33.8%) and coverage (99%) to Lampyris noctiluca toti-like virus 1 (QBP37034.1). Significant similarity has also been found to other toti-like viruses, which have been identified in terrestrial and aquatic animals, such as the horseshoe crab (Beihai toti-like virus 4), horseflies (Hubei toti-like virus 19), and Penaeid shrimp (Beihai toti-like virus 5). Generally, members of this group have two common ORFs in their genome sequence. We identified two extra small coding regions in 5′ and 3′ of the viral genome. It has been suggested these additional coding sequences potentially produce some proteins that facilitate more complicated infection mechanisms in higher order organisms, with large DNA (genome size) and complex developmental processes, that possess sophisticated immune systems . HoweverTraditionally, fungi and protozoa have been recognised as hosts for these viruses, but recent metagenomic analysis has revealed that they also infect higher-order organisms. Several unclassified toti-like viruses have been identified from arthropods, fish, worms, and insectivorous bats ,51,52. TReoviridae family and tentatively named it Dermolepida albohirtum reovirus (DaRV). The RNA genome consists of 4122 nucleotides and includes one open reading frame with 1274 aa and the predicted molecular weight of 145.502 kDa with an Isoelectric point 7.32  from the int 7.32 . The dedbohirtum .Reoviridae  can replicate in both plants and arthropod vectors, and their main insect vectors are planthoppers and leafhoppers. Sugarcane Fiji disease virus is a type species of Fijivirus [Plant viruses from the family ijivirus . Currentijivirus . These cFijiviruses, known pathogens of some agricultural crops, are multisegmented and have a double-shelled and icosahedral structure [tructure ,55. The tructure  whereas tructure  and showReoviridae that infect insects, and they are mainly classified under the genera Orbivirus and Cypovirus. Orbiviruses, such as the Umatilla virus [Aedes orbi-like virus [Anopheles hinesorum orbivirus [Cypovirus is another genus in this family that infects insects, such as the Heliothis armigera cypovirus [There are some other members of the la virus , Aedes oke virus , Anophelrbivirus , and blurbivirus , are preypovirus  and Bombypovirus , which aypovirus . The phyD. albohirtum) and French’s canegrubs (L. frenchi) that showed significant similarity to sobemo-like viruses. The Dermolepida albohirtum sobemo-like viruses’ (DaSV) complete genome sequence consists of a single segment of 2761 nucleotides (We identified two sequences in greyback (leotides A with thleotides B. A putasouthern bean mosaic virus (SBMV) [rice yellow mottle virus (RYMV), southern cowpea mosaic virus (SCPMV), and turnip rosette virus (TRoV), are transmitted by beetles [Diabrotica undecimpunctata, after they have fed on an infected plant material [Sobemoviruses are groups of plant viruses with linear and nonsegmented positive sense ssRNA genomes, named after their type species, s (SBMV)  Generalls (SBMV) . It has  beetles . Some plmaterial .Penaeus monodon, in Thailand; however, this virus has been also detected in healthy shrimps in other countries [Aedes albopictus from southern Switzerland [Ae. vexans nipponii from the Republic of Korea [Armigeres subalbatus from Thailand [There is no report on the pathogenicity of these viruses in insects. Previous studies proposed that the Laem-Singh virus (LSNV) can be a potential cause of retarded growth syndrome in the giant tiger shrimp, ountries . Recent ountries . These utzerland , Ae. vexof Korea , and ArmThailand . They arThailand . TherefoDendrocopos leucotos) and spirurian nematodes are also clustered under the same clades with these novel viruses , a 6371-nucleotide (nt) contig was assembled corresponding to the benyvirus and tentatively named Lepidiota negatoria beny-like virus (LnBV). The draft genome of this novel virus contains two open reading frames, one encoding a nonstructural protein, which is located between 146 and 3571 nucleotides (1142 aa) from the 5′ end of the genomic RNA, and another one encoding a structural protein with an RdRp-conserved domain located between 3586 and 5664 nucleotides (693 aa) from the 5′ end of the genomic RNA. Only 1350 reads from the total RNA library mapped to this sequence, and we could not recover this contig from the poly(A)- enriched RNAseq library data  in China. The predicted molecular weight of the structural protein with a conserved RdRp domain is 77.95 kDa (isoelectric point of 7.96). The translated open reading frame of the structural protein aligned by 51.2% to RdRp of the Sanya benyvirus 1, which reported from the Asiatic pink stem borer, Sesamia inferens, in China  tract [Rhodnius prolixu [A) tract . Beet ne prolixu ,72.Benyvirus. The other unassigned members of this family have infected or currently infect a wide range of hosts, including insects. The maximum likelihood phylogeny classified this novel virus with other insect-specific beny-like viruses and separated them from plant and fungi viruses  could be important components of pest management in the future. This study provided the complete genome sequences of some novel viruses from canegrubs, but their mode of transmission and pathogenicity are completely unknown. Since these recently identified novel viruses only originated from a metagenomics study, the method would not differentiate sequences from the insect host, its previous ingested meal, or a contaminating parasite or fungus. Incorrect host assignment is the major challenge and limitation of this sort of metagenomic analysis. Future studies are essential to determine the natural incidences of these viruses in their different life stages of the hosts, biodiversity, geographical distributions, and potential as biological control agents for these important pests of sugarcane.


According to the 2018 FAO report on aquaculture, there are 598 species of finfish, molluscs, crustaceans, and other organisms used in aquafarming around the world. Thus, the number of viruses potentially infecting these species is huge [The overall economic impact due to virus infections in aquaculture can amount to a loss of up to 20% of biomass in a production cycle . Aquacul0) of the virus infection amongst the fish in the cage easily shoots beyond the threshold where only the host number can limit the R0.Aquatic viruses drift through the water passively, and their chance of hitting something in which to amplify is very low. Therefore, not surprisingly und unlike terrestrial viruses, aquatic viruses are very promiscuous, with a huge host range. Infectious pancreatic necrosis virus (IPNV), for example, is replicated in about 50 finfish species. A penned-in finfish shoal in a cage is an ideal situation for an aquatic virus. Once the infection establishes a hold, the basic reproduction number (RThe aim of this Special Issue was to collect peer-reviewed reports, perspectives, reviews, and research articles focusing on recent advances in the study of known and emerging aquaculture viruses.The papers submitted reflect the diversity of aquaculture across freshwater and seawater. In keeping with the biggest output of 60% of total worldwide aquaculture production ,4 a thir


DPW containing AHP, ethanol, NaOCl, or single or dual QAC as active microbicidal ingredients removed/inactivated ~6 log10 of the virus, with minimal EBOV or no VSV virus transfer to a secondary surface observed. In Ebola virus outbreaks, a DPW with demonstrated virucidal efficacy, used as directed, may help to mitigate the unintended spread of the infectious virus while performing surface cleaning.The authors evaluated four disinfectant pre-impregnated wipes (DPW) for efficacy against Ebola virus Makona variant (EBOV) and vesicular stomatitis virus (VSV), Indiana serotype. Steel carriers were inoculated with the infectious virus and then were wiped with DPW in the Wiperator instrument per ASTM E2967-15. Following the use of J-Cloth impregnated with medium (negative control wipes) or the use of activated hydrogen peroxide (AHP)-, ethanol-, sodium hypochlorite (NaOCl)-, or single or dual quaternary ammonium compound (QAC)-based DPW, virus recovery from the carriers was assayed by titration assay and by two passages on Vero E6 cells in 6-well plates. The Wiperator also enabled the measurement of potential transfer of the virus from the inoculated carrier to a secondary carrier by the DPW or control wipes. The J-Cloth wipes wetted with medium alone  removed 1.9–3.5 log Disinfectant pre-impregnated wipes (DPW) are an optional intervention for disrupting the cycle of infection transmission since these wipes may remove and inactivate pathogens from contaminated high-touch environmental surfaces (HITES) during use . Sattar We have characterized the removal, transfer, and inactivation of infectious Ebola virus Makona variant (EBOV) or vesicular stomatitis virus (VSV) by a control wipe and two DPW wipes from stainless steel carriers experimentally contaminated with the viruses in a previous study . Our intFiloviridae family  containing 10% fetal bovine serum  and 10 units/mL penicillin/streptomycin (Gibco). EBOV  was obtained from a clinical isolate. The virus was subsequently engineered to express a green fluorescent protein (GFP). A stock of VSV, Indiana serotype, was prepared from a reverse genetics construct  were incubated at 37°C/5% COonstruct  to be ≥8The negative control wipe used in this study was the “J-Cloth,” a representative material composed of cellulosic fibers from wood pulp that has been used earlier in studies of this type , 10. The“AHP wipes” were prepared by impregnating sterile J-Cloth wipes with a 1:40 solution of accelerated hydrogen peroxide , as described previously . “SingleThe testing of wipes for the ability to decontaminate a prototype environmental surface (stainless steel carriers) was performed as per ASTM 2967-15 . EBOV an50 assay in a 96-well plate format on Vero E6 cells  and 4 days (VSV), after which the culture supernatant was harvested and used to inoculate fresh Vero E6 cultures. This was used to ensure that a negative result in the TCID50 assay was truly negative. The limit of detection of the plate safety test was determined to be 1–2 TCID50 per inoculation  were 3.1– 4.1 loging time . In the 10 TCID50/mL; n = 9 replicates/time point) (10 decontamination of EBOV from the inoculated carriers for the various wiping times ranged from 5.5 to 6.4 log10. The results of the plate safety assay (10 TCID50/mL and 0.1 log10 TCID50/mL (n = 9 replicates) following transfer from the inoculated carriers wiped for 5 and 30 s, respectively. The virus was transferred from three of the nine replicates of the inoculated carriers wiped for 5 s and from a single replicate of the inoculated carriers wiped for 30 s. No detectable infectious EBOV was transferred from the inoculated carriers wiped for 15 or 60 s.Ebola virus Makona variant titers recovered from the inoculated carriers after use of the AHP DPW ranged from 0.20 to 1.1 loge point) . The logty assay  confirme10 TCID50/mL; n = 9 replicates) was recovered from carriers after 5-s and 30-s wiping with the single QAC DPW, respectively . No infectious EBOV was recovered from the secondary carriers following the transfer from carriers wiped for 15, 30, or 60 s.Ebola virus Makona variant  was recovered after use of the NaOCl DPW from one of nine replicates evaluated after 5-s wiping (10) decontamination for this condition. This result was confirmed in the plate safety test  was recovered after use of the ethanol DPW from carriers evaluated after 5-s wiping . No infectious VSV was recovered from the secondary carriers following transfer from the inoculated carriers wiped for 5, 15, 30, or 60 s.For each of these three DPW, VSV was not able to be recovered from the inoculated carriers after the 5-, 15-, 30-, or 60-s wiping times . These rDisposable DPW represent an option for reducing pathogen loads on HITES in healthcare and home settings , 19. The10, equating to ~1500 infectious units) from the contaminated HITES to a new surface could represent a significant opportunity for transmitting infectious Ebola virus.For an emerging virus such as those causing hemorrhagic fever, what level of decontamination efficacy is required? For such viruses, a low level of virus transferred from a contaminated surface to a secondary surface may result in the spread of infection to otherwise healthy individuals coming in contact with contaminated HITES. For instance, the infectious dose for the Ebola virus has been estimated to be 1–10 infectious units , 25. If In the present study, we used the ASTM-2967-15 method to evaluate the efficacy of DPW containing one of five actives  against EBOV and VSV. These enveloped viruses were expected to be readily inactivated by each of the formulated actives tested. The comparative efficacy of DPW containing different microbicidal actives for decontaminating prototypic HITES inoculated with Ebola virus or VSV has not previously been explored. Our recent article . In that10 decontamination after wiping times of 5–60 s). The single QAC DPW required 60 s to cause complete decontamination (6.0 to 6.6 log10), while the AHP DPW failed to cause complete decontamination after wiping for up to 60 s. The recovery of infectious EBOV following 30-s wiping with the single QAC DPW, but not following 15-s wiping, was unexpected. This may represent sampling issues  as the titers of the virus approached the limit of detection.In the case of EBOV, the dual QAC DPW and ethanol DPW were the most effective DPW   whereas We acknowledge the following limitations to our study, which should be kept in mind when considering these results. First, we evaluated viruses deposited on stainless steel carriers only. There are other prototypic HITES surfaces, such as plastic, tile/ceramic, wood, or laminate counter tops, that could potentially be disinfected using DPW. It may not be appropriate to extrapolate our findings using non-porous stainless steel carriers to each of the other types of HITES surfaces. Second, we were able to evaluate wipes composed of cellulose and cellulose/polypropylene only. There may be DPW composed of other fabric types. Our results using cellulose or cellulose/polypropylene wipes should be extrapolated to DPW composed of other fabric types with caution. Finally, we evaluated two enveloped RNA viruses in this study, as our primary interest was in the Ebola virus and a surrogate, VSV. We did not challenge the DPW with an enveloped DNA virus or with any non-enveloped viruses. We expect that the DPW should have a similar efficacy for any enveloped virus on the basis of the known hierarchy of susceptibility of viruses to microbicidal active ingredients –24. Effi10 inactivation. NaOCl DPW and single QAC DPW required 30 s and 60 s, respectively, to cause complete inactivation of EBOV. The AHP DPW did not cause complete decontamination of EBOV after ≤ 60-s wiping. Each of the DPW were effective against VSV, causing complete (5.5 to 6.2 log10) inactivation at 15-, 30-, and 60-s wiping. During outbreaks involving emerging/re-emerging viruses such as Ebola virus, a DPW with demonstrated virucidal efficacy, used as directed, may help to mitigate the unintended spread of the infectious virus while performing cleaning of HITES.In this investigation, we have identified differences in decontamination efficacy among five DPW with differing microbicidal actives. The dual QAC DPW and ethanol DPW were the most effective for EBOV, causing 6.0 to 6.6 logThe original contributions presented in the study are included in the article's MI, TC, and ST conceived and designed the experiments. RN, MI, and TC analyzed the data. RN, MI, JR, TC, ST, JK, and SK each contributed to the writing of the manuscript. All authors reviewed and approved the manuscript.JR and MI are employed by RB, who also provided partial funding support for this study. RN provided data analysis and manuscript preparation as a paid consultant employed by RMC Pharmaceutical Solutions, Inc. The remaining 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.


Introduction: Frontotemporal dementia (FTD) is a complex syndrome characterized by changes in behavior, language, executive control, and motor symptoms. Its annual economic burden per patient in developed countries has been classified as considerable, amounting to US$119,654 per patient, almost double the patient costs reported for Alzheimer's disease. However, there is little information regarding cost-of-illness (COI) for FTD in Latin-America (LA).Aim: To describe the costs related to FTD in LA.Methods: We included COI studies on FTD conducted in LA published in English, Spanish, or Portuguese from inception to September 2020. We carried out a systematic search in Pubmed/Medline, Scopus, Web of Science, Scielo, Cochrane, and gray literature. For quality assessment, we used a COI assessment tool available in the literature. All costs were reported in USD for 1 year and adjusted for inflation.Results: We included three studies from Argentina, Brazil, and Peru. Direct costs (DCs) included medication , health care costs , and caregiver costs . Indirect costs (ICs) amounted to US$43,076.88.Conclusions: In LA countries, the reporting of costs related to FTD continues to be oriented toward DCs. They remain lower than in developed countries, possibly due to the limited health budget allocated. Only one Brazilian report analyzed ICs, representing the highest percentage of the total costs. Therefore, studies on the COI of this disease in LA are essential, focusing on both out-of-pocket spending and the potential economic loss to patients' homes and families. Frontotemporal dementia (FTD) is a complex syndrome characterized by clinical disorders that include progressive changes in the functions of behavior, language, executive control, and motor symptoms associated with anterior and frontal temporal lobe degeneration , 2. ThisWorldwide, FTD represents up to 5–6% of all dementias. Although less frequent than Alzheimer's disease (AD) , 4, 5, iAccording to the World Health Organization (WHO), in 2015 the societal cost of dementia worldwide was US$818 billion, being equivalent to 1.1% of the global gross domestic product. Consequently, some reports classify the annual economic burden per FTD patient as considerable, amounting to US$119,654 per patient, being almost double the patient costs reported for AD (The cost of illness (COI) is the value of resources spent or abandoned because of a health problem. It includes the costs of the health sector (direct costs [DCs]), the value of productivity diminished or lost by the patient (indirect costs [ICs]), and the cost of pain and suffering (intangible costs) , 11. DCsAlthough its calculation is complicated, COI analysis provides essential information on the financial impact of the disease in order to make more efficient use of resources  by health managers, researchers, and medical specialists. However, most studies on the economic burden of disease focus only on direct medical costs, as they are the easiest for the health sector to identify, and this underestimates the total costs (TCs) of the disease. Therefore, the objective in this systematic review was to describe the costs related to FTD in LA countries.10.6084/m9.figshare.14100797  from inception to September 2020. Additionally, we explored records of the Health Technology Assessment and assessed economic evaluation databases through We developed the search strategy from Medical Subject Headings–MeSH (Pubmed) for “Frontotemporal dementia” and related words for “Cost of the illness,” “Cost-Benefit Analysis,” and “Economics,” employing a PICO structure approach as much as possible as there was no intervention in our research question. Additionally, we developed a search filter for LA countries. The complete search strategy is shown in Two independent authors (MM & ARC) performed title and abstract screening, and ambiguities were discussed until consensus was reached. This was repeated for full-text selection, and a third author (CAD) participated if there was any discrepancy through discussion and consensus. Consensus was reached in every case.We included studies conducted in LA countries published in English, Spanish or Portuguese. We considered studies of socioeconomic evaluation that provided data on the disease costs of FTD. Studies had to include a population diagnosed as FTD in accordance to the Lund and Manchester criteria . The aimTwo authors (MM & ARC) carried out the data collection independently using a standardized form in Microsoft Excel. A third author (VVR) verified the quality of the data before analysis. Additionally, authors were contacted to request unreported data.The data extracted was made up of antecedents: author, year of publication, country, number of patients included in the study and the analysis, data collection method, calculation of costs, and quality of the articles. We categorized the cost components into direct medical costs and ICs. The first was made up of medication costs, health care services, direct social care costs, and caregiver expenses, while ICs included loss of productivity , 11.Finally, we adjusted the costs for inflation using the Consumer Price Index inflation calculator of the US Bureau of Labor Statistics for all costs reported in US dollars (US$) by similar studies . We perfFor quality assessment, we adapted a tool developed for COI studies designed initially for diabetes by Afroz et al. . This toWe identified 920 studies in the databases. We eliminated 145 duplicates and screened a total of 775 by title and abstract. Of these, we assessed 20 studies in the full-text phase and selected three studies from four records for qualitative synthesis –19.One study was developed in a Brazilian population  and the The median score was 9 (range 9–9.5). In the general domain, only one study specifically defined FTD and the criteria employed for its diagnosis. Additionally, two of the studies , 19 collAlthough the reported costs and their components were not similar among studies, they were included individually as they agreed with the study objective. Additionally, not all studies discussed limitations.As FTD patients were a minority of patients in all of the studies, none presented a sample representative of the population. However, the cost calculation approach was adequate in all studies, using a bottom-up approach, meaning that costs were obtained at the service provider level. Moreover, all the studies reported deviation standards and means and adequately performed and described the statistical analyses. Two studies , 18 perfThe criteria to define medication costs in the studies of Custodio et al.  and RojaThus, medical costs, which had the most homogeneous definition across studies, amounted to a total of US$5,423.00, US$6,555.00, and US$10,166.64 per year per patient in Argentina, Peru, and Brazil, respectively –19.Social care costs were not reported by Rojas et al. (18). Custodio et al.  only repCaregiver costs were analyzed separately as the definition varied across studies. Ferreti et al. reported the cost of informal caregivers, calculated using the time spent in patient care and the minimum wage the caregiver would receive . Rojas eOnly the Brazilian study by Ferreti et al.  describeEach study calculated TCs differently. In Brazil, Ferreti et al.  includedOut-of-pocket expenses could not be quantitatively summarized for all studies, as the authors did not report them directly. Rojas et al. mentioned that all social-care costs were considered out-of-pocket expenses, but did not report them . HoweverIn this qualitative review, we describe the costs related to FTD in LA countries. We identified three studies from Peru, Argentina, and Brazil –19. In tThe majority of LA studies focused their analysis on DCs related to medication and health service acquisition. These costs contrast to the experience in high-income countries, in which the focus of the ICs are on evaluating the economic impact by unemployment, decreased productivity, or individual-family care expenses . Most ofMedication expenses, from $ 5,423.00 to $ 10,166.64 annually, are still lower than those reported in countries with a higher development index, with DCs associated with annual dementia being from US$10,000 to US$60,000 –23. ThisAdditionally, the reported caregiver costs ranged from $ 677.64 to $ 10,980.72. These DCs related to patient care are the highest, since the expenses related to the management of disability and dependency in FTD are as high as in other dementia conditions , 28. TheFerreti et al.  describeWe critically evaluated the studies included using a COI-specific assessment tool . OverallThe main limitation of this review was the small number of patients included in the studies from only three countries, which affects the extrapolation of the results in the LA region. Furthermore, poor comparability due to different definitions and classifications of FTD patients produced poor comparability among studies. Additionally, the heterogeneity due to different data sources, different study design and different component of the costs should be considered. Therefore, the establishment of guidelines for COI studies in dementias other than AD would homogenize published information and future reviews. However, our report of the different types of costs was exhaustive. We adjusted the costs for inflation and for an annual period, which allows comparison of the costs reported in this work with other studies from different regions and countries.With moderate quality studies, we estimated a range of $ 6,100.64 to $ 71,807.16 in TCs and $ 43,076.88 in ICs. In LA countries, the reporting of costs related to FTD continues to be focused on DCs. These costs remain lower than in developed countries, possibly due to the limited health budgets allocated. Only one Brazilian report analyzed ICs, representing the highest percentage of the TCs. Therefore, studies on the COI of this disease in LA are essential and should be focused on both out-of-pocket spending and the potential economic losses to patients and families.Expenditures should be appropriately distributed at public and individual health levels so that managers and specialists can provide efficient treatment options and well-being to patients with FTD. The knowledge gap related to indirect and intangible cost expenses in FTD creates an opportunity for interventions by interest groups in research and public managers.The original contributions presented in the study are included in the article/CA-D, MM, AR-C, CM-M, and VV-R planned and designed the protocol. NC and CA-D reviewed the methodological aspects of economic synthesis. VV-R, CM-M, MM, and AR-C wrote the manuscript. All authors designed the study and approved the final version of the manuscript.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.


Infectivity and neutralizing antibody titers of flavivirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are frequently measured using the conventional plaque assay. While the assay is useful in the determination of infectivity, conventional plaque assays generally possess lower sensitivity and are time-consuming compared to nucleic acid amplification tests. In this study, a microcrystalline cellulose (MCC), Avicel, was evaluated as an alternative to the conventional virus overlay medium, methylcellulose, for a plaque assay. The plaque assay was performed using dengue and COVID-19 clinical samples and laboratory-established flavivirus and SARS-CoV-2 strains. In virus titration of clinical samples, the plaques were significantly larger, and the virus titers were higher when Avicel MCC-containing overlay medium was used than with conventional methylcellulose overlay medium. In addition, for some clinical samples and laboratory virus strains, infectious particles were detected as plaques in the Avicel MCC-containing medium, but not in the conventional methylcellulose medium. The results suggest that the viremia titer determined using the new overlay medium containing Avicel MCC may better reflect the innate infectious and plaque-forming capabilities of clinical samples and better reflect virus infectivity. Virus shedding during acute viral infection has important implications for the transmission and control of infectious disease . In addiDuring the course of SARS-CoV-2 infection, individuals may remain PCR-positive for several weeks . Early d2. FcγRIIA-expressing BHK-21 cell lines were cultured in E-MEM supplemented with heat-inactivated 10% FBS and 0.5 mg/mL neomycin  at 37 °C in 5% CO2 [Baby hamster kidney cells , African green monkey kidney Vero cells , and FcγRIIA-expressing BHK-21  were usen 5% CO2 .2 for 5–7 days or until CPE formation was observed. Cell culture supernatant was then collected, clarified by centrifugation, aliquoted and, stored at −80 °C until use.The following viruses and strains were used in this study: DENV-1 (01-44-1 HuNIID and 99St12A strains); DENV-2 ; DENV-3 (CH53469 strain); DENV4 (SLMC318 and TVP360 strains); Zika virus ; Japanese encephalitis virus ; yellow fever virus ; SARS-CoV-2 . Laboratory virus strains were propagated on Vero and BHK cells at 37 °C in 5% COA total of 27 serum samples obtained from DENV-1 patients during a DENV outbreak in Northern Vietnam in 2017 were used in this study. All patients were confirmed to be infected with DENV-1 by RT-PCR . PrimaryThree types of overlay media to facilitate plaque formation for infectious virus detection were used in this study: (1) Avicel medium (Av), (2) Avicel and methylcellulose medium (AvMc), and (3) methylcellulose medium (Mc). To prepare the Av medium, a total of 12 g of Avicel  and 9.4 g of E-MEM powder  were dissolved in 1 L of double distilled water (DDW) and stirred with a magnetic bar until all reagents were dissolved. To prepare the AvMc medium, a total of 5 g of Mc , 6 g of Avicel, and 9.4 g of E-MEM powder were dissolved in 1 L of DDW. The Mc medium was prepared with 10 g of Mc and 9.4 g of E-MEM powder in 1 L of DDW and, the reagents were then autoclaved, cooled, and stored at 4 °C until use. For complete overlay medium, a total of 10 mL of 100 × L-glutamine , 20 mL of heat-inactivated FBS (Sigma), and 31.5 mL of 7.5% sodium bicarbonate  were supplemented. Each mixture was stirred for consistency prior to use.5 cells per well) with 1 mL growth medium E-MEM supplemented with 10% FBS, and incubated at 37 °C in 5% CO2 overnight to allow the formation of a cell monolayer. Infectious cell culture fluids and patient samples were serially diluted in E-MEM from 1:10 to 1:106 in 10-fold dilutions. A total of 100 µL of serially diluted sample was added to each well. At least two replicates of each sample were inoculated. The plates were then incubated at 37 °C in 5% CO2 for 60 min. After incubation, 2 mL of overlay medium was added to each well. For titrations of virus stock, the three types of overlay media  were used. After 4 to 5 days of incubation, the cells were fixed with 4% paraformaldehyde for 1 h at room temperature and stained with 0.25% crystal violet . The number of plaques was counted with the naked eye, and plaques were captured using an Automatic Colony Counter PSF-1000 . Virus titer was defined as the number of plaque-forming units per mL (PFU/mL). The size (mm) of 10 randomly selected plaques was measured using a Fluorescence Microscope BZ–X700 . Briefly, 10 plaques were randomly selected from one to three wells under the microscope with a magnifying power of 40. Each well was divided into four sections, and 2–3 plaques were randomly chosen from each section for measurement. The diameter line was placed at the major axis and measured according to manufacturer’s instructions. Flavivirus infection experiment was performed in a biosafety level 2 (BSL-2) facility, whereas SARS-CoV-2 infection experiment was performed in the biosafety level 3 (BSL-3) facility at the Institute of Tropical Medicine, Nagasaki University, according to local biosafety guidelines and regulations.Virus titration was performed using a conventional plaque assay . BHK-21,p-value was calculated using two-tailed t tests for the viral titer and Mann–Whitney U tests for the plaque size. Statistical analysis was performed using GraphPad Prism version 6.07 .The mean and standard deviation (SD) for virus titers and plaque size for each overlay medium in each cell line was determined by using GraphPad . Virus titer results were transformed to base-10 logarithm values for analysis. For comparison of each overlay medium, the 10 viral titer (PFU/mL) was significantly different between cultures grown in Av and Mc ).Of the 15 flavivirus strains used and among the three cell lines tested, up to one log higher virus titer was detected in 10 (66.7%), 4 (28.5%), and 14 (93.3%) flavivirus strains for BHK, Vero, and FcγR-BHK cells, respectively, when Av medium was used compared to Mc medium . The log10 virus titer (PFU/mL) differences between Av-containing media and Mc medium were statistically significant (p AvMc vs. Mc < 0.0001), the detection limit of virus titer by plaque assay was log10 2.0 PFU/mL. While a total of 42 COVID-19 samples were positive for the SARS-CoV-2 genome by RT-PCR, the samples were negative for plaque formation (p < 0.05).A total of 27 samples were obtained from patients with DENV-1 infection, and virus titers in each clinical sample were determined using three different cell lines and overlay media . In thesormation . These rormation . In addiDetection of infectious particles remains a challenge as live virus particles decrease concurrently with decreased virus shedding or viremia. In this study, higher virus titers and clearer plaque formation were detected in flavivirus and SARS-CoV-2 laboratory-established strains and clinical samples by a conventional plaque titration method using an improved overlay medium. As high virus titers have been associated with severe clinical outcomes ,4,5, assPlaque formation using Av has also been reported for enterovirus , Rift VaBecause of clearer plaque formation, plaques were consistently detected using Avicel overlay medium even when there was no plaque formation using the conventional Mc method. One of the challenges encountered in textbook descriptions of flavivirus viremia levels and SARS-CoV-2 virus shedding is that the conventional titration method uses Mc as an overlay medium. Our results demonstrated that this classical description of virus titer may have resulted in the infectious capacity of these viruses not being robustly evaluated, because of an underestimation of the infectious particle levels. In addition, clearer plaque formation allows the use of a wider range of clinical isolates for plaque-based assays, including virological and anti-viral drug studies and neutralization antibody titration for flavivirus and SARS-CoV-2 ,33,34,35


Resolving the Coronavirus Disease 2019 (COVID-19) pandemic is hamstrung by emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants with the potential to evade both natural and vaccine-induced immunity. Variants occur naturally from replication errors by viral polymerases and escape variants arise when virus mutations allow evasion of recognition by the immune system or therapeutic treatment. The selective pressure of vaccines, innate immune responses, and clinical interventions fosters the emergence of mutations that confer the capacity to continue infecting new hosts , and madRapid characterization of variants for future viruses is therefore essential to an effective pandemic response. If we can predict and characterize VOCs before they arise, monoclonal antibodies and potential vaccine antigens could be developed to proactively neutralize these variants. Furthermore, better understanding of the molecular characteristics of each variant could guide more precise public health policies.Currently, variants are identified and functionally tested using a combination of viral sequencing, cell-based assays, protein engineering methods, and computational tools. Sequencing of viral genomes from infected hosts identifies mutations away from the earliest reported genome of the virus, called the reference genome, highlighting possible variants. After identifying these mutations, cell and protein-based assays are used in the lab to test whether these mutations enable escape from neutralizing antibodies, which are antibodies created by the immune system from previous infection or vaccination ,5. MutatAs methods for variant characterization become increasingly powerful, we have yet to see effective coordination of the research groups conducting these studies across disciplines. Although the United States spends billions of dollars annually on infectious disease research and development, including genomic surveillance for identifying variants , this inIn preparation for future pandemics, we propose establishing a consortium of research groups with expertise in both computational and experimental techniques to more exhaustively map the landscape of variants upon the emergence of a new virus and predict which variants are most likely to arise. This would enable a rigorous set of standard analyses for assessing potential VOCs, as well as allow for early design of vaccines with efficacy against the variants deemed most likely to arise. While this work is currently being done for SARS-CoV-2 at multiple universities, companies, and government agencies, we can use lessons from this pandemic to propose a more rapid and coordinated response for future emerging viruses. Below, we highlight recent technological advances in these fields. By combining existing technology and new technologies, we envision a collaboration of groups employing computational and experimental tools to quickly predict and characterize likely VOCs, which could be used to aid initial identification of neutralizing monoclonal antibodies and vaccine design when a new virus emerges and inform policy decisions as variants arise.An efficient approach to predict viral variants would be for the US to invest in a new system for predicting and testing likely VOCs immediately upon identification of a new virus. This workflow would be handled by experts in the individual technologies working collaboratively and sharing data in real time. While we propose technologies that already exist, they are implemented in different labs around the world with different levels of expertise, requiring communication, collaboration, and sharing of information. One delay in the US response is bringing together these experts, allowing access to clinical surveillance samples and coordinating efforts between labs in a meaningful manner. The current variant response approach in the US is reactive since these collaborations take time to develop after a new virus becomes a concern to public health, increase the administrative burden for hospitals and diagnostic laboratories who are already overstretched in a pandemic and have to ensure compliant sample handling and patient consent to surveillance studies, and could ultimately cost thousands of lives, billions of dollars, and extend a pandemic because the nascent collaboration is not fast enough to prevent the spread of new variants.Instead, this proposed workflow, described in Computational approaches can rapidly identify VOCs early in a pandemic, especially given the reduced need for time, resources, and safety regulations associated with many computational approaches. Computational tools are a necessity since there are approximately 20^n single coding mutations, let alone multiple mutations and indels. While experimental tools are still the gold standard, the best experimental approaches, including pooled screening experiments, would not be able to handle every possible mutation.We envision this coordinated response would be activated upon obtaining the genetic sequence for a new virus. With only the genome, computational tools such as BLAST  and otheOne notable recent study implemented a deep learning algorithm known as a language model to build a powerful predictor of variants . The modAnother promising computational technique for early interrogation and prediction of viral changes are models based on nucleotide composition. It is known that nucleotide and dinucleotide compositions of viruses evolve to match their host species to evade detection and subsequent clearance by cellular defense systems ,21. AccoD of interaction [Computational modeling of the structural effects of mutations is another approach that provides rapid molecular characterization of a large set of viral variants. For example, molecular dynamics (MD) simulations have characterized the change in binding affinity of the SARS-CoV-2 spike receptor-binding domain to the human entry receptor angiotensin converting enzyme 2 (ACE2) upon mutation , and in eraction , achievieraction  and the eraction  were avaAs the pandemic progresses and novel variants arise, their genomes will be collected into databases  and fed In sum, these computational methods provide an enormous opportunity to support efforts to predict and identify VOCs, traditionally driven by experimental techniques. For example, finding suitable animal models posed an initial challenge for studying SARS-CoV-2 because it was unable to infect mice due to differences in the ACE2 receptor protein used for viral entry. Computational techniques allow for molecular insight independent of the access to these and other resource-intensive experimental techniques. Furthermore, computational approaches offer the unique opportunity to integrate information on the virus sourced from many different types of analyses, such as incorporating observed VOC frequencies from genomic surveillance to refine the predictions made by the models, thus accounting for human-specific factors that are not captured by the molecular mechanisms that form the basis of the computational techniques. Importantly, incorporating continuous low-level surveillance in the proposed consortium model will allow continuous refinement of computational methods, across several families of viruses that occur seasonally. This could also help better model seasonal influenza strain development.Complementing the computational tools, lab-based technologies could simultaneously predict variants and feed additional information to the computational tools. The most straightforward approach to mapping the mutational landscape is to simply test each one in turn, using a technique known as deep mutational scanning or saturation mutagenesis. In a series of studies, the group of Dr. Jesse Bloom at the University of Washington used an exhaustive library of single nucleotide variants of the SARS-CoV-2 spike protein receptor-binding domain expressed on the surface of yeast to test for binding to the entry receptor ACE2 , for escTo map the escape landscape of a whole viral genome, laboratory researchers incubate the virus with cells and potential treatments to examine how the virus mutates to adapt to selective pressures . This apLab-based assays can also be performed to identify epitopes for vaccine targets that would be less prone to mutations by finding epitopes and antibodies that have high binding affinities across related viral species. Recently, Wang and colleagues identified the main antigens of the SARS-CoV-2 spike (S) protein targeted by the immune system, which were distinct from antigens that are more conserved between SARS-CoV-2 and SARS-CoV-1 . By elimTo implement this system, strong computational expertise, tools, and access to computing power are needed within the consortium so that results can be quickly utilized by all assays to predict variants and vaccine candidates. The consortium would be active at a “maintenance” level continuously, characterizing the yearly influenza pandemic as well as several cold virus strains, conducting surveillance and optimizing training algorithms on the emergence of variants. This could improve the influenza vaccine selection and our response to other seasonal pathogens. We could also learn a lot about virus surveillance, transmission and mutagenic behavior of viruses, potentially informing public health recommendations for nonpandemic viruses that still cause significant morbidity . A consortium conducting continuous low-level virus surveillance in the population could easily ramp up activities when a virus reaches Phase IV of WHO’s pandemic phase designation, which includes sustained human to human transmission. The sequence of this virus will be immediately fed into the computational models above  while simultaneously being used in the lab-based assays (by other labs in the consortium with appropriate biosafety labs and expertise), with results being shared automatically among all members. These results will be fed into a central database maintained by the consortium and distributed to each member to update their respective work. Ultimately, a list of most likely variants and potential phenotypes associated  will be output along with potential vaccine and therapeutic targets within a few weeks to months.There are examples of many successful consortia, including The Broad Institute’s Interdisciplinary Research Consortium, the Enzyme Function Initiative funded by the National Institute of General Medical Sciences, and many others. The Viral Hemorrhagic Fever Consortium (VHFC) is a great example of a nonprofit consortium starting from an initial grant, spanning multiple universities, countries, and agencies and has since expanded to include many additional partners. The majority of existing consortia focus on a single virus or group of viruses, while this consortium could be expanded to include a broad range of experts spanning all viral families. Using lessons learned from these successful consortia as well as best practices for governance, reward structures, and implementation of consortia ,45, we bThe ability to centralize consent forms, IRBs, and other documentation while gaining access to large data sets will entice many labs to join this consortium. This consortium would be formed with NIH support, which could help develop “blanket” IRBs and consent forms to increase sequencing of samples throughout future pandemics by clinical and diagnostic labs. By working with these labs, the consortium could facilitate increased sequencing, improving surveillance for VOCs while providing additional data for the computational models and increasing efficacy of the consortium. These computational methods were applied to SARS-CoV-2 even at the poor sequencing rates in the country, so the additional sequencing would not be critical, but would certainly help policy makers and the consortium.There are many documented benefits to collaborative research, and this consortium would improve access to different resources, increase professional networks, minimize burdens on individual labs, provide additional funding for labs, provide opportunities for publishing, and ultimately speed up research that has the potential to impact the entire world. However, the consortium needs to be managed and funded well, be ready to act when called upon, and established structures and workflows  need to be sufficiently robust to support a rapid expansion of activities in case of a pandemic. Best practices mentioned above and a significant amount of learnings from the COVID-19 pandemic can help implement a system that helps prepare for and mitigate future pandemics.Additionally, virology and basic biology expertise is required, especially related to the lab-based assays working with high containment pathogens at Biosafety Level 3. The verification experiments we propose would comprise in vivo lab technologies requiring a Biosafety Level 3 and use mainly pseudovirus assays to avoid working with dangerous live viruses. It is of utmost importance that more virulent virus strains are only generated using precautions such as pseudovirions and appropriate containment and that once the computational tools are trained to be highly predictive, safety measures against abuse are taken, and both details of the tools themselves and sequences of highly virulent proposed viruses are appropriately safeguarded together with NIH and other government institutions. However, if the consortium is successful in predicting early variants that are moderately efficient at evading selective pressure and this informs improved vaccine design with future variants in mind, the risk of developing a highly virulent strain is in practice significantly reduced. Allowing a virus to “fester” with only partially effective vaccines over long periods of time will increase risk for more virulent variants.Another important consideration for VOC prediction is the different selective pressures experienced by the virus in parallel as it infects different hosts. For example, variants of SARS-CoV-2 are thought to have arisen in farmed minks before transmitting back to humans in Denmark  and PolaAdditionally, computational modeling and prediction efforts separate from the broader workflow may be warranted: While the computational models described above showed success in separating viral sequences from different host species for both influenza viruses and coronaviruses , the abiThe vision we present for an ideal response to the identification and sequencing of a new virus is to maintain at a low level and in case of pandemic, rapidly deploy both a computational and a lab-based assay pipeline to predict and characterize the most likely VOCs, and define optimal monoclonal antibody and vaccine targets. While many of these technologies were created for previous viruses and pandemics, they have been updated and now include many more computational aspects because of SARS-CoV-2. We have highlighted many older and newer techniques that would benefit from integration in a consortium of experts that could be activated at a specific time after new a virus is identified. This coordinated approach would combine the power and speed of computational methods with the accuracy and translation of lab-based assays. By preemptively organizing this group around recurring influenza and other seasonal virus outbreaks, algorithms could be trained and workflows optimized for continuous low-level virus surveillance, allowing a rapid and efficient ramp-up during outbreaks that could become pandemics. A coordinated effort could predict VOCs early to aid vaccine design and guide public health policies. These candidate vaccines would have a greater likelihood of being able to prevent and treat variants that arise or even prevent variants from arising by squashing transmission early in a pandemic. This approach could also be used to design booster shots for a pandemic even after initial vaccines are available. Additionally, the characterization of each viral variant can also provide critical data to guide public health messaging as new variants do begin circulation. This broadly applicable workflow would ultimately cost a fraction of what the US is spending on infectious disease work in addition to saving lives during future outbreaks and generate unprecedented insight into virus transmission and evolution.


Plasmodium spp., is still causing serious problems in endemic regions in the world. Although the WHO recommends artemisinin combination therapies for the treatment of malaria patients, the emergence of artemisinin-resistant parasites has become a serious issue and underscores the need for the development of new antimalarial drugs. On the other hand, new and re-emergences of infectious diseases, such as the influenza pandemic, Ebola virus disease, and COVID-19, are urging the world to develop effective chemotherapeutic agents against the causative viruses, which are not achieved to the desired level yet. In this review article, we describe existing drugs which are active against both Plasmodium spp. and microorganisms including viruses, bacteria, and fungi. We also focus on the current knowledge about the mechanism of actions of these drugs. Our major aims of this article are to describe examples of drugs that kill both Plasmodium parasites and other microbes and to provide valuable information to help find new ideas for developing novel drugs, rather than merely augmenting already existing drug repurposing efforts.Malaria, a disease caused by the protozoan parasites  Plasmodium spp. which infect humans, Plasmodium falciparum has the highest pathogenicity and accounts for most of the cases in the African region , enlarged spleen and liver, and hematuria, all of which can lead to unconsciousness followed by death . These serious symptoms are the results of sequestration of the parasite on the epithelial cells in the microvasculature, which makes P. falciparum the most virulent among the human malaria parasites , there were 229 million sick people and 409,000 deaths including 67% of children aged under 5 years old, due to malaria in the world in 2019 . Despiten region . Other tby death . Also, Parasites .Plasmodium spp. has been one of the most serious problems necessary to be addressed. Drug-resistant P. falciparum have emerged for virtually all antimalarial drugs, including chloroquine, quinoline, sulfadoxine–pyrimethamine, mefloquine, or quinoline, and also artemisinin (ART), the essential drug in the current first-line treatment of ART combination therapies (ACTs)  . To copete drugs .Antiviral chemotherapeutics have been one of the subjects which have been focused in the field of public health. Transboundary infectious diseases are among the most serious issues not just in developing countries but also in economically developed countries and include diseases caused by human immunodeficiency virus (HIV), pandemic H1N1 influenza A virus, Zika virus, Ebola virus, dengue fever virus, West Nile virus, severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, and currently, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although there have been developments of some antiviral drugs in practice such as adamantane derivatives and neuraminidase inhibitors for influenza A virus  and a grRepurposing of already approved drugs for drug treatment different from the original purpose is an alternative approach compared with the discovery of entirely new drugs and often beneficial since the toxicity risk of the drug can be eliminated and the required time and costs for the required human trial processes can be substantially reduced; the average cost for a repurposed drug is US$300 million, compared with an estimated $2–3 billion for a new chemical entity with success rates of less than 4% in the top 3 areas of drug development between 1990 and 2004 . HoweverIn the current situation of COVID-19, the antiviral effects of some antimalarial drugs have been drawing attention, leading to an increase of related studies . Also, cP. falciparum, the spread of drug-resistant parasites in the world forced halting its use in practice. The antimalarial action of CQ is related to hemoglobin digestion of malaria parasites. CQ exerts its antimalarial activity by inhibiting hemoglobin digestion in the digestive vacuole (DV) in the parasite, a lysosomal acidic organelle, in which the parasite digests hemoglobin transferred from the host red blood cell to heme and converts into non-toxic hemozoin  is an FDA-approved drug used for the treatment of malaria for decades . Althoughemozoin . CQ is ahemozoin . Due to vailable .P. falciparum has been studied  on the DV, which interferes CQ concentration inside the DV , leading to interference in the binding of ACE2 and the spike (S)-protein of SARS-CoV-2 upon the virus entry in the host cells . Such intivities .Lentivirus in the family Retroviridae, binds to its target CD4+ T cells through binding of the gp120 protein of the virus to the CD4 molecule, and loss of infectivity with the virus collected from HIV-infected cells with CQ treatment was reported in comparison to the virus from the untreated cells, suggesting that CQ changed the structure of HIV gp120 which bound to the host cell receptor  released through the digestion reactions cleave the endoperoxide bridge of ART. Only after this cleavage of the endoperoxide bridge, ART exhibits its antimalarial activity with the carbon-centered free radicals produced by the endoperoxide bridge cleavage  and its fast-acting killing of the intraerythrocytic stages of arasites . The endarasites . The erycleavage . Activatcleavage . Recent cleavage . Also, se to ROS . The quie to ROS .P. falciparum, manifested by slower rates of parasite clearance, was reported in 2014 in South Asian countries , hepatitis B virus (HBV), hepatitis C virus (HCV), HIV, and SARS-CoV-2  of ART against HCV-infected cells was 2- to 5-fold reduced by the treatment with L-N-acetylcysteine (L-NAC), which inhibits free radical generation  pathway by targeting RelA/p65 was reported as a mechanism of artesunate action on HCMV-infected cells . RelA/p6in vitro study , which are known to inhibit the growth of malaria parasites  with the active sites of P. falciparum plasmepsins, multiple-copy aspartic proteases involved in hemoglobin digestion of the parasite, suggesting potential targets of ARPIs against Plasmodium spp. in their hemoglobin digestion pathways which are essential for the antimalarial activities of both ART and CQ  than the untreated ones  and glutathione S-transferase (GST), respectively, in arasites . In thisted ones . Since dPlasmodium spp. are quite common in endemic areas , while others target bacterial metabolic pathways , protein synthesis , and nucleic acid synthesis  . Due to tivities . Here, wPlasmodium spp., respectively, which are the enzymes catalyzing two reactions in the series in FSP (Inhibitors of folate synthesis pathway (FSP), such as sulfonamides, are widely used antibacterial agents and inhibit the synthesis of folate, a major precursor of nucleic acids of bacteria. Sulfadoxine–pyrimethamine (SP) targets dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) of s in FSP . Though s in FSP .Plasmodium spp. infections compared with the untreated ones. Similar to SP, the targets for sulfamethoxazole and trimethoprim are the enzymes in the series of FSP, DHPS, and DHFR, respectively  are a group of antibiotics effective against a wide range of bacteria, including Gram-positive and Gram-negative bacteria, which have been used for the treatment and prophylaxis of um vivax .in vitro assay with a clinical isolate of P. falciparum, the IC50 values for tigecycline and doxycycline were 699 and 4,276 nM, respectively, suggesting faster action of tigecycline than tetracyclines of the older generations  was evaluated as a potential target of new antimalarial compounds. However, some studies reported high sequence identities between PfGSK-3 and its human homologs GSK-3α or GSK-3β (P. berghei-infected hosts (Manzamines are inhibitors of glycogen synthase kinase-3 (GSK-3), which is also thought to be the target for their antimalarial activities ; therefor GSK-3β  and the ed hosts ; 50 to 1ed hosts . On the ed hosts , suggestAxinella cannabina  and its inhibition of the heme-synthesizing ability of ferrochelatase, and despite its failure in clinical trial, this unique mode of action of griseofulvin demonstrated a new potential lead chemical to develop new antimalarial drugs  3A4 and is used to treat a number of fungal infections (P. falciparum strains in vitro and with the multidrug-resistant Plasmodium yoelii nigeriensis in mice in vivo assay (Ketoconazole interferes with ergosterol synthesis by inhibition of cytochrome Pfections . The comvo assay . Artemisvo assay .Due to the impact of COVID-19, significant effort has been taken for the discovery of antiviral drugs since the need for serious countermeasures was recognized when the virus was spreading throughout the world. At the same time, antibacterial, antifungal, or antiparasitic drugs with promising antiviral activities were sought more than ever. The drugs described in this article are summarized in TF: manuscript writing, editing, and figure and table production. TE, HT, and IS: manuscript writing. All authors contributed to the article and approved the submitted version.This study was supported by Japan Society for the Promotion of Science, Grant-in Aid for Scientific Program 20K06389.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.


Background: With the increasing disease burden of cancer worldwide, more and more anticancer drugs have been approved in many countries, and the results of budget impact analyses (BIAs) have become important evidence for related reimbursement decisions.Objectives: We systematically reviewed whether BIAs for anticancer drugs consider the scope of costs rationally and compared the results of different cost scopes to provide suggestions for future analyses and decision-making.Methods: Eligible BIAs published in PubMed, Embase, Web of Science, and the Cochrane Library from 2016 to 2021 were identified based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We extracted 15 terms from the included studies and analyzed how they considered the scope of costs. In addition, a budget impact model was developed for the introduction of geptanolimab to China's National Reimbursement Drug List to enable a comparison of two cost-scope scenarios.Results: A total of 29 studies were included in the systematic review. All 29 studies considered the costs of anticancer drugs, and 25 (86%) also considered condition-related costs, but only 11 (38%) considered subsequent treatment costs. In the comparative study, the predicted budget impacts from 2022 to 2024 were significantly impacted by subsequent treatment costs, with annual differences between the two cost-scope scenarios of $39,546,664, $65,866,161, and $86,577,386, respectively.Conclusions: The scope of costs considered in some existing BIAs for anticancer drugs are not rational. The variations between different cost scopes in terms of budget impact were significant. Thus, BIAs for anticancer drugs should consider a rational scope of costs that adheres to BIA guidelines. Researchers and decision-makers should pay more attention to the scope of costs to achieve better-quality BIAs for anticancer drugs and enhance reimbursement decision-making. Cancer comprises a large group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body uncontrollably . Given iTo prolong the lives of cancer patients, an increasing number of anticancer drugs have been authorized in many countries. For example, the United States Food and Drug Administration approved 17, 12, and 50 novel anticancer drugs in 2018, 2019, and 2020, respectively , while CBudget impact analysis (BIA), which supplements cost-effectiveness analysis (CEA), is a decision-making tool that can be used to predict the financial impact on reimbursement funds of the adoption of a new healthcare technology in a specific healthcare setting. The result of a BIA is generally used to determine the affordability of a new intervention for a specific payer , 10. ManThe BIA framework generally uses a simple cost-calculator approach that synthesizes costs and epidemiology parameters , 13. RegSome organizations, such as the International Society for Pharmacoeconomics and Outcomes Research (ISPOR), have published BIA guidelines in an effort to standardize the BIA calculation framework, and have provided advice on the scope of costs that need to be considered , 16. HowIn this study, we systematically reviewed a range of published BIAs for anticancer drugs, focusing on the scope of costs, and then compared the results of different cost scopes using an example. Our aim was to confirm the necessity of rationally considering the scope of costs used in BIAs for anticancer drugs and to provide guidance for BIAs and relevant decision-makers.Some guidelines for BIAs have been published in an effort to standardize research procedures. To obtain a better understanding of existing recommendations regarding the scope of costs used in BIAs, we searched for and summarized these guidelines. Based on a previous review , 10 BIA This systematic review was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . PubMed,Studies were included if they (1) were original BIAs pertaining to anticancer drugs used to treat cancer patients, (2) reported the scope of costs, and (3) were published in English. Studies were excluded if they (1) did not focus on cancer patients, (2) did not include the scope of costs, (3) calculated the budget impact of biosimilars compared with that of the original drug , or (4) were published in the form of a systematic review, meta-analysis, abstract, or dissertation. The literature search and screening were undertaken independently by two investigators. Any disagreements were adjudicated by senior investigators.On the basis of the ISPOR Task Force guidelines , 16, we To illustrate the influence of the scope of costs on BIA results, we developed a Microsoft Excel-based budget impact model for an anticancer drug and estimated two cost-scope scenarios: scenario 1, which did not consider subsequent treatment costs, and scenario 2, which considered subsequent treatment costs. We then compared the results of these two cost-scope scenarios. The model was developed based on ISPOR guidelines , 16.This model was built to estimate the budget impact of introducing geptanolimab as a treatment option for patients with relapsed or refractory peripheral T cell lymphoma (R/R PTCL) from the perspective of China's National Healthcare Security Administration. The target population was the annual number of new patients with R/R PTCL. The model conceptualized two distinct market scenarios: (1) a status quo scenario in which geptanolimab was not included in the National Reimbursement Drug List (NRDL), which only included chidamide for the treatment of R/R PTCL; and (2) an alternative scenario in which geptanolimab was included in the NRDL and offered as an alternative treatment to chidamide. The budget impact was the cost difference between the two market scenarios. The baseline year was 2021 and the time horizon was 3 years. The structure of the model is shown in Demographic and epidemiological data were obtained from published studies, statistical yearbooks, and expert interviews –34.As chidamide was the only drug listed in the NRDL for treatment of R/R PTCL, we assumed that the market share for chidamide was 100% in the scenario without geptanolimab, and in the scenario in which geptanolimab was included in the NRDL, we assumed that geptanolimab would gradually replace chidamide over time. The various market shares were based on available sales data for chidamide .Based on the study perspective, in scenario 1 we only considered drug costs and condition-related costs, which included drug acquisition costs, inspection and testing costs, disease management costs, and AE management costs. As mentioned above, in scenario 2, we also considered subsequent treatment costs. All interventions and condition-related costs and reimbursement-related parameters were based on the Menet Medical Information database, published literature, China's National Healthcare Security Administration website, expert interviews, and assumptions –39. All In addition, we assumed that the median progression-free survival rate was equal to the average treatment duration using geptanolimab and chidamide. We also used the rate of disease progression in 1 year under treatment with geptanolimab and chidamide to estimate the number of patients who were likely to experience further progression and require further treatment each year. Clinical efficacy data for geptanolimab and chidamide were obtained from published clinical trials , 39. SubA summary of all model inputs and their sources is presented in The study perspective is related to what resources should be assessed in BIA. The majority of the guidelines included recommend that the perspective should be that of the healthcare payer or the budget holder , 21–27. The existing recommendations regarding the scope of costs are summarized in A total of 1,367 articles were initially identified, of which 29 articles were included in the final analysis –70. Figun = 18, 62%)              , 64, 66,= 2, 7%) , 70 and = 2, 7%) , the Net= 2, 7%) , Japan  , Norway = 2, 7%) , Saudi A= 2, 7%) , Spain  , and Tha= 2, 7%) , as well= 2, 7%) . The stu=8, 28%) , 62, 65,=5, 17%) , 59, 64, 4, 14%) , 68, 70, 3, 10%) , 53, 63,= 2, 7%) , 61, mye= 2, 7%) , 58, mel= 1, 3%) , head an= 1, 3%) , cell ca= 1, 3%) , gastroe= 1, 3%) . Most of= 1, 3%) , 53, epi= 1, 3%) , 62, imm= 1, 3%) , and vas= 1, 3%) .n = 22, 76%) considered the budget-holder perspective  considered the societal perspective , 67, 69,spective –66, 68. spective , 55, 64,spective , 65, 68.spective , 58, 68.The focus of our study was the scope of costs included in the BIAs of the sample studies. All of the studies considered the costs of anticancer drugs, most of which were calculated based on the unit prices and treatment duration assumptions. In 12 studies (41%), the authors assumed a treatment duration on the basis of treatment effect data, including the median progression free survival, the average metastasis-free survival, the median time to treatment failure, and the time until relapse , 63, 69.Regarding condition-related costs, 25 studies (86%) considered these, most of which included administration costs , AE management costs , gene-testing costs , and hospitalization costs. Of these studies, 12 (41%) provided a clear reason why they considered condition-related costs, while the other 13 (48%) reported condition-related costs directly. For example, Stargardter et al.  considerFour studies (14%) did not consider condition-related costs. Monirul et al.  and GoldTwo studies (7%) considered indirect costs, which included loss of productivity or absence from work of either the patient or a caregiver , 70. TheBased on demographic and epidemiological data, it was estimated that there would be 7,885, 9,050, and 10,017 newly diagnosed patients with R/R PTCL in China in 2022, 2023, and 2024, respectively. These patients would receive treatment with either geptanolimab or chidamide. The resulting market shares are shown in When not considering subsequent treatment costs (scenario 1), comparing the two market scenarios (without/with geptanolimab in the NRDL), the model estimated that the total annual reimbursement budget would increase by $1,458,842, $1,844,493, and $2,190,023 in 2022, 2023, and 2024, respectively. These increases were mainly driven by the longer period for which patients receiving geptanolimab were progression-free compared with that for patients receiving chidamide.When considering subsequent treatment costs (scenario 2), there was a shift from cost increases to savings, with the model estimating that the total annual reimbursement budget would decrease by $38,087,822, $64,021,668, and $84,387,363 in 2022, 2023, and 2024, respectively. These decreases were mainly driven by (1) the higher annual disease progression rate in chidamide patients compared with that in geptanolimab patients, and (2) the higher average annual costs of subsequent treatment for chidamide patients compared with that for geptanolimab patients.The differences in the budget impact of the two cost-scope scenarios were significant, at $39,546,664, $65,866,161, and $86,577,386 in 2022, 2023 and 2024, respectively. The results of the comparative study are shown in We systematically reviewed BIAs for anticancer drugs conducted over the last 5 years and included 29 studies in our analysis. These studies were conducted in 11 countries and were performed from different perspectives including those of the payer, healthcare system, and society. They were representative of both the increasing number of BIAs for anticancer drugs and the main approaches used in BIAs for anticancer drugs. To the best of our knowledge, changes to the disease spectrum have resulted in increasing numbers of patients being diagnosed with cancer worldwide, leading to more and more innovative anticancer drugs being developed by pharmaceutical companies and approved by various governments , 72. BecThe results of our systematic review revealed that although BIA guidelines from the ISPOR, the NICE, and various countries indicate that costs related to decision-making should be included in BIAs , 19–27, The results of our comparative study showed that for some anticancer drugs, the inclusion of subsequent treatment costs can result in significant differences in the budget impact, even replacing costs with savings in some cases. There are two main reasons for this: first, innovative anticancer drugs usually have a better treatment effect, albeit over a longer treatment duration at a higher price . Thus, iBIAs for anticancer drugs need to rationally consider the scope of costs in accordance with BIA guidelines, for instance, the ISPOR good practice guidelines , 16, befIn addition, it should be noted that although a comprehensive scope of costs is important in BIAs for anticancer drugs, not all BIAs need to consider all related costs. When the target drug and its comparator(s) display no differences in terms of treatment effects and only differ in price, such as biosimilars, researchers do not need to consider a comprehensive scope of costs. Because the consumption of medical resources during subsequent treatment regimens are similar, this will not have a significant effect on the BIA. This is why we excluded BIAs for biosimilars from our systematic review –78.There is another important point that researchers need to ensure the transparency of BIAs for anticancer drugs, because some published BIAs are too simple and do not provide enough cost information, such as detailed parameters and assumptions related to costs . These wBIAs and affordability estimates for anticancer drugs, especially innovative drugs, have become increasingly important for reimbursement decision-makers in many countries in recent years. Our study is the first systematic review of BIAs for anticancer drugs and also the first to discuss the scope of costs in relation to BIAs. Among the previous studies, Jahn et al.  publisheOur systematic review has several limitations. First, BIAs for anticancer drugs are usually undertaken as a supplementary exercise in addition to the CEA that is submitted to budget holders, instead of being prepared independently. Therefore, the BIAs included in our systematic review might only represent a subset of all anticancer-drug BIAs, and some relevant studies may have been overlooked. Second, because the treatment regimens for various types of cancer differ and are generally complex, there is no optimal approach to calculating costs, and thus different approaches may have resulted in different outcomes in terms of budget impact. However, we did not consider this issue in our comparative study. Third, in addition to the scope of costs, there are several other factors influencing the accuracy and reliability of BIAs, such as uncertainty and scenario analyses, validation, and data sources. However, because these were not the focus of our study, we did not consider these factors. Fourth, although nowadays almost each jurisdiction has their own BIA guideline, we only included 10 guidelines as reference. Because some BIA guidelines are not available in public databases or published in other than English. This limitation restricted us from comprehensively reviewing all cost-scope recommendations.Most BIAs for anticancer drugs do not rationally consider the scope of costs, which is not in line with the recommendations of the BIA guidelines, and is unrealistic. Our comparative study showed that the difference in budget impact resulting from considering different cost scopes was significant, especially when the same payer was responsible for both the target intervention costs and condition-related costs. Thus, researchers undertaking BIAs for anticancer drugs and reimbursement decision-makers should pay more attention to the scope of costs to improve the rationality, accuracy, reliability, and equity of BIAs for anticancer drugs and the related reimbursement policies.The original contributions presented in the study are included in the article/HL: takes responsibility for the data source and the accuracy of the modeling analysis. YM and HL: study design. YM and YL: literature search and data extraction. YM: drafting of manuscript. AM: critical revision of the manuscript. All authors contributed to the article and approved the submitted version.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.