Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. Here, we identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although our knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using  In multicellular organisms, damaged cells are frequently removed via apoptosis, the major form of programmed cell death. Intriguingly, these apoptotic cells can emit signals to induce proliferation of their neighboring cells for the maintenance of tissue homeostasis, a phenomenon termed apoptosis-induced proliferation (AiP). Caspases, a family of cysteine proteases well known to execute apoptosis, also play a critical role to trigger AiP via activation of JNK, a stress response kinase. In this study, we identified the actin cytoskeleton, a dynamic structural network of the cell, as a key mediator of JNK activation in AiP. During this process, actin filaments undergo increased polymerization which depends on LIM domain kinase 1 (LIMK1). Genetically, caspases and LIMK1 work together to promote actin polymerization, which in turn drives production of reactive oxygen species (ROS) and the subsequent activation of JNK. Therefore, our study discovered a role of actin remodeling in dying cells which mediates the non-apoptotic roles of caspases in AiP and tissue repair. In multicellular organisms, damaged cells are frequently removed by apoptosis, the major form of programmed cell death. Intriguingly, these stress-induced dying cells, prior to their removal, can actively induce proliferation of neighboring cells to compensate for their loss and maintain tissue integrity –8. This Drosophila have revealed distinct mechanisms of AiP mediated by different groups of caspases. In differentiating tissues where cells have exited mitosis, the caspase-3-like effector caspases DrICE and Dcp-1 activate Hedgehog signaling to drive cell cycle re-entry [Caspases, an evolutionary conserved family of cysteine proteases, are essential for both apoptosis and AiP. Whilst caspases mediate execution of apoptosis, they can also promote AiP via activation of mitogenic signals such as cytokines or growth signaling pathways in a context-dependent manner , 14–16. re-entry , 17.Notably, in the proliferating larval eye and wing epithelia, Reactive Oxygen Species (ROS), the oxygen-containing free radicals produced during cellular metabolism, accumulate in apoptotic tissues and trigger activation of JNK , 19. Rechid, a pro-apoptotic gene, and p35, an inhibitor of effector caspases DrICE and Dcp-1, are simultaneously expressed under control of the ey-GAL4 driver  together with the tub-GAL80ts, a ubiquitously expressed and temperature sensitive (ts) inhibitor of GAL4 [tub-GAL80ts inhibits activity of DE-GAL4 at 18°C. A temperature shift to 30°C releases this inhibition and allows expression of hid, i.e. induction of apoptosis, in the dorsal half of the eye disc under the control of DE-GAL4. After a 12-hour period of hid expression, larvae were transferred back to 18°C for tissue recovery. Compared to the control where only GFP is expressed , a F-actin specific marker. Compared to the control ey>p35 , in ey>hosophila , to indimulation . To deteng discs . Therefohid-p35-expressing discs are overgrown, the actin accumulation we observed in these tissues can potentially be a secondary effect caused by tissue overgrowth. Therefore, in addition to the whole disc analysis, we also employed a clonal analysis to determine whether F-actin accumulates in AiP independently of tissue overgrowth. We co-expressed hid and p35 in heat-shock-induced clones (hid-p35 clones) and examined F-actin in these clones 48 hours after their induction  does not inhibit the F-actin accumulation in hid-p35 clones , moderate (M) and severe (S), as previously described [UAS-hid; ey-Gal4 UAS-p35/TM6B,tub-Gal80 was used for the analyses of larval eye discs because of the convenience to identify the correct larval genotypes. Accordingly, the ey>p35 controls used in each experiment are the ey-Gal4 UAS-p35 on either second or third chromosome as appropriate.Genetic crosses for all experiments were reared at 25°C unless otherwise noted. Two escribed . The stoUAS-LIMK1RNAi lines , LIMK12 (59033), UAS-LIMK1 (9117), UAS-cofilin (9235), UAS-droncRNAi (32963), UAS-Myo1DRNAi (33971), UAS-SOD (24750), UAS-mCherry (38424), T80-GAL4 (1878), nub-GAL4 (86108), TAK12527 (58809) and TRE-red (59012) were obtained from the Bloomington Drosophila Stock Center. Dorsal Eye-Gal4  [UAS-bskDN [UAS-dronc [UAS-DuoxRNAi [GMR-hid [DE-Gal4) , UAS-bskAS-bskDN , UAS-droAS-dronc , UAS-DuoDuoxRNAi  and GMR-UAS-SOD 250, UAS-mDEts>hid ; 2. DEts>LIMK1RNAi ; 3. DEts>hid-LIMK1RNAi ; 4. DEts>cofilin ; 5. DEts>hid-cofilin  were raised at 18°C. Expression of the UAS-constructs  was induced by a temporal temperature shift to 30°C for 12 hours. This was then followed by a 72 h recovery period at 18°C before the late 3rd instar eye discs of each genotype were dissected and analyzed. Full details of the DEts>hid assay have been described previously [Larvae of the following genotypes 1. eviously .rd instar stage. (1) Generation of the control in larval p35 expressing clones: hs-FLP/+; act>y+>GAL4 UAS-GFP/+; UAS-p35/+. (2) Generation of hid and p35 co-expressing clones: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/+; UAS-p35/+. (3) Generation of clones expressing hid, p35 and droncRNAi: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/+; UAS-p35/UAS-droncRNAi. (4) Generation of clones expressing hid, p35 and LIMK1RNAi: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/+; UAS-p35/UAS-LIMK1RNAi-57012. (5) Generation of clones expressing hid, p35 and cofilin: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/+; UAS-p35/UAS-cofilin. (6) Generation of clones expressing hid, p35 and bskDN: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/+; UAS-p35/UAS-bskDN. (7) Generation of clones expressing hid, p35 and SOD: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/+; UAS-p35/UAS-SOD. (8) Generation of clones expressing hid, p35 and DuoxRNAi: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/UAS-DuoxRNAi; UAS-p35/+. (9) Generation of clones expressing hid, p35 and Myo1DRNAi: hs-FLP/UAS-hid; act>y+>GAL4 UAS-GFP/+; UAS-p35/UAS-Myo1DRNAi. For mosaic analysis with hid-p35 clones in the LIMK or TAK1 hemizygous mutant background, late second instar larvae of the following genotype were heat shocked at 37°C for 1 hour and then grew at 25°C for 48 hours before they were dissected and analyzed: (1) LIMK2/Y; act>y+>GAL4 UAS-GFP/+; UAS-hid UAS-p35/hs-FLP and (2) TAK12527/Y; act>y+>GAL4 UAS-GFP/+; UAS-hid UAS-p35/hs-FLP. The same act>y+>GAL4 FLP-out assay was used to generate control clones without expression of hid and p35.For mosaic analysis with clones expressing transgenes, a heat shock inducible FLP-out assay combined with the GAL4-UAS system was used , 49. Latrd instar larval eye and wing discs were dissected, fixed , and then labeled with antibodies using standard protocols as described [Late 3escribed . Primaryescribed . Secondaescribed .LIMK1 and cofilin in ey>hid-p35  versus the control ey>p35-mCherry , 100 third instar larval eye discs of each genotype were used to extract total RNA with an RNeasy Plus Mini Kit (Qiagen). For the analysis of LIMK1 RNAi efficiency, T80-GAL4 was used to drive expression of LIMK1RNAi, 26294 or 57012, ubiquitously in all 3rd instar larval imaginal discs. Total RNA was then isolated from 100 imaginal discs collected from either the control T80-GAL4 or T80-GAL4 UAS-LIMK1RNAi 3rd instar larvae using the TRI Reagent Solution (Invitrogen). Following RNA extraction, cDNA was generated from 300ng of total RNA with the GoScript Reverse Transcription System (Promega) for each genotype. This was followed by the real-time PCR using the SensiFAST SYBR Hi-Rox kit (Bioline) with an ABI Prism7000 system (Life Technologies). LIMK1 or cofilin mRNA levels were normalized to the reference gene ribosomal protein L32 (RPL32) by using the ΔΔCt analysis. Three independent biological repeats were analyzed in triplicate for each experiment. The following primers suggested by the FlyPrimerBank [LIMK1 Fw, GTGAACGGCACACCAGTTAGT; LIMK1 Rv, ACTTGCACCGGATCATGCTC; RPL32 Fw, AGCATACAGGCCCAAGATCG; RPL32 Rv, TGTTGTCGATACCCTTGGGC. The primers used for cofilin were Fw, GTGAAAGAAGGCGGAAGGTTAA, and Rv, CACAGTTACACCAGAAGCCATT, as described [To measure expression of imerBank  were useescribed .DEts>hid regeneration assay, at least 10 representative eye discs from each genotype were measured for their sizes of dorsal versus ventral half of discs using the “histogram” function in Adobe Photoshop CS6, as previously described [Adult fly eye images were taken using a Zeiss stereomicroscope equipped with an AxioCam ICC1 camera. Fluorescent eye and wing disc images were taken with a Zeiss LSM 710 confocal microscope. For quantification of the number of PH3-positive cells  and F-acescribed . The staS1 Figrd instar eye discs expressing a GMR-hid transgene to induce apoptosis, anterior is to the left. DE-GAL4 was used to drive expression of various UAS-constructs in these discs. The cleaved Dcp-1 (cDcp1) antibodies label apoptotic cells. GMR-hid induces two waves of apoptosis as shown in the control . Expression of droncRNAi under the control of DE-GAL4 suppresses apoptosis in the dorsal half of the disc . In contrast, expression of LIMK1RNAi (C) or cofilin (D) does not affect GMR-hid-induced apoptosis. (E-H) Representative adult fly eye images of the indicated genotypes. GMR-hid induces an eye ablation phenotype as shown in the control (E). Expression of droncRNAi driven by DE-GAL4 partially rescues the dorsal part of the eye due to its suppression of GMR-hid-induced apoptosis in this region . However, expression of LIMK1RNAi (G) or cofilin (H) does not suppress GMR-hid-induced eye ablation phenotype.(A-D) Late 3(TIF)Click here for additional data file.S2 Figrd instar ey>hid-p35 disc labelled with PHN  and the cleaved caspase-3 antibodies , a marker of Dronc activity. B and B’ are enlarged images of the outlined area in A, which are shown together with their vertical and horizontal cross sections on right and bottom, respectively. The apical (a) and basal (b) surfaces of these cross sections are as indicated. Four representative F-actin aggregates on these images are indicated by arrows and arrowheads. F-actin accumulation and aggregates are present in the proliferating portion of the disc anterior to the MF . Despite a strong activation of Dronc in the same tissue , majority of the F-actin aggregates are not overlapping with or adjacent to the cCasp3-positive cells (B). Moreover, these F-actin aggregates localize apically in the disc (arrows and arrowheads in B and B’).A late 3(TIF)Click here for additional data file.S3 Figrd instar wing discs labelled with PHN. Anterior is to the left. Compared to the control nub>p35 (A), a massive increase of F-actin filaments was observed in the overgrown wing pouch area of the nub>hid-p35 discs . Such F-actin accumulation and tissue overgrowth were suppressed by a knockdown of LIMK1 with RNAi (C).(A-C) Late 3(TIF)Click here for additional data file.S4 Figrd instar wing discs with 48-hour old mosaic clones positively marked by GFP. PHN (magenta in A-F and grey in A’-F’) labels F-actin. Images of the apical part of the discs are shown. The clones expressing droncRNAi , LIMK1RNAi , or bskDN  have no effect on the cortical F-actin pattern in the discs. Similarly, the wildtype clones in the LIMK12  or Tak1  mutant background does not alter the F-actin pattern either. Notably, expression of cofilin  reduces the level of F-actin.Late 3(TIF)Click here for additional data file.S5 Figrd instar eye discs, anterior is to the left. TRE-red (magenta in A-E and grey in A’-E’) is a marker of JNK activity. ELAV (green in A-E) labels photoreceptor neurons therefore indicates the posterior differentiating portion of the eye discs. Compared to the control ey>p35 , TRE-red signals strongly increase in the ey>hid-p35 eye discs . This increase is suppressed by expressing droncRNAi , LIMK1RNAi  or cofilin . (F) Quantification of the TRE-red signal intensity in the eye discs of the indicated genotypes. Compared to the control ey>p35, the TRE-red signals are significantly (***p < 0.001) increased in the ey>hid-p35 discs. This increase is significantly (***p < 0.001) reduced in response to expression of droncRNAi, LIMK1RNAi or cofilin.(A-E’) Late 3(TIF)Click here for additional data file.S6 Figrd instar wing discs with mosaic clones positively marked by GFP. hid, p35 and SOD are simultaneously expressed in the clones for 48 hours. The JNK activity, indicated by the MMP1 labeling (magenta in A and grey in A’), is very low in these clones. (B) Quantification of the MMP1 signal intensity in the representative wing disc pouches carrying clones of the indicated genotypes. Compared to the discs with hid-p35 clones, the MMP1 signals are significantly (****p < 0.0001) reduced in response to expression of SOD in the clones. (C-D’) Late 3rd instar wing discs with either p35-expressing  or hid-p35-expressing  clones positively marked by GFP. Anti-Hemese  antibodies label hemocytes. Only a few hemocytes are attached to the wing discs. Their localizations do not correlate with the positions of the p35 or hid-p35 clones.(A-A’) Late 3(TIF)Click here for additional data file.


Medicago sativa L.) plants and western flower thrips in south-central Idaho and proposed to be a first flavi-like virus identified in a plant host. We argue that the SRAV, based on its prevalence in alfalfa plants, readily detectable dsRNA, genome structure, presence in alfalfa seeds, and seed-mediated transmission is a persistent new virus distantly resembling members of the family Endornaviridae.Here we report an occurrence of Snake River alfalfa virus (SRAV) in Washington state, USA. SRAV was recently identified in alfalfa (The online version contains supplementary material available at 10.1186/s12985-023-01991-7. Frankliniella occidentalis collected in the Minidoka and Twin Falls counties of Idaho, USA [Snake River alfalfa virus (SRAV) was recently identified from alfalfa plants and thrips aho, USA . Based oaho, USA . The SRATo date, no occurrences of SRAV have been reported in alfalfa or on different hosts from other locations. In this work, applying high-throughput sequencing (HTS), we detected SRAV in 50 individual alfalfa plant samples collected from 10 commercial fields in Grant County, WA. Plants used for RNA extraction displayed a diverse symptomatology that occasionally correlated with the symptoms allegedly reported for SRAV, such as yellowing and vein clearing .Total RNA was extracted using Promega Maxwell® RSC Plant RNA Kit . Library preparation was performed with Illumina TruSeq Stranded Total RNA with Ribo-Zero kit , and the sequencing platform used was HiseqX10 (PE150) . Bioinformatic pipeline included adapter trimming followed by de-novo assembly of reads, unmapped to g SPAdes  in HMM-gg SPAdes  using MaAll 50 unique alfalfa plant samples contained viral reads, varying in quantity from 46 to 71,267 .One of the de novo-assembled contigs recovered from the individual library and sample containing 8,079 viral reads, was 11,811 nt in length and had 100% base coverage with the reference, thus representing a complete genome of the virus. It was 7 nt longer at the 5' end than ON669064, which was confirmed by 5' RACE using SMARTer® RACE 5'/3' Kit . The 3’ end of the sequence was 59 nt longer than that of the reference ON669064 and matched another isolate of the virus, SRAV_ALF1071, found in GenBank under accession number ON669090.1 [viridae  was used to detect protein sequence similarities.At the nucleotide level, the SRAV-WA1 (for Washington State) was 99.8% identical to the reference genome ON669064 with 18 SNPs between the two, therefore depicting an isolate of the same virus. The genome of SRAV-WA1 encoded a single 3,835 amino acid (aa) polyprotein 99.9% identical to the reference. BLASTP and PSI-BLAST searchers of the SRAV-WA1 polyprotein against the GenBank database identified no related sequences except existing SRAV submissions. In silico analyses of the viral polyprotein using Pfam, InterPro and CDD databases revealed the presence of the conserved RdRp domain . No other domains were reliably identified. A weak relation to the superfamily of trypsin-like serine proteases  was found in the 1855–1910 aa region of the polyprotein when Superfamily database . One set was ANPV_3 derived from Dahan et al. , and anoEndornaviridae family were often reported as double-stranded RNA viruses [The omnipresence of the virus in all analyzed samples indicated the possibility of persistent infection like those caused by partitiviruses and endornaviruses . Conside viruses , 16, cur viruses .Endornaviridae, and members of the family Flaviviridae, placed both SRAV isolates within Endornaviridae, although SRAV isolates formed a separate cluster , SRAV clustered with the former as well, again forming a distinct grouping , it sho2SO4, surface-sterilized with 70% ethanol, and rinsed with sterile water [Since plant endornaviruses are transmitted through seeds via the gametes , 13, 19,le water . Total Rle water .One of the characteristic features of all endornaviruses is readily detectable viral replicative form, double-stranded RNAs (dsRNAs), that accumulates in the host tissues in high quantities . To extrAdditionally, given that many endornavirus RNAs have a site-specific discontinuity (nick) on 5’ terminus of the coding strand, we have attempted, but failed, to determine its presence in the genome of SRAV-WA1 using 5'RACE approach .Ilarvirus, Carmovirus, Sobemovirus and Machlomovirus genera [Dahan et al.  detecteds genera . Based os genera , 13, 19,Alternaria spp., Bipolaris spp., Stemphylium spp., Fusarium spp., Pseudomonas spp., Erwinia spp. etc. were also detected. These findings suggested that traditional Koch’s postulate of “one microbe—one disease” should be broadened into the principle of a pathobiome, when disease symptoms are attributed to a diverse community of pathogenic organisms within the plant, rather than to a single infectious agent [Other viruses found in samples infected with SRAV-WA1 included alfalfa mosaic virus, pea streak virus, lucerne transient streak virus, bean leaf roll virus, partitiviruses, and amalgavirus. Fungal and bacterial pathogens, described in alfalfa, like us agent .Overall, our research confirmed association of SRAV with alfalfa and, for the first time, identified an extensive occurrence of this virus in Washington State. The importance of this work also relies on the hypothesis that placement of SRAV within the flavi-like lineage, as suggested by Dahan et al. , may notHowever, the low percent identity of SRAV with endornaviruses and flaviviruses, absence of the poly (C) and presence of the poly (A) tract at the 3’ terminus of the genome, and lack of the site-specific nick at the 5’ end, indicate that SRAV may represent an entirely new taxonomic group of persistent viruses that does not belong to either of the two families. Altogether, more data are needed to assess taxonomy, biology, and economic importance of the virus.Additional file 1: Phylogenetic relationship of SRAV with members of the families Endornaviridae and Flaviviridae. The original unrooted tree was deduced from MUSCLE alignment [lignment  of the vAdditional file 2: Phylogenetic relationship of SRAV with members of the families Endornaviridae and Flaviviridae. The unrooted tree was deduced from MUSCLE alignment of the viral RdRP domains and built using MEGA software with Maximum Likelihood method and bootstrap analysis of 1000 replicates.Additional file 3: Color coded matrix of pairwise similarity scores obtained with Sequence Demarcation Tool Version 1.2 (SDTv1.2). Polyproteins of the representative endorna- and flaviviruses were aligned using MUSCLE program [Additional file 4: Color coded matrix of pairwise similarity scores obtained with Sequence Demarcation Tool Version 1.2 (SDTv1.2). RdRp domains of the representative endorna- and flaviviruses were aligned using MUSCLE program [


Medicago sativa amalgavirus, two alphapartitiviruses, and one deltapartitivirus. Additionally, a new flavi-like virus with an unusual genome organization was discovered, dubbed Snake River alfalfa virus (SRAV). The 11,745 nt, positive-sense (+) RNA genome of SRAV encodes a single 3835 aa polyprotein with only two identifiable conserved domains, an RNA-dependent RNA polymerase (RdRP) and a predicted serine protease. Notably, unlike all +RNA virus genomes in the similar size range, the SRAV polyprotein contained no predicted helicase domain. In the RdRP phylogeny, SRAV was placed inside the flavi-like lineage as a sister clade to a branch consisting of hepaci-, and pegiviruses. To the best of our knowledge, SRAV is the first flavi-like virus identified in a plant host. Although commonly detected in alfalfa crops in southern Idaho, SRAV sequences were also amplified from thrips feeding in alfalfa stands in the area, suggesting a possible role of Frankliniella occidentalis in virus transmission.Alfalfa is an important perennial forage crop in Idaho supporting dairy and cattle industries that is typically grown in the same field for as many as 4 years. Alfalfa stands of different ages were subjected to screening for viruses using high-throughput sequencing and RT-PCR. The two most common viruses found were alfalfa mosaic virus and bean leafroll virus, along with  Medicago sativa L.) is an important forage crop grown on more than 400,000 ha in the State of Idaho in support of the burgeoning cattle and dairy industries. In terms of acreage, it is the second most widely grown of all Idaho crops, behind wheat [Alfalfa (nd wheat . In IdahAlfamovirus), bean leafroll virus , pea streak virus , and red clover vein mosaic virus , all of them single-stranded (ss), positive-sense (+) RNA viruses that are aphid-transmitted [Nucleorhabdovirus) and lucerne transient streak virus , which are transmitted by aphids and beetles, respectively [Iflaviridae and Solemoviridae [Prior to the introduction of HTS methodologies, the six most common viruses were reported to affect alfalfa production worldwide, with another 14 occasionally found to infect alfalfa but not having a significant impact on the crop . The founsmitted . Two othectively ; the latectively ,4. With ectively ,12,13,14ectively ,16,17,18ectively ,11,12,13ectively . AdditioAllexivirus) in the State of Washington [In the United States, studies of the alfalfa virome using HTS have led to the identification of new viruses, such as alfalfa virus S  in Minidoka County represented a single standard, 50-ha irrigated production field with an alfalfa stand planted in Spring 2017. It was in its fourth growing season and was sampled in July 2020. Location 2 in Twin Falls County represented experimental grounds of the Kimberly Research and Extension Center, University of Idaho, and included five relatively small, 0.4–2.2 ha fields planted with alfalfa between 2013 and 2020; this location was sampled twice: in July 2021 and again in August 2021.Samples were collected randomly in the field from alfalfa plants visually assessed to exhibit mosaic, yellowing, vein clearing, leaf distortions, or growth retardation. A sample represented one complete alfalfa stem with all leaves intact but without roots; this entire stem was placed in a plastic bag and kept in a cooler on ice for transport to the laboratory. The typical time between alfalfa leaf tissue collection and nucleic acid extraction was between 3 and 7 days. Tissue aliquots for each sample were stored at −80 °C until further use.Frankinella occidentalis) were collected from two alfalfa fields, alongside three pea aphids (Acyrthosiphon pisum). A summary of all the samples collected and subjected to analysis is presented in In August 2021, concomitantly with the collection of alfalfa samples, 20 thrips  and 8 weeks (N. benthamiana) post-inoculation (wpi) .Alfalfa samples collected in August 2021 were tested for the presence of alfalfa viruses using RT-PCR (see below) and were also used as a source of inoculum of laboratory plants in the greenhouse. Each of the ten collected alfalfa samples was mechanically inoculated into three About 200 mg of each of the alfalfa samples collected in July 2020 was ground using a mortar and pestle in liquid nitrogen, and total RNA was extracted with a Spectrum Plant total RNA kit . Resulting RNAs were subjected to DNase treatment using a DNAse Max kit  and subsequently concentrated using an RNA clean and concentration kit—5 . Ribosomal RNAs were then depleted using a RiboMinus plant ribodepletion kit , and resulting ribodepleted RNAs were concentrated by ethanol precipitation. Between 340 and 420 ng of ribodepleted RNA per sample was then subjected to strand-specific library preparation using a Kapa RNA HyperPrep kit  and sequencing in paired-end 300 bp read format on a MiSeq platform .Resulting paired-end reads were cleaned using Trimmomatic v0.38 in ILLUMINACLIP mode with the following settings: 2:30:10:8:TRUE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:100 . Paired,E. coli using a pGEM-T Easy vector system (Promega) and sequenced using M13 primers. The 3′ terminus was amplified using SP5 primer .Visual assessments of alfalfa stands during the summers of 2020 and 2021 were conducted randomly. Only plants exhibiting visible foliar symptoms of mosaic, vein clearing, flecking, leaf distortions, and overall growth retardation were sampled for further laboratory studies. Examples of the symptoms observed are presented in Five of the six samples collected from location 1 in July 2020 were subjected to HTS analysis on the MiSeq platform. Between 6702 and 7560 contigs over 1 kb per sample were assembled from each of the five samples  and analHowever, there were three exceptions. Among the various virus-related sequences, an 11,803 nt contig was identified in sample ALF1060 see a, and a −4; 21% identity over an amino acid sequence alignment containing 265 positions with a 450 amino acid C-terminal region of the polyprotein of Bole tick virus 4 (GenBank Accession number QUJ17980 [A BLASTn search of the GenBank database returned no similar nucleotide sequences. A BLASTp search of the +RNA virus subset of the NCBI non-redundant protein sequence database (as of 20 April 2022) yielded a significant alignment ) contaiQUJ17980 . A more In addition to the RdRP, the HHPred search detected an approximately 100 amino acid region in the middle of the polyprotein with moderate similarity to the serine proteases of arteriviruses and flaviviruses . Despite the limited sequence conservation, this region of the new virus polyprotein contained conserved histidine and serine residues corresponding to the catalytic residues of the virus proteases, with the catalytic serine embedded with a characteristic GxSGG motif. The secondary structure prediction for this portion of the polyprotein was also fully compatible with the known protease structures , indicatTo confirm and validate the presence of the SRAV sequences in the original samples collected in July 2020, RT-PCR assays were conducted on the six original alfalfa samples using SRAV-specific primers  designedWe then undertook a more thorough molecular and bioinformatics analysis of the SRAV genome and polyprotein. The conceptual translation of the complete SRAV genome produced a single, large open reading frame encoding a 3835 aa polyprotein .The relative positions of the two identified conserved domains, Tryp_SPc and RdRp, within the 3835 aa polyprotein resembled the genome architecture of flavi-like viruses see . The genHepacivirus, Pegivirus, and an unclassified virus from the Wenling moray eel. Together with SRAV, this branch was the sister group of another large branch that included the genera Flavivirus and Pestivirus grouped with two yet unclassified clades of ‘Jingmen’ and ‘Hermitage’ viruses  where foliar samples were procured in August 2021; three pea aphids and 20 western flower thrips feeding on alfalfa were collected at the same time. All three aphids were bulked together into a composite sample, and the thrips were bulked into two composite samples according to the collection field: nine thrips from field 4B and eleven thrips from field 14B. Total nucleic acids were extracted from each of the composite samples according to the protocol described by Dellaporta et al.  and subjM. sativa (L.), such as AMV and BLRV with positive-sense ssRNA genomes, and four persistent viruses with dsRNA genomes: MsAPV1, MsAPV2, MsDPV1, and MsAV1  , flavi-lPestivirus [The host range of flavi-like viruses is currently almost exclusively confined to vertebrates and arthropods . Some flstivirus . HoweverOf interest is an apparently wide prevalence of SRAV in alfalfa fields along the Snake River valley in the two counties of Idaho sampled over a period of two years, which are located on the opposite sides of the river, approximately 80 km apart . This wiN. benthamiana in a greenhouse experiment were unsuccessful but should be extended to other receptor hosts. In a preliminary experiment, SRAV was detected by RT-PCR in Western flower thrips collected in the same alfalfa fields where SRAV-positive plants were found (The effects of viruses on the yield and quality of the alfalfa hay and on the overall economics of hay production is an understudied area ,5. All Sre found . This RTIn this work, we reported the genome sequences and some biological characteristics of a highly unusual +RNA virus, SRAV, that commonly infects alfalfa in south-central Idaho. The overall organization of the 11,745 nt genome of SRAV and phylogenetic analysis of the RdRP suggest an evolutionary relationship between SRAV and flavi-like viruses. However, SRAV is only distantly related to these viruses and is, so far, unique among +RNA viruses with genomes larger than 6 kb apparently lacking a helicase domain. Thus, SRAV is likely to become the founding member of a new virus family or order once additional related viruses are discovered. To the best of our knowledge, SRAV is the first flavi-like virus detected in plants. SRAV sequences were also identified in thrips, suggesting that these insects might serve as vectors for this virus. The unique features of SRAV might have evolved as an adaptation to reproduction in plants or in both plants and insects.


Viral infections can lead to the generation of new virus particles, whereas other viruses behave as chameleons by camouflaging themselves to evade or mislead the immune system of the host, thereby establishing a latent infection. Yet, other viral infections may cause transformation of the host cell. No matter the outcome of a viral infection, viruses are masters of usurping the host cell’s machinery to facilitate their life cycle. In the case of a productive infection, these pathogens may avail themselves of cellular proteins to enter the host cell, to support the replication and expression of their genome, to produce new, mature virus particles and to be released. Viruses will also utilize cellular proteins to establish a latent or persistent infection, and even to transform the cell. One of the mechanisms by which viruses appropriate the cellular machinery is by manipulating signal transduction pathways, which control numerous cellular processes .This Special Issue, “Role of Signaling Pathways in the Viral Life Cycle 2.0”, compiles nine contributions; three original articles and six reviews. Three papers describe the interaction of specific signal transduction pathways with the DNA viruses human papillomavirus (HPV), Merkel cell polyomavirus (MCPyV) and hepatitis B virus (HBV), respectively, whereas four manuscripts focus on signaling pathways and the RNA viruses influenza A virus (IAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and neurotropic RNA viruses. Finally, two papers discuss the role of nucleoporin RanBP2/Nup358 and tetraspanins in viral infection in general.E6 and E7 by interacting with YY1-binding sites in the HPV transcriptional control region (LCR) and by mediating epigenetic changes. In differentiated keratinocytes, however, repression is relieved because YY1 levels become reduced. Interestingly, YY1-binding sites in the HPV16 LCR are often mutated in cervical cancer, allowing increased expression of the E6 and E7 oncoproteins. The authors also explored the role of YY1 in virus-independent cancers and in the transcriptional regulation of other human viruses, such as retroviruses, herpesviruses, and HBV [Warowicka and colleagues reviewed the role of the transcription factor Yin Yang 1 (YY1) in the life cycle of HPVs and in cancer development . BesidesEpigenetic changes such as DNA methylation, histone modifications, chromatin remodeling, and the expression of noncoding RNA are a feature of tumorigenesis . The revAnother virus associated with cancer is Merkel cell polyomavirus. The virus is the cause of approximately 80% of all Merkel cell carcinomas (MCCs), an aggressive skin cancer . The oriBoth the paper by Harris and colleges and the paper by Gerlt and coworkers deal with the respiratory pathogen IAV ,10. The +/H+ exchanger regulator factor 1 (NHERF1), a PDZ domain-containing protein, and β-arrestin are components of the GPCR signalosome and can be hijacked by SARS-CoV-2, thereby causing dysfunctional GPCR signaling. β-arrestin can also activate G protein-independent signaling and regulate multiple signaling cascades, include the MAPK pathway. Disrupting interactions between viral proteins and PDZ domain-containing proteins may be a strategy to treat infected patients [Another respiratory virus that has not been out of the news in the last few years is SARS-CoV-2. In their review, Zhang and Friedman discuss the role of PDZ domain-containing proteins in viral entry and how SARS-CoV-2 can disrupt G-protein coupled receptor (GPCR) signaling . The PDZpatients .The review by Vazquez and Jurado elaborates on mechanisms that different viruses infecting the central nervous system (CNS) use to modulate the immune response . One majThe review by Jiang and colleges discusses how mutations in the Ran binding protein 2 (RanBP2 or nucleoporin 358 KDa), a component of the nuclear pore complex, interacts with distinct viruses and affects infection . RanBP2 New et al. provide an update on the dual role of tetraspanins, a family of transmembrane glycoproteins, in the life cycle of HPV, IAV, human immunodeficiency virus type 1 (HIV-1), Zika virus (ZIKV), and coronavirus . VirusesIn conclusion, viruses usurp different signaling pathways to facilitate different stages in their life cycle, but also to contribute to the pathogenicity of viruses. Preventing viruses from taking advantage of signaling pathways may be used to tailor specific antiviral strategies. However, the importance of these pathways for normal host cell function may lead to off-target effects and harm the treated host when signaling cascades are inhibited.


Its triphosphate derivative can be incorporated into viral RNA and extended by the coronavirus RNA polymerase. Incorrect base pairing and inefficient extension by the polymerase promote mutagenesis by increasing the G→A and C→U transition frequencies. Despite having remarkable antiviral action and resilience to drug resistance, carcinogenic risks and genotoxicity are important concerns limiting their extended use in antiviral therapy.In RNA viruses, a small increase in their mutation rates can be sufficient to exceed their threshold of viability. Lethal mutagenesis is a therapeutic strategy based on the use of mutagens, driving viral populations to extinction. Extinction catastrophe can be experimentally induced by promutagenic nucleosides in cell culture models. The loss of HIV infectivity has been observed after passage in 5-hydroxydeoxycytidine or 5,6-dihydro-5-aza-2′-deoxycytidine while producing a two-fold increase in the viral mutation frequency. Among approved nucleoside analogs, experiments with polioviruses and other RNA viruses suggested that ribavirin can be mutagenic, although its mechanism of action is not clear. Favipiravir and molnupiravir exert an antiviral effect through lethal mutagenesis. Both drugs are broad-spectrum antiviral agents active against RNA viruses. Favipiravir incorporates into viral RNA, affecting the G→A and C→U transition rates. Molnupiravir (a prodrug of β- Many viruses evolve rapidly . Their cEscherichia coli bacteriophage Qβ RNA using a cell-free system   or can be incorporated (as RMP) into the viral genome, leading to viral mutagenesis . RibavirThe early studies with poliovirus suggested that the virus could develop resistance to ribavirin. However, the analysis of the polymerase-coding sequence of virus grown in the presence of 0.8 mM ribavirin showed that conserved motifs C–E remained unchanged, while some variability was observed at motifs A and B . The pasUnexpectedly, the equivalent substitution in the RNA polymerase of foot-and-mouth disease virus  was never selected when the virus was passaged in the presence of ribavirin and was not detected as a minority variant in the mutant spectra of the virus that replicated in the absence or presence of ribavirin or other mutagenic agents ,77. InstApart from the structural constraints and mechanisms affecting ribavirin incorporation and nucleotide selectivity by the viral RdRp, it should be noted that viral fitness might be an important factor contributing to the viral response to lethal mutagenesis. Cell culture studies with hepatitis C virus showed that high-fitness viral quasispecies showed resistance to ribavirin and favipiravir without modifying the mutation-type bias typical of those mutagens, probably by limiting the expansion of their mutational spectra . AlthougFavipiravir   is an anEnzyme kinetic analysis with influenza A virus RdRp demonstrated that favipiravir-ribofuranosyl-5′-triphosphate inhibited the incorporation of ATP and GTP in a competitive manner ,126. In Evidence of the mutagenic effects of favipiravir in infected animals was reported after studying norovirus infections in a mouse model . Viral RThe heterotrimeric influenza virus polymerase, containing the PA, PB1, and PB2 proteins, catalyzes viral RNA replication and transcription in the nucleus of infected cells. Studies with H1N1, H3N2, and H7N9 strains of influenza A virus showed that K229R in the catalytic PB1 subunit confers favipiravir resistance while impairing viral replication . K229R rFavipiravir resistance has also been mapped in the RNA polymerases of chikungunya virus   and enteClinicaltrials.gov (accessed on 10 April 2022) currently includes more than one hundred clinical trials testing the efficacy of favipiravir alone or in combination with other drugs . Most of these trials (>85%) evaluate the efficacy of favipiravir as a drug against SARS-CoV-2, although there are trials where the compound has been tested against influenza, Ebola, or Lassa virus infections. One of the major limitations to the use of favipiravir is its relatively low bioavailability, resulting in relatively low plasma concentrations of the drug. This is apparently due to its short half-life caused by rapid renal elimination. Strategies to increase its potency are needed, particularly considering its strong antiviral effect against different viruses and in different animal models  (reviewed in ). Despitd-N4-hydroxycytidine   is the isopropylester prodrug of the nucleoside derivative β-DD-1931) . The triaviruses ,96,99. Caviruses . Evidenccytidine  and infld-N4-hydroxycytidine triphosphate competes effectively with cytidine triphosphate (CTP) for incorporation into the nascent RNA. Molnupiravir is then effectively used as a template in the next round of viral RNA synthesis. β-d-N4-hydroxycytidine (NHC) forms base pairs with A and G due to the tautomerization of the cytosine analog [Two papers published in 2021 described the biochemical and structural bases of how molnupiravir impairs the fidelity of SARS-CoV-2 replication and provokes error catastrophe ,137. Bote analog . The fore analog , but NHCe analog ,135. d-N4-hydroxycytidine in the template strand [d-N4-hydroxycytidine triphosphate also competes weakly with UTP during RNA polymerization [High-resolution cryo-EM structures showed that SARS-CoV-2 RdRp can accommodate NHC:G and NHC:A mispairs without introducing major distortions in the enzyme’s active site or the nucleic acid scaffold . Althouge strand , the lowe strand  could berization . d-N4-hydroxycytidine is a potent inhibitor of its exonuclease activity [The biochemical experiments described above were carried out with the RdRp holoenzyme, without the intervention of the exonuclease protein (nsp14) found in the SARS-CoV-2 replicase-transcriptase complex. However, previous studies with the related complexes of murine hepatitis virus and MERS coronaviruses revealed that β-activity .d-N4-hydroxycytidine to infected ferrets prevented SARS-CoV-2 transmission in untreated and uninfected animals [The efficacy of molnupiravir against SARS-CoV-2 has been observed in animal models and humanized mice ,139, whi animals . In Octo animals . The int animals . In addi animals ,144,145. animals ,99.FiguA synergistic effect has been reported for molnupiravir and favipiravir in the hamster model . The synd-N4-hydroxycytidine can be metabolized by the host cell to the 2′-deoxyribonucleoside form by the ribonucleoside reductase and then incorporated into the host cell’s DNA. The mutagenic effect of β-d-N4-hydroxycytidine has been shown in animal cell cultures [Despite evidence showing the efficacy of molnupiravir, there are still concerns about its use. Getting molnupiravir to patients in a timely way is likely to be problematic, and its mutagenic potential is a matter of concern. β-cultures . However® (cII locus), or MutaTMMouse LacZ are examples of well-established genotoxicity assays. The reported genotoxicity profile for molnupiravir was negative with the Big Blue® (cII locus) assay and inconclusive with Pig-a [Candidate drugs are subjected to genotoxicity assays with somatic cells to determine the possibility of mutations or chromosomal abnormalities that could pose a risk to human health. Pig-a, Big Blueth Pig-a . The Pigth Pig-a . Furtherth Pig-a .The development of lethal mutagens faces several challenges. Most notably, the cell toxicity needs to be very low, particularly in relation to their carcinogenic potential. Similar to the case of other antivirals, drug resistance is a fundamental issue, not only because it could lead to drug inactivation, but also because a mutagen could spur the emergence of novel virus variants with potentially increased pathogenicity and transmissibility. Attempts to design and develop bona fide lethal mutagens  have not been successful and issues related to their efficacy and selectivity need to be addressed. However, lethal mutagens, such as ribavirin or favipiravir, were approved for clinical use based on their antiviral efficacy before the demonstration of their mutagenic effect. In the case of ribavirin, the drug has been used for decades to treat chronic hepatitis C, although with limited efficacy and adverse side effects. The mutagenic effect of ribavirin has been demonstrated with several viruses, including hepatitis C virus . HoweverInterestingly, favipiravir and molnupiravir act mainly by driving viruses to error catastrophe. Both compounds have broad-spectrum activity and show some resilience to drug resistance. Molnupiravir shows a very high genetic barrier to resistance and the selection of mutants conferring drug resistance has not been successful. However, learning from experience with HIV and other viruses ,152, comConcerns about the potential genotoxicity of lethal mutagens are justified and have to be carefully addressed. However, in acute infections leading to COVID-19 or flu, the relatively short incubation time limits the extent of the medication and, therefore, the exposure to the mutagenic compounds. Reliable assessment of mutagenicity and carcinogenicity is still complex, since the results obtained in current tests  are difficult to confirm in primate models or humans. Another issue that is not always considered in genotoxicity assays is the impact of the drug exposure time. This could be short in acute viral infections, but long and probably hazardous in chronic infections. Finally, the prescription of mutagenic nucleosides should take into account long-term pharmacovigilance. A registry of patients treated with those drugs would be very helpful for a long-term follow-up of potential adverse effects, including genetic, carcinogenic, teratogenic, and embryotoxic damage.


The coronavirus disease caused by the SARS-CoV-2 virus has affected people worldwide for more than two years. Here we present a new diagnostic method based on nonlinear dielectric spectroscopy to detect the presence of the SARS-CoV-2 virus in swab samples. A known current is injected into the virus sample suspension, and the biomarker is the third harmonic detected in the power spectrum of the recorded signal. Computational modeling of harmonic production supports the hypothesis of ion channels (the E-protein) with nonlinear current–voltage characteristics being present on the virus envelope as a possible origin of harmonics. The developed system is able to distinguish between positive and negative samples with 5–10 dBc (decibels relative to the carrier) higher third harmonic ratios in positive samples, in agreement with the computational estimation. Our early results demonstrate that this method can detect the virus in solution. This is the first time harmonic signatures are used to detect SARS-CoV-2 in swab samples. The World Health Organization (WHO) has recommended PCR to be considered as the golden standard method for coronavirus detection and COVID-19 diagnosis. Though this method has sufficient sensitivity, it comes with roughly ten percent false-negative or false-positive results2. Furthermore, this laboratory-based technique is time-consuming, expensive, and requires advanced tools and skilled clinical lab technicians3. Due to the fast spread of the virus and the consequent high infectivity and mortality rates, it is vital to innovate fast-response, low-cost, and user-friendly SARS-CoV-2 detection techniques2. Therefore, alternative rapid POC diagnostic kits are extremely desired.It has been more than two years since the outbreak of the SARS-CoV-2 virus in 2019 and the announcement of the Covid-19 Pandemic. Although nowadays vaccines are being used globally to harness the epidemic, their efficiency is not ideal, and the pandemic is yet taking victims. Since then, various biomarkers have been used to diagnose the disease. The virus genome is the most potent biomarker used in most point-of-care (POC) diagnostic kits. There are several routine techniques being used to detect the virus, including; polymerase chain reaction (PCR), qRT-PCR, RT-LAMP, CRISPR, and NGS (next-generation genome sequencing)4. A biosensor is usually composed of a sensing agent that interacts with the biological molecules to produce a meaningful signal for detection. This signal is known as a biomarker for the presence of pathogens. The monoclonal antibodies, nucleic acids, glycan, lectin, and enzymes are the most frequently used sensing agents in biosensors. The response of the biosensor, i.e., the signal produced from the pathogen interacting with the receptor, is converted into a measurable signal by a transducer. Accordingly, the presence of the pathogen can be revealed after thorough processing of the recorded signals4. Different types of biosensors based on electrochemical, optical, and microfluidics approaches have been developed for the COVID-19 diagnosis, and some even made their way to the market. Viral proteins, viral genome, and antibodies specific to viral components generated by the host immune system are the three most used biomarkers in these biosensors1. Nevertheless, to harness the widespread of different lethal strains of viruses across the globe, research and development of yet even more efficient, sensitive, accurate, instant, economic, and, if possible real-time biosensors are extremely desired.Biosensors are simple, real-time, and effective devices capable of detecting various infectious diseases5. Recent studies have indicated that several SARS-CoV-2 proteins, including E, ORF3a, and ORF8a, can self-assemble into oligomers and generate ion channels (ICs)6. Kern et al. strongly suggest that 3a proteins are non-selective cation channels7. Numerous studies have established the relevance of the E protein and protein 3a as fundamental pro-inflammatory SARS-CoV virulence factors. They have suggested that these proteins act as ion channel proteins5. The unique distribution of different transmembrane proteins in the SARS-CoV-2 demonstrates a more complex structure than integrated solid-state semiconductors. The virus possesses a capacitive membrane formed by arrays of parallel lipid molecules. This lipid membrane is formed in a polar and highly dynamic water medium and holds different proteins, including rectifying ion-channel forming proteins, ORF3a.Physically, coronavirus is a spherical particle with a diameter of 100–160 nm enclosed in an envelope lipid bilayer, in which the membrane (M), envelope (E), and spike (S) structural proteins are embedded8. The relevant statistical signal analysis aspects have been applied to detect single nanoparticles and biological molecules such as yeast ribosomes floating in fluids exposed to laser beams9. Furthermore, Dielectric spectroscopy has been utilized to measure the size and distribution of oil-in-water particles in fluids10. However, we think nonlinear dielectric spectroscopy has been originally applied to detect a biological organism, yeast, by Woodwards and Kell11. The results of theoretical, statistical and practical studies of biological and non-biological material conducted here is consistent with the relevant aspects considered in the mentioned literatures and well approves the capability and credibility of our method towards detection of viruses in solution.Power Spectrum Analysis has been a major tool to detect particular characteristics of the event(s), including amplitudes, energy ratio, frequency, noise, correlation, probability density, cumulative distribution function, etc. occurred in the recorded signal from different sources through the Fast Fourier Transform (FFT)12. However, to our best knowledge, no study has yet used this method to detect the presence of virus in the solution. Also, most ion channels on the membrane do not have ohmic behaviour and instead show a nonlinear I-V relation which consequently results in harmonics generation.Nonlinear dielectric spectroscopy (NLDS) has been shown to be capable of detecting some bacteria in suspension based on the harmonic pattern observed in the Fourier transform of their corresponding voltage signalEspecially because of nonlinear ion channels on the SARS-CoV-2 viral particles, we hypothesize that power spectrum harmonics can be used as a signature for the electronic detection of coronavirus. The sputum samples from patients were used to detect the presence of the virus through a novel biosensor based on the harmonic changes in the Fourier spectrum of the recorded voltage. By detecting the harmonic content in a patient's sample, we were able to develop new diagnostic criteria for the early detection of SARS-CoV-2. The virus detecting biosensor constructed here does not require any labeling scheme, is simple, inexpensive, non-invasive, and easy to use by patients, and can be further advanced to respond in real-time.The samples therein were throat swabs taken from patients and each kept in two separate vials in viral transport medium (VTM). One of the two vials was used for the RT-PCR test, and the result was labeled as positive or negative accordingly. The samples were loaded in our tailored airtight water-proof recording chamber, and remained tightly closed all throughout the experiments in BSL-2 labs. A virus-free VTM was used as the reference in all experiments conducted in this study. The samples were de-identified at the clinical laboratory prior to handing over for electrical experiments, with no numbers or labels (except for the positive or negative PCR results), and were numbered by the second hand operator. Electrical measurements described herein, were performed on the samples and the samples were safely discarded. All protocols of the clinical experiments were approved by the Research Center for Clinical Virology of Tehran University of Medical Sciences according to the obtained informed consent of all subjects and/or their legal committee under the Ethical License of IR.TUMS.VCR.REC.1399.599, issued in compliance with relevant laws and institutional guidelines in accordance with the ethical standards of the Declaration of Helsinki, by the Biomedical Research Ethics Committee of the Tehran University of Medical Sciences, Tehran, Iran.The NLDS technique was used to apply a single frequency sinusoidal current/voltage excitation to a bio-suspension solution loaded in the recording chamber of the biosensor. The power spectrum of the recorded voltage signals from the bio-suspension at different current levels was then calculated. The harmonics pattern observed in the power spectrum was further analyzed and interpreted in terms of membrane surface proteins. It should be noted that the electrodes were washed three times before each test with 70% ethanol, followed by pure deionized water. Figure Here, we aim to propose an innovative detection method for SARS-CoV-2 through a coherent combination of theoretical and experimental approaches. First, we theoretically estimate the power spectrum obtained from a virus solution using a mathematical model for the nonlinear flow of ion currents on the membrane surface. Second, the power spectrum of the virus-containing VTM solution is measured experimentally. Finally, the theoretical and experimental results are compared against each other to check for the possible existence of the SARS-CoV-2. Also, Statistical analyses were performed using GraphPad Prism version 9.3.1 and drawings were performed using Inkscape version 1.1 and GIMP version 2.10.28.14 containing spike and channel-forming proteins. The virus genome encodes three ion channels . The E protein is the smallest of the major structural proteins in the virus. The 8a protein has been found in the membrane of the mitochondria as well as the endoplasmic reticulum, though it has not yet been established whether it forms an ion channel5. However, the 3a protein  forms an ion channel and is involved in virus release, inflammasome activation, and eventual cell death7. Accordingly, we studied the activity of the 3a protein, the main ion channel of the virus, and modeled its I-V relation.The SARS-CoV-2 virus consists of a lipid membrane with a thickness of 5–7 nm7, the only membrane ion channel examined in the virus is the 3a channel. In this section, we use the current–voltage diagrams obtained from the study of SARS-CoV-2 protein 3a in a nanodisk of phosphatidylcholine lipids based on the currents recorded across excised patches pulled from proteoliposome blisters7 to develop a mathematical model of the nonlinear channel response. Figure According to a study reported by Kern et al.2 > 0.99, and the current To obtain the mathematical model for the ionic currents of the virus membrane, the data for each ion type was fitted by a third-order polynomial with R1-n, current coefficient of individual ions, and x is indexing K, Na, Ca, and NMDG ions. Further, C = Cm.A, where Cm is the membrane capacitance per unit area By assuming a capacitance for the membrane, Eq. . Models Step 1:When a sinusoidal voltage (V) is applied to parallel electrodes, a uniform electric field will be created between them, des Fig. a.Figure Step 2:15:In the presence of this electric field, positive and negative ions move in the solution. The Accumulation of ions in the non-conductive membrane site leads to the creation of a potential difference across the membrane, the maximum induced membrane voltage entclass1pt{minimaStep 3:16. Simulation of the membrane voltage caused by the virus-containing solution was calculated in MATLAB software.This potential difference across the membrane leads to the activation of ion channels. Due to the activity of the channels and the movement of ions through them, a nonlinear current is generated. Thereafter, the membrane channels are modeled as dipole currentsThe detailed mathematical model of ion currents is presented in “Step 4:This nonlinear current flows through homogenous and linear volume conduction space (solution) and will receive at the electrode site Fig. d.Step 5:Finally, we measure the nonlinear voltage between two electrodes. This voltage is related to ion channel currents with volume conduction theory. This theory described more in the rest of the paper. This voltage is nonlinear because the ion channel current is nonlinear and this leads to the presence of harmonics in the power spectrum Fig. e.The voltage across the virus membrane was calculated by volume-conductor theory using a mathematical model for the membrane. Here in five steps, we describe how the recorded nonlinear voltage on the electrodes related to ion channel’s nonlinear currents.According to the volume-conductor theory, it is possible to calculate the potential recorded on the sensing electrodes using a quasi-static version of Maxwell's Equations. The volume-conductor theory relates the recorded potential to the current dipole on the membrane through Eq. ,3\documeApplication of the electric field induces a voltage across the membrane and causes depolarization and hyperpolarization Fig. b. The sumentclass2pt{minimmentclass2pt{minimConventional dielectric spectroscopy techniques use two-, three-, or four-electrode systems to stimulate and record signals. Here, we used a four-electrode system in an especially designed chamber with a total volume of about 2 ml to stimulate and record the nonlinear signal, as seen in Fig. in in Fig. The nonlinear dielectric spectrum was recorded after injecting an AC current of 20 mA and a frequency of 10 or 100 kHz into the solution and then recording the resulted voltage at the electrodes using a custom electronic module Fig. a. We useThere is a trade-off in choosing the input impedance of the differential amplifier between the electrode polarization effect and THD of the recording system. If the input impedance becomes less than a threshold, the polarization effect can disturb the recorded voltage. On the other hand, if it gets larger, the THD of the operational amplifier in the recording system will exceed the − 60 dB level, which is more than the required system THD threshold. The measured impedance of the virus solution was about 100 ohms, therefore, the value of 20 KOhms was chosen to be larger than the solution and still not too large to cause electrode polarization affect the readout, so that the best performance is achieved.The electronic system plays a fundamental role in receiving the signal. For this reason, low harmonics and low noise was taken into consideration in the design of electronic circuit, printed circuit and selection of electronic components. The spectrum analyzer, a low-noise device with about − 100 dBm was used to increase the sensitivity and accuracy. Thorough attention was paid to the electrodes and chamber design to minimize their artefacts and intrinsic effects on the applied field and recorded signals. However, in order to characterize the system and detect possible Noise/Harmonic distortion, a series of separate tests were conducted before measuring the viral samples including: (i) Source Test, where Spectrum analyzer was connected directly to the signal generator and the harmonics of the signal generator were measured for values of frequency, output voltage and load comparable to actual measurement. The source harmonics were about − 60 dBc in our experiment. (ii) Resistance test, by which the overall harmonics of the system consisting of signal generator, measurement system and differential amplifiers see Fig.  was deteThe simulation result for the presence of a virus between two electrode plates using the mathematical model for the ionic currents of the membrane is shown in Fig. carrier . A third harmonic given as − 10 dBc is 10 dB smaller than the main harmonic. Theoretically, a third harmonic level of − 40 to − 60 dBc was estimated.The ratio of the third harmonic to the main harmonic amplitudes depends on the frequency and the amplitude of the electric field stimulation. At higher frequencies and amplitudes, the presence of the third harmonic was stronger. The harmonic levels in this paper are given in units of dBc, which stands for dB relative to Harmonic measurement was performed for all patient samples, including positive and negative PCR labels. Each sample was measured twice. A 20 mA sinusoidal current was applied to the samples at 10 and 100 kHz frequencies. The resulting voltage signal between the sample and the reference solution was fed into the spectrum analyzer. The harmonic pattern was obtained, and the amplitudes of the measured harmonics were compared with the main harmonic in GraphPad Prism Software version 9.3.1 as reported in Fig. 5–107 Viron/ml in the solution17. The positive samples had a third harmonic strength equal to approximately − 50 dBm (about 0.01 microwatts). This corresponds to a sensitivity of approx. 10–14 watts per virus particle. Also considering the 2 ml volume of the chamber, this results in a minimum concentration of the virus of about 104/ml, that is needed to produce a reliable detection for our system which has a noise level of − 100 dBm. Under the conditions of our preliminary experiments, a rough estimation of accuracy is about 6 virions/ml or higher.The sensitivity of the method presented here was based on the Ct value immerged from rtPCR. Accordingly, a rough idea of the number of viruses in the sample was worked out. The sensitivity of the method presented here, i.e., the signal strength per virus particle, was estimated as follows. The positive samples possessed a Ct value of at most 35 which was an indication of about 10The theoretical results showed that the application of electrical stimulation caused a change in the membrane's voltage and resulted in nonlinear ion currents flowing in the membrane. This event was detected as a harmonic pattern in the voltage power spectrum recorded at the electrodes site.The measurements involved several challenges and there are certain noise effects that have to be considered. In nonlinear spectroscopy, as for detecting the harmonic changes caused by the bio-solution, the undesired harmonics caused by the voltage source and the electronic measurement circuits shall be carefully analyzed and removed. The total harmonic distortion (THD) level should be less than 60 dB based on theory. Therefore, first, with a resistance test, i.e., using specific resistance elements instead of the bio-solution, the system and the source THD levels were measured, and the deriving circuit was carefully redesigned, and op-amps were reselected to have a THD level of less than 60 dB with high accuracy and validity in the absence of the bio-solution.11, a reference solution is utilized to remove the nonlinear effect of the electrode. In this way, in addition to the patient sample solution, the harmonic spectrum was also recorded from the VTM reference solution, i.e., the virus-free solution. The common factors are eliminated by subtracting the reference signal from the sampled signal. The reason for choosing VTM is that it is a solution with electrode polarization harmonics close to positive and negative samples. By subtracting the VTM-only signal from the positive or negative virus containing sample signals, the virus-specific signal will present dominant harmonics. Furthermore, it is possible to set up two identical chambers, one including the sample solutions and the other as VTM-only. These two chambers can be connected by special membranes that allow the fluids to equilibrate in terms of ionic strength, pH, etc. without exchanging viral particles. The signals from these chambers can then be subtracted from each other to cancel non-virus harmonics.With the bio-solution in place, it is known that the polarization of the electrode in contact with the electrolyte has a nonlinear nature and could be considered as another significant source of undesired harmonics. Thus, a four-electrode system was used to eliminate this effect. In other words, based on Woodward's ideaAs a result, the harmonic pattern obtained from the difference between these two signals is only due to the presence of the virus in the solution. It shall be emphasized that the accuracy of the detection depends on careful sample handling, thus to prevent damaging the virus or its RNA before performing the measurements.Thus, it becomes possible to verify the appropriateness of the harmonics of the system and also to eliminate the harmonic effects of electrode polarization. Accordingly, we expect the positive samples to dominantly show harmonics caused by the viruses in the solution. We believe the critical precaution taken in the design, utilization and analysis of the results of the current approach and technique, paves the path for the definition and fabrication of the next generation techniques needed to detect various microorganisms.In the recorded pattern, the third harmonic amplitude was strongly correlated with the presence of virus particles. The measured third harmonic level showed an overall agreement with the theoretically estimated level of − 60 to − 40 dBc. Furthermore, applying T-Test analysis on the values  showed that the positive and negative samples are distinguishable from each other significantly with a p-value of 0.018. Therefore, we believe our proposed NLDS technique has the capability of detecting the presence of SARS-CoV-2 particles.18. This method interacts with released material from the microorganisms, whereas the proposed method here interacts directly with microorganism structure. The promising preliminary results paves the path and encourages our ongoing research to improve its specific sensitivity, accuracy and reliability not only for the detection of viruses but also to distinguish between different strains of viruses and also other microorganisms in future.This is the first time that nonlinear dielectric spectroscopy is being utilized towards the detection of viruses, in particular for the corona virus whose fast, cheap, and real time detection is vital. The method presented here is based on the electronic and electrical aspects of the microorganism studied and does not require any sample preparation and labeling nor relies on the expensive chemical and/or molecular ingredients used in routine methods such as rtPCR. Furthermore, possibilities of tailoring the method to detect in a fast, real time and portable manner at low expenses indicates its superiority over current detection methods. Another newly-emerging technology to detect pathogenic microorganisms, is based on electrochemical sensing of Reactive oxygen species (ROS) released by microorganisms5–107 Viron/ml in the solution17. At low concentrations, the relationship between the harmonic amplitude and the virus concentration was expected to be linear11. Accordingly, for a 10 time increase in the number of viruses in the solution, the harmonic level would increase by 20 dB and vice versa. The most nonlinear effect was caused when high number of viruses were present on the surface of the electrodes due to the stronger electric field. Thus, at high concentrations, the surface of the electrodes would saturate with virus particles and the harmonic strength does not increase with increasing viral content. Further to the RT-PCR, other techniques such as Dynamic Light Scattering (DLS) and Quantal Assays, TCID50, LD50, EID50 are being used to identify the actual number of the viruses in the sample, with less than 50% accuracy.The concentration of the virus copies in the sample was worked out based on the cycle threshold value  that emerged from the RT-PCR tests that depends on different factors considered and applied at test centers. The positive samples possessed a Ct value of at most 35 which was an indication of about 1019. Therefore, more extensive data can possibly be obtained by using a digital lock-in amplifier or phase-sensitive signal analyzer as the output device.Phase information is also useful for the analysis of impedance spectra of biological systems20 that makes them susceptible to external electric fields. The amplitude and frequency of the external field contribute to major electrostatic forces on the membrane proteins. The stationary status of the proteins in the membrane hiders their conformation change or rotation across membrane to dissipate the imposed electrical energy by simple Debye-like rotation11 causing a signature response that is manifested by the intensity, frequency and/or harmonics of the resulting signal. The membrane is excited by the applied field and the dielectric moment of the membrane proteins together with membrane surface potential caused by charges of both membrane constituent lipids and proteins as well as the dispersion and relaxation of the counter ions arranged tangential to the membrane surface in the surrounding electrolyte indicate the significant generation of a non-linear dielectricity21. The arrangement that is manifested by electric double layers (EDL) and potentials such as zeta potential, make a unique bioelectrical status that is deviated by the applied field and responds in an exceptional manner. The spatial and temporal diversity of the distribution, arrangement and dynamics of the membrane constituent polyelectrolyte molecules as well as surrounding charges makes the rather dynamic and complex bioelectric and bioelectronics profile of system that responds in a nonlinear manner to the frequency, intensity and harmonies of the applied signals.We now turn our attention from technical aspects to a short discussion of the fundamental mechanisms: Membrane proteins possess significant dielectric moments11, as well as the membrane activation and rearrangement of the counter ions on the membrane surface. These mechanisms are well known. But our paper suggests a novel mechanism for the harmonics in SARS-Cov-2, which is the ion channel on the viral membrane with nonlinear IV characteristic. The overall agreement obtained between a mathematical model based on this assumption and our experimental results might support this mechanism. More detailed biomolecular studies are necessary, however, to understand the real mechanism(s) involved. We suggest these as future work in this next-generation technology.Based on the resulting entropy level, and the odd or even harmonic produced one might work out the amount of the energy spent to change the conformation of the channel, membrane part of the spike as the major source of the resulted effect on the signalFinally, our experimental observations supported a third harmonic growth in solutions with positive PCR tests compared to the negative PCR tests at both selected frequencies of 10 and 100 kHz. The presence and activity of the Orf3a ion channel in the SARS-CoV-2 virus membrane is hypothesized to be the main source of harmonic generation. Accordingly, our NLDS-based detection system might be further developed at different pH and ionic conditions to distinguish between different virus strains. The method presented here is being further developed to differentiate between virus strains. As the bioelectric and bioelectronics of individual strains of viruses are dependent on the pH and ionic strength, these factors clearly play a major role. Different measures can be extracted from the data. For example, the ratios between harmonics can be one way to eliminate common factors (such as ionic strength and pH) that influence every harmonic amplitude. Accordingly, we consider that absolute harmonic amplitudes are affected by various factors including, (i) ionic strength and counter ion type and concentration (ii) pH of the virus containing medium, (iii) temperature, (iv) surface charges of the virus formed by its membrane lipids, proteins and peripheral sugars and/or linking molecules, (v) ionic permeability through either channels or lipid layers in the first place. Thus, in order to tailor an efficient method to detect viruses or other microorganisms, relative measures, such as the ratio between the third and first harmonics (h3/h1) should be considered. We anticipate that harmonic ratios tend to cancel out the common effects of the medium ionic strength and conductivity, and be more sensitive to biological nonlinearities. Such measures can be obtained experimentally for different virus strains under various pH and ionic strength conditions. Subsequently classification, and pattern recognition techniques can be used to define criteria for distinguishing different viruses, as the next generation techniques used to detect different microorganisms.The worldwide prevalence of COVID-19 disease and the need for early and PoC type diagnosis have led to numerous efforts in this area through inventing new techniques for virus detection. In this study, changes in the third harmonic pattern of the recorded power spectrum resulting from electrical excitation of the patient sample solution are introduced as new tentative biomarkers for virus detection. It was demonstrated that the third to first harmonic amplitude ratio can be significantly different between positive and negative samples. This difference is promising as it can potentially be used as a diagnostic tool. Using this biomarker alone or in combination with other methods, like electrochemical methods or through electrode surface functionalization for virus-specific binding, we can conveniently and efficiently improve the SARS-CoV-2 detection.We strongly believe that the proposed NLDS technique, by careful choice of the optimal frequency and the voltage range to detect the presence of the virus in a patient sample, regardless of its genetic content, could open a new avenue in designing SARS-CoV-2 diagnostic kits. Although the setup presented here still needs further development in order to be used as a stand-alone diagnostic kit, we hope the outcomes of our ongoing experiments will form a basis for the detection of all pathogenic microorganisms, including different kinds of viral agents, with high accuracy in near future.Supplementary Table S1.


Screening for colorectal cancer (CRC) has considerably contributed to declining CRC incidence and mortality rates in Germany over the last two decades. However, evidence on the cost-effectiveness of this public health remains scarce, mostly due to the lack of detailed sex- and age-specific estimates on CRC treatment costs over time. Using a large research database on insurance claims data, we analyzed CRC-related inpatient, outpatient as well as medication costs up to five years after diagnosis and prior to death. Our findings show that costs in the terminal phase of care were consistently on a high level even several years preceding death, mostly driven by high inpatient and medication costs, and substantially higher as compared to the initial phase of care. As well, we observed a consistent pattern of higher costs in men versus women, most markedly in the first year of diagnosis and the final year of life, and strongly varying costs by age. Our findings could be highly useful in informing cost-effectiveness analyses e.g., to further optimize strategies to screen for CRC.Objective: Evidence on the cost-effectiveness of screening for colorectal cancer (CRC) in the German general population remains scarce as key input parameters, the costs to treat CRC, are largely unknown. Here, we provide detailed estimates on CRC treatment costs over time. Methods: Using insurance claims data from the Vilua healthcare research database, we included subjects with newly diagnosed CRC and subjects who died of CRC between 2012 and 2016. We assessed annualized CRC-related inpatient, outpatient and medication costs for up to five years after first diagnosis and prior to death, stratified by sex and age. Findings: We identified 1748 and 1117 subjects with follow-up data for at least 1 year after diagnosis and prior to death, respectively. In those newly diagnosed, average costs were highest in the first year after diagnosis  and dropped steeply in the following years, with no consistent pattern of differences with respect to age. Costs prior to death were substantially higher as compared to the initial phase of care and consistently on a high level even several years before death, peaking in the final year of life, with strong differences by sex and age . Conclusion: Once clinically manifest, CRC causes substantial treatment costs over time, particularly in the palliative care setting. Strong differences in treatment costs by sex and age warrant further investigation. Since the introduction of screening colonoscopy in Germany in the year 2002, the incidence and mortality of colorectal cancer (CRC) have declined by more than 20% and more than 35%, respectively , and nowHowever, even though screening has been remarkably successful in reducing the CRC burden, evidence on the cost-effectiveness of this public health measure in the German population remains scarce ,5,6. ThiIn this retrospective study, we, thus, sought to assess the sex- and age-specific treatment costs for CRC over time in the German statutory health care system, firstly in subjects newly diagnosed with CRC and secondly in subjects who died of CRC.For this study, we used healthcare insurance claims data obtained from the Vilua Healthcare research database. The database contains entries for approximately 3.5 million individuals across Germany, which represents approximately 4% of the German population covered by statutory health insurance . Information on in- and outpatient diagnoses, medication, costs, procedures, diagnosis-related groups (DRGs) and demographics is regularly collected and inspected for outliers, errors and changes over time. As all data were anonymized and deidentified prior to analysis, no approval by an institutional review board or informed consent was required. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines .This analysis was performed on consecutive insurance years from 2007 to 2016 in subjects > 18 years of age with the presence of an ICD-10-GM  code for CRC (C18.*–C20.*) [Subjects newly diagnosed with CRC;Subjects who died of CRC.To reflect the initial and end-of-life phases of care, we assessed two groups of subjects:In population (1), to ensure that cost estimates reflected state-of-the-art treatments, we included subjects with new CRC diagnoses (two consecutive outpatient diagnoses or at least one inpatient diagnosis) from the years 2012 onwards who did not die during the observation period. Subjects with an ICD-10 code for CRC within the five years preceding the diagnosis were excluded to ensure that covered cancer cases were truly de novo. Population (2) included all subjects with prevalent CRC from 2007 onwards who died in 2012 or later to reflect recent developments in treatment schemes. Notably, population (2) also included individuals with new CRC diagnoses and death between 2012 and 2016, who, in the absence of death by CRC, would have been allocated to population (1). To assess the influence of such individuals with poor prognosis, we excluded these subjects in a sensitivity analysis.We defined a positive list of all potentially CRC-related cost positions, broken down into inpatient, outpatient and medication costs, and assessed these costs for all populations. To be considered CRC-related, all inpatient, outpatient and medication costs required coding of CRC as primary or secondary diagnosis. Additionally, total healthcare costs, e.g., all incurring costs regardless of the origin, were assessed. In terms of costing, the Vilua Research Database contains per-case cost position entries for inpatient, outpatient or medication intake. Cost positions was then multiplied with the actual cost as incurred and reported by the collaborating health insurance in their corresponding year of origin .Inpatient costs, including all partial and full hospital procedures, such as surgeries, diagnostic procedures, provision of medicines, remedies and aids during the hospital stay, nursing care and expenses for room and board, were based on the German DRG model, which simplified works by case-based flat rates depending on the medical condition. Outpatient treatment costs were based on the Uniform Assessment Standard , a catalogue which regulates invoicing for all ambulatory procedures. For this study, ambulatory medication costs were assessed separately (based on the ATC nomenclature), and stratified by chemotherapeutic drugs , innovative medicines , possibly CRC-related medication and further medicines (explicitly not related to CRC). Hospital and outpatient costs were considered CRC-related when the ICD-10 diagnosis for CRC was coded in the hospital stay (either primary or secondary diagnosis) or the outpatient case (only confirmed (“gesicherte”)) diagnoses. Further details are provided in Of note, the interpretation of health care costs typically depends on whose perspective is considered , with varying terms and definitions . For thiIn newly diagnosed patients (population 1), annual costs were assessed for up to five years after first diagnosis ), and in patients who died of CRC (population 2), for up to five years prior to death . For each population, all cost positions were assessed separately for men and women and stratified by age (<70 years vs. ≥70 years). We then calculated the mean and median costs with associated parameters of dispersion, both in terms of total CRC-related costs as well as separately for each cost category. For the total CRC-related costs in the first year after diagnosis and the final year before death, where the highest costs were to be expected ,7, diffeFor patients with newly detected cancers (population 1), we identified 1748, 1275, 503 and 359 subjects  with follow-up data for 1, 2, 3 and 4 years, respectively see .p-value < 0.001). A breakdown by cost category revealed higher inpatient costs in men to be the key driver for this pattern  and slightly higher in those ≥70 years (53–54%).The distribution of CRC treatment costs in the terminal phase of care was also strongly skewed, with maximum costs for individual patients exceeding EUR 250,000, EUR 100,000, EUR 100,000, EUR 150,000 and EUR 60,000 1–5 years prior to death, respectively. Average treatment costs followed a growth trajectory towards the end-of-life, with differences by sex and age. In the final year of life, costs peaked at EUR 34,351 in men and EUR 31,417 in women < 70 years, and at EUR 14,463 in men and EUR 9930 in women ≥ 70 years. In contrast to the years after a new diagnosis , costs in the terminal phase of care were consistently on a comparably high level , even several years before death .Differences by sex were most marked for those ≥70 years, with average treatment costs in men vs. women being 41% and 46% higher in the penultimate and last year, respectively. The key determinant for this difference was costs for innovative medicines .Differences by age, observed both for men and women, were even more pronounced than the differences by sex. Average total CRC treatments for those <70 years were at least twice the costs for those ≥70 years in each year of follow-up. These strong differences were mostly driven by substantially higher costs in the younger age group for inpatient care (approximately two times higher costs), chemotherapeutic drugs as well as novel medicines (approximately three times higher costs) . NotablyAverage total healthcare costs were consistently several thousand EUR higher than CRC-related costs, with the largest differences observed in the final year of death. This was mostly driven by inpatient costs not unambiguously identifiable as related to a CRC treatment .A sensitivity analysis excluding individuals with poor prognosis (diagnosed and died within 2012–2016) found lower estimates, but was overall consistent with the patterns seen in the main analyses . SeparatBased on claims data, this study provided detailed sex- and age-specific estimates on average CRC treatment costs over time in Germany, both in newly detected cases as well as prior to death. In those newly diagnosed, costs were highest in the first year after diagnosis and dropped steeply in the following years. Costs in the terminal phase of care were consistently on a high level even several years preceding death, mostly driven by high inpatient and medication costs, and substantially higher as compared to the initial phase of care. We observed a consistent pattern of higher costs in men versus women, most markedly in the first year of diagnosis and the penultimate and final year of life. Finally, the costs prior to death varied strongly by age, with average costs to treat individuals < 70 years at age of death amounting to at least twice the costs of those ≥70 years.Our findings were in agreement with previous evidence, in that highest costs arose in the first and final year(s) of care ,18,19,20Our cost estimates for the initial and final 12 months of care were notably lower than those recently reported in a claim-based study from Saxony, one of the Eastern states of Germany . This maThirdly, the initial and end-of-life phases in previous studies quantifying CRC treatment costs in Germany covered only 12-month periods ,7. With Notably, screening for CRC is generally accepted as cost-effective or even cost-saving ,27 by reFinally, the observed differences by sex and age warrant further investigation. Similar differences were also seen in previous studies, mostly in US American populations ,10,11,13The limitations of this study included the small sample size, the retrospective nature, the limited observation period and the reliance on insurance data, implying that incorrect reporting could not be ruled out. For instance, the quality of death information in our dataset was not guaranteed, and CRC deaths could not be reliably separated from deaths for other reasons. However, potential bias by costs other than for CRC treatment was minimized by specifically designed eligibility criteria  as well as by a positive list of costs excluding non-CRC-related positions.Furthermore, subjects who developed metastatic disease may have been slightly underrepresented in our sample, as distant metastases are sometimes incorrectly coded as new primary cancer, which were excluded in our study. Our cost estimates may, therefore, have been slightly underestimated, as metastatic cancers require more intensive treatment.Treatment costs may have been further underestimated by the increased use of immuno-oncological drugs, such as nivolumab or pembrolizumab, which were only authorized in early 2021 for specific patients with metastatic CRC in most European countries, including Germany. As these rather cost-intensive drugs were not yet part of standard clinical practice for CRC during the observation period of this study (2007–2016), possibly associated spikes in treatment costs since their market authorization were not reflected in our estimates.As well, the relatively small number of patients included in our analyses, in particular for >1 year after diagnosis or >1 year prior to death, implied that the generalizability of our findings may need to be interpreted with caution. Ideally, costs should be assessed with a prospectively planned incidence-based approach, e.g., with a long-term survival follow-up of a cohort of incident CRC cases linked to health insurance data, which may allow to compare costs from diagnosis to end of life. However, as this approach is highly complex ,31, the Finally, while we stratified for sex and age, a large heterogeneity between these predefined subgroups, e.g., with respect to the distribution of comorbidities, could not be ruled out, which caused intergroup comparisons to be more difficult. We purposely abstained from matching individuals between these groups as this would have further reduced the already small sample size. On the one hand, the use of a positive list of costs likely limited the impact of the heterogeneity between groups, as strictly only CRC-related costs were considered. On the other hand, this approach may also imply that some expenses which in fact occurred for the treatment of CRC were missed, e.g., if a procedure was incorrectly claimed. Though we believe this to have occurred very rarely, it may contribute to a possible underestimation of costs.Once clinically manifest, CRC causes substantial treatment costs over time, particularly in the palliative care setting. Observed higher costs in men versus women as well as strong differences by age in the terminal phase of care warrant further investigation, which should also additionally address indirect costs.