Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. HID-1 is a trans-Golgi network (TGN) localized peripheral membrane protein contributing to LDCV formation. There is no information about HID-1 structure or domain architecture, and thus it remains unknown how HID-1 binds to the TGN and performs its function. We report that the N-terminus of HID-1 mediates membrane binding through a myristoyl group with a polybasic amino acid patch but lacks specificity for the TGN. In addition, we show that the C-terminus serves as the functional domain. Indeed, this isolated domain, when tethered to the TGN, can rescue the neuroendocrine secretion and sorting defects observed in HID-1 KO cells. Finally, we report that a point mutation within that domain, identified in patients with endocrine and neurological deficits, leads to loss of function. The regulated release of polypeptides is essential for many biological processes, such as feeding, sleep, and physiological homeostasis –3. SecreC. elegans. Worms lacking HID-1 display reduced levels of LDCV soluble cargo and impaired neurosecretion [Recently, several studies have shown that the formation of LDCVs relies on a variety of cytosolic factors. Rab2 and its interactors orchestrate cargo sorting to LDCVs –17. The ecretion ,26. Inteecretion , suggestecretion . In neurecretion .Initial bioinformatic analysis of HID-1 amino acid sequence predicted the presence of several transmembrane spanning domains . HoweverHere we show that an N-myristoyl and a polybasic patch within the first 17aa of the N-terminus of HID-1 are required to increase its affinity for membranes but fail to provide TGN specificity. We further demonstrate that the highly conserved C-terminus serves as the functional domain of HID-1. We show that artificial tethering of this domain to the TGN is sufficient to rescue the phenotype observed in HID-1 KO PC12 cells. Finally, we report that a point mutation within that domain, identified in patients with endocrine and neurological deficits , leads tS1 Table) were verified by Sanger sequencing (Quintarabio). Primers were obtained from IDT and restriction enzymes from NEB. HID-1 lentiviral plasmid was generated by amplifying HID-1 from rat PC12 cell cDNA using the following primers: P1_F and P1_R. The main isoform amplified from these cells corresponds to isoform #2, which has been reported for mouse (Uniprot: Q8R1F6-2) and for human (Uniprot: Q8IV36-2). The PCR products were then subcloned into pLenti-puro using the restriction enzymes XbaI and BstEII. The G2A and Δ22 HID-1 mutant plasmids were constructed by amplifying from the WT HID-1 pLenti-puro plasmid using the following primers: G2A Forward: P2_F and P2_R; P3_F and P3_R. The PCR products were subcloned into pLenti-puro using the restriction enzymes XbaI and BstEII. HID-1 C-terminus human mutation was amplified from WT HID-1 and cloned in pLenti-puro by Gibson using the following primers: P4_F and P4_R. The GFP-TGN38-HID-1 C-terminus fusion was made by amplifying GFP-TGN38 from a premade plasmid using the primers: p5_F and P5_R. The HID-1 C-terminus was amplified from WT HID-1 using the primers: P6_F and P6_R. These PCRs were cloned into the pCAGG vector by Gibson. HID-1-GFP tail fusions were amplified with the following primers: pEGFP-N1-HID-1-N22: P7_F and P7_R; pEGFP-N1-HID-1-N22(G2A): P8_F and P8_R; pEGFP-N1-HID-1-N17: P9_F and P9_R; pEGFP-N1-HID-1-N17(FVL): P10_F and P10_R; pEGFP-N1-HID-1-N17(KRK): P11_F and P11_R. PCRs were subcloned into pEGFP-N1 using BamHI and AgeI. The HID-1 truncation constructs were amplified using the following primers: FUGW-HID-1(1–583)-HA: P14_F and P14_R; FUGW-HID-1(1–686)-HA: P15_F and P15_R. PCRs were subcloned into FUGW or pLenti-puro using Gibson reaction. pEGFP-C3 plasmid was digested with XhoI and BamHI and then Gibson assembled using TGN38-HID-1 C-terminus (HID-1687−788) gene block (Twist Bioscience).All constructs , were maintained in DMEM supplemented with 10% horse serum and 5% calf serum under 5% CO2 at 37°C. Transfection of PC12 and INS-1 cells was performed using Fugene HD (Promega) or Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. HEK293T cells were maintained in DMEM with 10% fetal bovine serum under 5% CO2 at 37°C. Lentivirus was produced by transfecting HEK293T cells with FUGW or pLenti-puro, psPAX2, and pVSVG using PEI at 1μg/μL in final reaction volume or Fugene HD according to the manufacturer’s instructions. 24hr after virus application, stably transduced cells were then selected for ~24hr using 3ug/mL puromycin.Rat INS-1 cells, originally obtained from the laboratory of Dr. Peter Arvan (University of Michigan), were maintained in RPMI supplemented with 1mM sodium pyruvate, 10% fetal bovine serum and 50μM β-mercaptoethanol under 5% COCACCGAGCCACCGACAATGCTTTCT-3′; Reverse2: 5′-AAACAGAAAGCA-TTGTCGGTGGCTC-3′ to generate HID-1 KO. The following primers were used to genotype HID-1 KO cells: P1F: 5′-GCACTAAACGGGGAGTCC-3′; P1R 5′-GCATAATGGACACTGAAGGTAGGAGG-3′; P2F: 5′-GGCATGGACGAGCTGTACAAG-3′; P2R: 5′- CCAGTCCACTGGGATGGC-3′; P3F: 5′-GTGTAACTCTGGCTACTCGATTCC-3′; P3R: 5′-CTTGTAGCACAGGGTGGCC-3′. To test for the presence of indels, primers P3F/P3R were used. The resulting PCR products were ligated into pBluesecript II KS. Isolated plasmids from 24 random colonies were then analyzed for the presence of indels by sequencing.The human codon-optimized Cas9 and chimeric guide RNA expression plasmid (pX330) developed by the Zhang lab were obtained from Addgene . To geneINS-1 HID-1 KO cells were transfected with either GFP, N22-GFP, G2A-GFP, N17-GFP, N17FVL-GFP, or N17KRK-GFP. Transfected cells were either untreated or treated for 50 seconds in 0.1% saponin in PBS and then immediately fixed with 4% paraformaldehyde. Quantitative analysis of fluorescence was performed using flow cytometry to determine the percent decrease in cells with a positive GFP signal in saponin-treated cells compared to untreated cells.HA (3F10) rat mAb was obtained from Roche, SgII rabbit antibody from Meridian Life Science (USA), TGN38 mouse mAb from BD Biosciences (USA), tubulin and actin mouse mAb from Developmental Hybridoma Bank Studies (USA), HID-1 mouse mAb from Novus Biologicals (USA), GFP mouse mAb from Proteintech (USA), and goat anti-rabbit Alexa Fluor 647, anti-mouse Alexa Fluor 488, anti-mouse Alexa Fluor 647, and anti-rat Alexa Fluor 488 secondary antibodies from ThermoFisher (USA).+  or 90 mM K+ (stimulated) for 30 min at 37°C. The supernatant was then collected, cell lysates prepared as previously described [+  or 90 mM K+ (stimulated) for 30 min at 37°C. The supernatant was collected and cell lysates prepared as described above. The amount of cellular and secreted NPY-sfCherry3 was determined using a plate reader (Tecan).PC12 cells were plated on poly-l-lysine, washed, and incubated in Tyrode’s buffer containing 2.5 mM Kescribed , and the4Cl in PBS. For saponin extraction experiments, prior to fixation, cells were treated with 0.1% saponin in PBS for 50s and washed with PBS. Cells were blocked in PBS containing 2% BSA, 1% fish skin gelatin, and 0.02% saponin. Primary antibodies were diluted in blocking solution at 1:1000 (TGN38), 1:1000 (SgII) and 1:500 (HA.11). The secondary goat anti-rat and donkey anti-mouse antibodies were diluted in blocking solution at 1:1000. Images were collected with a 63× objective (Oil Plan Apo NA 1.49) and an ImageEM X2 EM-CCD camera . For quantification of SgII, Z-stack were converted into a max intensity projection. ROIs were drawn around cells in the GFP channel which were then used to determine the background subtracted mean fluorescence of SgII in these GFP positive cells.PC12 and INS-1 cells were rinsed with PBS and fixed in 4% paraformaldehyde in PBS and incubated for 20 min at room temperature followed by a 20min quenching step with 100mM NHINS-1 HID-1 KO cells stably expressing HID-1-GFP or G2A-GFP were transfected with sialyltransferase-pTAG-RFP657. The following day, transfected cells were plated on poly-L-lysine coated coverslips. Two days after transfection, coverslips were placed in an open imaging chamber (Thermofisher) and imaged using an Olympus Fluoview scanning confocal microscope and a 63× oil objective (NA 1.42) at a resolution of 512 × 512 pixels with a sampling speed of 8.0 μs/pixel with Kalman filter (integration count 5). The region of interest for photobleaching was selected based on proximity to the TGN and was positioned at the periphery of the cell. This was to ensure our primary objective of specifically photobleaching the cytosolic pool was met, while minimizing the risk of inadvertently affecting the Golgi-bound pool during the experiment. Cells were bleached for ~1sec at 100% laser output and imaged followed by a 15sec recovery period. Movies were taken for a total of 5min. Analysis of fluorescence intensity was restricted to the Golgi area by using the sialyltransferase-pTAG-RFP657 to generate an ROI within ImageJ.t test. Statistical analyses were conducted using Excel or Prism.Unless indicated otherwise, all statistical analysis was performed using the two-tailed Student’s Images were processed using ImageJ; any changes in brightness and contrast were identical between samples meant for comparison.Fig 1A). We next tested the role of N-myristoylation by generating a G2A N22 mutant fused to GFP (N22G2A-GFP) and found that it behaved similarly to cytosolic GFP, suggesting decreased membrane affinity .We have previously generated HID-1 KO PC12 cells using a genome-editing strategy that led to the fusion of tdTomato in frame directly after the first 22 amino acids (N22) of HID-1 followed by an early stop codon . InteresS1A Fig). In other cases, the myristate moiety increases membrane affinity by rendering an N-terminal alpha-helix more amphipathic, as it has been shown for both Arf and Arl proteins [Fig 1A). To distinguish between the two possibilities, we reasoned that replacement of positively charged amino acids with alanine  should affect both motifs, whereas substitution of hydrophobic residues for polar residues  should only affect a putative amphipathic helix. Strikingly, the N17KRK mutant washed away completely during cytosolic extraction, whereas the N17FVL construct was resistant to saponin extraction . To quantify these observations, we repeated the experiment to determine the percent change in the number of GFP positive cells in presence or absence of saponin by flow cytometry. Consistent with our qualitative assessment, we observed little to no change in the percent of GFP positive cells transfected with N22-GFP, N17-GFP, or N17FVL-GFP. Conversely, the large majority of cells transfected with GFP alone, N22G2A-GFP, or N17KRK-GFP lost their fluorescence signal with saponin treatment . We obtained similar results in PC12 cells . Altogether, these results suggest that a myristoyl group and a polybasic patch within the first 17 amino acids of the N-terminus of HID-1 contribute to membrane binding.Typically, N-myristoylation per se is not sufficient for membrane targeting and only promotes weak interaction with the membrane. This type of lipidation is thus often found in combination with other motifs or modifications . In someproteins ,36. Of nFig 1A). To directly test whether the N-terminus is required for HID-1 localization, we generated HID-1 KO INS-1 cells using CRISPR/Cas9. As there are currently no commercially available antibodies for HID-1 western blotting, we validated our HID-1 KO by indel sequencing and immunofluorescence . We then used lentivirus to stably reintroduce HA tagged wildtype (WT), myristoylation deficient (G2A), or N-terminal deletion (Δ22) versions of HID-1. Although the G2A and Δ22 mutants displayed a greater amount of cytosolic signal at steady-state when compared to WT HID-1, pre-fixation saponin extraction revealed the presence of a pool co-localizing with a TGN marker . These observations suggest that the N-terminus of HID-1 increases its affinity for membrane but is not strictly required for TGN localization.Although the N-terminus of HID-1 is capable of membrane binding, our reporter did not show enrichment at the TGN as evidenced by the lack of co-localization with a co-transfected TGN marker  .To further investigate these findings, we conducted a series of experiments relying on fluorescence loss in photobleaching (FLIP). FLIP provides information about dynamics of fluorescently labeled molecules in various cellular structures. In our case, we reasoned that photobleaching the cytosolic pool of HID-1 while measuring the change in HID-1 signal at the TGN should enable us to quantify the difference in binding behavior of WT vs G2A HID-1. Using INS-1 cells expressing WT or G2A HID-1-GFP, we selected defined cytosolic regions at the periphery of cells and continuously (every 15s) photobleached the HID-1-GFP molecules undergoing passive diffusion within said area. We concomitantly determined HID-1-GFP fluorescence over time at the TGN determined by co-transfected sialyltransferase-RFP657. Comparison of WT and G2A HID-1-GFP confirmed that the absence of the myristoyl moiety only partially decreased HID-1 TGN immobile pool from 66.3±4.6% for WT to 30.3±3.1% for G2A HID-1-GFP and led to a faster cycling off the TGN as evidenced by analysis of the initial decay slope between WT and G2A  and overexpressed WT or G2A HID-1-HA using lentivirus in HID-1 KO cells. We found that under conditions of overexpression G2A was indeed sufficient to fully rescue the defect in SgII storage and secretion . While we have not tested the rescue ability of G2A at endogenous levels, these data suggest that myristoylation increases membrane affinity, but it is not an absolute requirement for HID-1 function. It further indicates that an additional domain within HID-1 is responsible for its function.We have previously observed that loss of HID-1 in PC12 cells significantly reduced basal cellular levels of the LDCV soluble neuropeptide secretogranin II (SgII) and impaired the stimulation of SgII release by depolarization . To deteS3 Fig) and is preceded by a predicted disordered region (amino acids 584–788). We hypothesized that the C-terminus with or without this disordered region might act as the functional domain. To test this directly, we stably expressed HA-tagged C-terminal truncations (1–686 and 1–583) in HID-1 KO PC12 cells for functional assays and found that they both failed to rescue the defect in SgII regulated secretion . In the case of proteins that are not purely cytosolic, such as peripheral membrane proteins, a lack of functional rescue by mutants could be an indirect consequence of their inability to localize to the proper compartment (TGN in the case of HID-1). However, we observed that, when these same C-terminal truncations were stably expressed in HID-1 KO INS-1 cells and immunostained for HA, they still localized to the TGN (. These data indicate that HID-1 C-terminus is required for function.The C-terminus of HID-1 (amino acids 686–788) displays a particularly high degree of conservation across species , we decided to artificially tether this domain to the TGN. For this, we fused HID-1687−788 (HID-1 C-terminus) to the C-terminus of GFP-TGN38 to constitutively localize it to the TGN. Transient expression of this fusion protein in HID-1 KO PC12 cells increased regulated secretion of a co-transfected regulated secretory cargo (NPY-sfCherry) compared to GFP-TGN38. The extent of the rescue observed for the C-terminus construct was similar to that of full-length HID-1-GFP . Although the cellular content of NPY-sfCherry tended to be higher in cells expressing the C-terminal domain, it did not reach significance (p = 0.09). We also looked at the effect on endogenous SgII storage using immunofluorescence. Expression of the C-terminal domain of HID-1 tethered to the TGN (GFP-TGN38-C-term) in HID-1 KO PC12 cells rescued the levels of SgII to levels similar to WT PC12 cells, whereas expression of GFP didn’t rescue SgII levels . Altogether these data suggest that the C-terminus of HID-1 (amino acids 687–788) contains the functional domain.We next tested if the C-terminal domain (amino acids 687–788) can be sufficient for function by itself. As stable expression of this HA-tagged domain in HID-1 KO INS-1 cells revealed a diffuse distribution across the cells without obvious enrichment at the TGN . The result of this mutation is predicted to substitute the last 15 amino acids (IWYDTDVKLFEIQRV) for 10 amino acids (CLVRHRREAV). Stable expression of this mutant in HID-1 KO PC12 cells led to proper TGN localization . However, this mutant completely failed to rescue the secretion phenotype observed in HID-1 KO cells . These data suggest that a defect in LDCV formation is the underlying cause of the symptoms associated with this patient. Recent work also reported that mutations in HID-1 patients cause an early infantile encephalopathy with hypopituitarism [A homozygous mutation within HID-1 has been found in human patients with severe endocrine and neurological deficits ,37. ThisS1 FigA) Protein sequence alignment of HID-1 N-terminus. (B) PC12 cells were transfected with the indicated GFP constructs. Cells were treated or not with saponin were fixed and immunostained for TGN38. Scale bars indicate 10μm.((TIF)Click here for additional data file.S2 FigA) Genomic DNA sequence of rat HID-1. The predicted Cas9 cleavage site is indicated. (B) Indels (shown in red) detected in INS-1 HID-1 KO cells. (C) Indels (shown in red) detected in PC12 HID-1 KO cells. (D-E) HID-1 KO PC12 and INS-1 immunostained for endogenous HID-1. Scale bars indicate 10μm.((TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigTubulin is shown as a loading control.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


The spread of Chikungunya virus is a major public health concern in the Americas. There were >120,000 cases and 51 deaths in 2023, of which 46 occurred in Paraguay. Using a suite of genomic, phylodynamic, and epidemiologic techniques, we characterized the ongoing large chikungunya epidemic in Paraguay. Togaviridae , a single-stranded positive-sense RNA virus belonging to the family CHIKV can be classified into 4 distinct genotypes: West African, East/Central/South African (ECSA), Asian, and Indian Ocean lineages  Ethics Review Committee (PAHO no. 2016-08-0029) and by the Paraguayan Ministry of Public Health and Social Welfare (MSPyBS/SG no. 0944/18). Samples used in this study were deidentified residual samples from routine diagnosis of arboviruses in the Paraguayan Public Health Laboratory, which is part of the public network within the Paraguayan Ministry of Health.<35 and availability of epidemiologic metadata, generating 179 viral genomes deposited in GenBank under accession nos. OQ775394‒567 and OQ567722‒5). We performed sequencing by using Nanopore technology  to proceed with library preparation, and their PCR Ct values were a mean of 21 (range 9‒34) (Appendix 2). Samples had good spatial representation of southern Paraguay (10/17 districts) (>90% was achieved (We screened 179 quantitative reverse transcription PCR‒positive samples for CHIKV. All contained sufficient DNA (stricts) , panel Bstricts)  and captstricts) , panel Cachieved . Most gege 9‒34) . SamplesMost (58%) genomes were from CHIKV infection outcomes in outpatients, followed by fatal (18%), intensive care unit (17%) and inpatient (7%) infections . CompareTo determine the dynamics of the CHIKV ECSA in Paraguay, we performed phylodynamic analysis of a dataset comprising 715 available representative genomes combined with viruses sequenced in this study  . A date-2 = 0.40, correlation coefficient = 0.60), indicating relatively clock-like virus evolution  representing this virus clade in isolation. We found a relatively strong correlation between sampling date and root-to-tip genetic divergence in this dataset (rvolution . Phylogevolution  and suggvolution .Virus transmission dynamics roughly followed patterns of population density, moving most often between the most populous urban localities . BecauseThis study highlights the resurgence of CHIKV ECSA in Paraguay during 2022–2023. Our findings provide evidence of lineage persistence over a period of 11 months preceding resurgence and report the notable coincidence of virus resurgence and the highest mean temperatures recorded in Paraguay. Those 2 factors, combined with presence of the vectors and a large proportion of the population susceptible to CHIKV probably generated an ideal scenario for the observed fast and large CHIKV epidemic wave that started at the end of 2022. Given the association of ongoing resurgence with a specific lineage of CHIKV ECSA with 2 synonymous changes in nonstructural proteins 3 and 4 and uncertainty of how the ongoing epidemic will unfold, genomic surveillance should remain active to track real-time evolution and spatial spread, contributing to public health risk assessments in Paraguay and other countries in South America.Additional information on rapid epidemic expansion of chikungunya virus East/Central/South African lineage, Paraguay.Virus strains used in study of rapid epidemic expansion of chikungunya virus East/Central/South African lineage, Paraguay.


Barley yellow striate mosaic virus (BYSMV) is transmitted by the small brown planthopper . Here, we show that BYSMV infects the central nervous system (CNS) of SBPHs, induces insect hyperactivity, and prolongs phloem feeding duration. The BYSMV accessory protein P6 interacts with the COP9 signalosome subunit 5 (LsCSN5) of SBPHs and suppresses LsCSN5-regulated de-neddylation from the Cullin 1 (CUL1), hereby inhibiting CUL1-based E3 ligases-mediated degradation of the circadian clock protein Timeless (TIM). Thus, virus infection or knockdown of LsCSN5 compromises TIM oscillation and induces high insect locomotor activity for transmission. Additionally, expression of BYSMV P6 in the CNS of transgenic Drosophila melanogaster disturbs circadian rhythm and induces high locomotor activity. Together, our results suggest the molecular mechanisms whereby BYSMV modulates locomotor activity of insect vectors for transmission.Transmission of many plant viruses relies on phloem-feeding insect vectors. However, how plant viruses directly modulate insect behavior is largely unknown.  Most plant viruses are transmitted by insect vectors in finely regulatory mechanisms. Here, the authors show that a plant rhabdovirus can modify circadian rhythm of its insect vectors and enhances locomotor activity for efficient transmission. Transmission of insect-borne plant pathogens including viruses, fungi, bacteria, and phytoplasmas heavily relies on their insect vectors1. It is well known that plant viruses directly or indirectly modulate behavior of their insect vectors for efficient transmission3. During indirect modulation, plant viruses render infected plants to become more attractive to insect vectors by changing volatile odor compounds, nutrition, visual appearance, or defense profiles4. Although it is a widespread phenomenon, we are just beginning to understand the underlying mechanisms how plant viruses modify plant physiological states to attract insect vectors indirectly3. For instance, the C2 protein of tomato yellow leaf curl virus (TYLCV) and the NSs protein of tomato spotted wilt tospovirus (TSWV) inhibit MYC-regulated jasmonic acid (JA) signaling to enhance vector attraction6. The cucumber mosaic virus 2b protein directly interacts with JAZ proteins to inhibit JA-mediated herbivore defense of host plants7. The begomovirus βC1 protein modulates plant immunity, which is beneficial for infection of begomovirus and its whitefly vectors but deters non-vector insects8. These findings represent indirect effects of virus components on insect behavior manipulation through changing plant phenotypes and inducing insects to preferentially settle on infected plants for virus acquisition9. However, it is a far less described and functional explanation for how persistently transmitted plant viruses directly regulate insect behaviors when they propagate within the vectors.Insects are vectors of many parasites causing various diseases in animals and plants. Increasing evidence has demonstrated that these parasites have evolved diverse effectors to modulate insect behaviors for their horizontal spread in nature10. For instance, the uridine 5′-diphosphate (UDP)–glucosyltransferase of baculoviruses render infected gypsy moths to climb to tree foliages, facilitating virions release from insect cadavers and are acquired by new insect vectors12. Besides, Kakugo RNA, a novel picorna-like virus, was only detected in the brains of aggressive honey bees13, implying that honeybee aggressive behaviors probably enhance virus horizontal transmission. Most insect-borne plant viruses are transmitted along with salivary secretion during insect feeding on host plants. Therefore, longer feeding time and more food intake can enhance virus transmission probability. For instance, TSWV drives thrips to take more food than healthy thrips, which facilitate virus transmission along feeding14. Similarly, viruliferous whiteflies with TYLCV have more food intake than healthy whiteflies for virus transmission15. Besides, TYLCV can cause apoptotic neurodegeneration in whitefly, and drive the movement of viruliferous whiteflies from infected plants to surrounding healthy plants for virus spread16. Rice gall dwarf virus (RGDV), a devastating rice reovirus, inhibits the secretion of a calcium-binding protein, leading to more saliva secretion into host plants for virus transmission17. However, to date, the mechanisms of directly modulation of insect feeding behavior and the responsible virus components have not yet been studied in previous studies.Some parasites can spread into the central and peripheral nervous systems of insect vectors to regulate insect behaviors and performances, termed neuroparasitology18. The molecular machinery of circadian clock has been extensively studied in Drosophila melanogaster19. Two transcription factors, CLOCK (CLK) and CYCLE (CYC), form a heterodimer and promote transcription of period (per) and timeless (tim) genes in late day to early night22. Subsequently, the PER and TIM proteins gradually accumulate during the night, form heterodimers late night, and move into the nucleus to repress transcription of CLK-CYC heterodimer21. Light can entrain the clock system through degrading TIM by light-activated CRY and JETLAG (JET)26. The F-box protein, JET, mediates light-induced TIM degradation through triggering ubiquitination of TIM, which requires the COP9 signalosome (CSN) to remove Nedd8 moiety from the scaffold protein cullin1 of the SCFJET complex27. The per/tim oscillatory loop triggers a rhythmic expression of various clock-related genes for modulation of insect behavior and physiological functions28. However, whether and how insect-borne plant viruses modify circadian rhythms of insect vectors remains elusive.Most insects exhibit high locomotor activity before sunrise and sunset (termed as morning and evening peaks), which is regulated by endogenous circadian clocks29. Two model rhabdoviruses, Vesicular stomatitis virus (VSV) and Rabies virus (RABV), can invade the central nervous system (CNS) and induce hyperactivity in infected animals33. Most plant rhabdoviruses are transmitted by insect vectors in a circulative-propagative manner. Several plant rhabdoviruses, including rice yellow stunt virus (RYSV), rice stripe mosaic virus (RSMV), maize mosaic virus (MMV), and maize fine stripe virus (MFSV), have been shown to spread into the CNS of insect vectors37. Recently, reverse genetic systems been developed in several plant rhabdoviruses, including sonchus yellow net virus (SYNV)38, barley yellow striate mosaic virus (BYSMV)39, northern cereal mosaic virus (NCMV)40, and MMV41. These systems provide genetic approaches for investigating virus–insect–plant interactions43.The family Rhabdoviridae consists of 20 genera with ecologically diverse members infecting mammals, plants, insects, fishes, birds, and reptilesBarley yellow striate mosaic virus (BYSMV), an important member of the Cytorhabdovirus genus, infects >25 species of monocots worldwide. BYSMV is persistently transmitted by the small brown planthoppers . The BYSMV genome encodes five accessory proteins  interspersing into five conserved structural proteins 44. We have established reverse genetics systems of BYSMV and applied these systems to studies of the tripartite interactions of virus–insect–plant48. Recently, we found that BYSMV P6 inhibits JA signaling in host plants and indirectly affects insect attractiveness49. Here, we undertook experiments to identify a new function of BYSMV P6 in regulating locomotor activity of their insect vectors. We here show that BYSMV P6 interferes with degradation of TIM and directly modulates locomotor activity of insect vectors for virus transmission.51. Here, we determined whether BYSMV spread into the CNS of SBPHs. To this end, we used a recombinant BYSMV expressing red fluorescent protein (BY-RFP), in which the RFP gene with the gene junction sequences was inserted between the N and P genes of BYSMV  after a 2-d-inoculation period on BY-RFP-infected barley plants52, intense RFP fluorescence was observed in ~40% of inoculated SBPHs, but not in non-inoculated controls  system to monitor the locomotor activity of non-inoculated or viruliferous SBPHs under light–dark (LD) cycles. Notably, the locomotor activity of BY-RFP-infected SBPHs was higher than that of non-inoculated controls , penetration , phloem-feeding (N4a and N4b), and xylem feeding (N5)  technique is a powerful tool for recording feeding behaviors of sap-sucking insects on host plantsN5) Fig. . The EPGN5) Fig. .Fig. 2BY54. To test whether virus infection influenced food intake of SBPHs, we incubated healthy or BY-RFP-infected SBPHs within an inverted transparent plastic cup over a filter paper  and timeless (tim), through RT-qPCR to measure their relative transcripts at five time points  under LD cycles. Notably, oscillation of tim and per transcripts was significantly inhibited in viruliferous SBPHs compared to robust oscillation in mock-treated SBPHs  and cycle (cyc), another two core transcription factors, were also attenuated in BY-RFP-infected SBPHs but maintained in mock-treated SBPHs  of barley to subvert plant JA signaling49. Moreover, the 16th Ile (Ile16) residue of BYSMV P6 is required for the specific interaction with HvCSN549. Since COP9 and CSN5 are conserved in all eukaryotes57, we assumed that BYSMV P6 may interact with the CSN5 protein (LsCSN5) of SBPHs, which shares 60.0% identity with HvCSN5 . All yeasts expressed protein combinations grew on the SD/-Trp-Leu medium. Only yeasts expressing AD-P6 and BK-LsCSN5, rather than other protein combinations, were able to proliferate on SD/-Trp-Leu-His-Ade selection medium with 5 mM 3-amino-1,2,4-triazole (3-AT)  Fig. , indicatE. coli were incubated with total protein extracts from BY-RFP-infected SPBHs and precipitated with anti-His agarose beads for immunoblotting analysis with anti-P6 and anti-His antibodies. The results show that only LsCSN5-His, rather than GFP-His, can be co-precipitated with the BYSMV P6 protein expressed from BY-RFP infections  to CUL1  E3 ligase complexesUL1 Fig. , indicatLsCSN5 dsRNA (dscsn5) were synthesized in vitro and injected into the thorax of SBPHs to interfere with endogenous LsCSN5, and the gfp dsRNA (dsgfp) served as a negative control. As expected, SBPHs injected with dscsn5 accumulated relative higher ratio of CULNedd to CUL1 compared with the dsgfp control  share 75.9% identity in amino acid sequence  flies, display strong morning/evening peaks of locomotor activity and normal rhythm in LD cycles  . As expected, the amounts of per and tim mRNAs exhibited a daily oscillation in inactivated transgene lines , while oscillation of per and tim mRNAs were attenuated in heterologous expression flies of P6-GFP   Fig. . By contNAs Fig. . ConsistCollectively, the results demonstrate that BYSMV P6 interacts with DmCSN5, and heterologous expression of BYSMV P6 in neurons disturbs the circadian rhythm and prolongs the high activity of morning and evening peaks.Lepidoptera), which is induced by the conserved protein tyrosine phosphatase (ptp) gene in a subset of baculoviruses59. Virus infection in brain of aggressive bees indicates that virus infection was associated with aggressive trait60. Since most insect-borne plant viruses do not cause obvious cytopathology, it remains technically challenging to select alive viruliferous insects from healthy controls for comparison of their behavior. To circumvent this problem, we deployed a recombinant BYSMV-RFP (BY-RFP) expressing an RFP reporter gene to easily differentiate viruliferous SBPHs from healthy SBPHs, which is crucial for monitoring virus-modulated feeding behavior accurately. As shown in Fig. Many economically important plant viruses are transmitted by insect vectors. The feeding behavior of insects is a key determinant of plant virus transmission. It has long been known but is still at the beginning of our understanding how viruses induce hyperactive behavior in their insect hosts. Baculoviruses induce hyperactive trait in their caterpillar hosts  through transmission by the small brown planthoppers  as described previously50. Healthy and viruliferous SBPHs were reared on rice seedlings in chambers at 25°C with 12 h: 12 h (light: dark) photoperiod. Flies were raised and crossed at 25°C on standard cornmeal agar media. Transgenic flies were generated in the background of Drosophila strain w1118 using the GAL4/UAS system. We cloned the ORFs of P6 or P6I16A into the pUAS vector, in which the ORFs were fused between upstream activating sequence (UAS) of yeast transcription factor GAL4 and GFP. The UAS-P6-GFP and UAS-P6I16A-GFP transgenic flies were generated by the Tsinghua Fly Center . Then the transgenic male flies were crossed with the virgin flies with Elav-Gal4 driver to generate heterologous expression flies of P6-GFP or P6I16A-GFP in the nervous systems.The recombinant BYSMV-RFP (BY-RFP) virus was maintained in barley plants . The detailed localization in brains was acquired with an optical slice thickness of 0.3 μm using a ×100, 1.44 N.A. oil objective (Leica HC PL APO). The signals were recorded by a camera (Andor Zyla 4.2) of scientific complementary metal-oxide semiconductor (sCMOS). DAPI and RFP were visualized at excitation of 405- and 568- nm, respectively. In detail, images were acquired with 405-nm (30–35%), exposure time 300 milliseconds (ms) and 568 nm (20–25%), exposure time 100–200 ms. Images were acquired by the Fusion 2.1 software (https://andor.oxinst.com/products/dragonfly#fusion), and processed by the Imaris software . The positions of SBPH neuropil were according to the three-dimensional reconstruction of planthoppers as described previously64.Viruliferous and healthy SBPHs were fixed in 4% paraformaldehyde overnight at 4°C followed by removal of all the legs. Samples were dehydrated in a series of ethanol, cleared in xylene, embedded in paraffin. The samples were sectioned serially at 8 μm in a microtome and collected on adhesion microscope slides as described previouslyWhole bodies of Mock or BY-RFP-infected SBPHs were photographed with an Olympus stereomicroscope SEX16 . Dissected brains of SBPHs and transgenic flies were photographed with the BX53 microscope using the DP72 digital camera . Some dissected brains were observed with a Leica TCS-SP8 laser scanning confocal microscope. For Immunological detection of N protein in brain tissues of SBPHs, Heads of non-inoculated and BY-RFP-infected SBPHs at 12 dpi were dissected and placed in 4% paraformaldehyde for fixation, followed by the added of rabbit antibodies to the N protein, and incubation with alex-488-coupled rabbit secondary antibody (GFP), observed under the BX53 microscope. GFP and RFP were visualized, respectively, at excitation of 488 and 543 nm and detected at 500–540, 585–625 nm, respectively.65. Briefly, the assays were performed in a chamber with a condition at 25 ± 2°C and 50% humidity. Insects were placed into glass tubes individually and exposed to a 12 h: 12 h (Light: Dark) LD cycles for 5 d. The light onset was taken as Zeitgeber time 0 h (ZT0). The infrared beam passing through the middle of tubes was interrupted by insect movement and was automatically recorded every 30-min. The results of locomotor activity were analyzed using the ActogramJ software in actogram format66. The FaasX software was used for analysis of fly rhythm67. Rhythmic flies were defined with the following criteria of χ2 periodogram analysis: power ≥20 and width ≥1.5. The constant darkness (DD) or light (LL) was settled for more than 5 d following 2-d LD cycles.Locomotor activity of planthoppers or flies was detected using the Drosophila Activity Monitor 2 (DAM2) system  as described previously68. Briefly, total RNA was extracted using TRIzol reagent  and treated with DNase I  to remove contaminated genome DNA. Next, 1–2 μg total RNA was used for reverse transcription with oligo (dT) using HiScript® II Reverse Transcriptase . Quantitative PCR was carried out on the Bio-Rad CFX96 Real-Time PCR System using 2× SsoFast EvaGreen Supermix . SBPH β-actin1 and Drosophila tubulin served as endogenous controls. Three independent experiments were carried out for biological statistics. All primers were listed in Supplementary Table RT-qPCR assays were performed as described previouslyCUL1 fragment (aa 354-813) and TIM fragment (aa 1-338) of SBPHs were introduced into the pET-30a (+) vector, respectively. The resultant plasmids were transformed into the E. coli strain BL21 for expression of LsCUL1-6×His and LsTIM-6×His protein. The 6×His-tagged proteins were purified using the Ni-NTA agarose  and used to immunize rabbits for anti-LsCUL1 and anti-LsTIM antibodies . Immunoblotting analysis was carried out as previously69. Briefly, total proteins were extracted from SBPHs in RIPA Lysis Buffer with phenylmethylsulfonyl fluoride (PMSF) using a TGrinder high-speed tissue grinder . After incubating on ice for 30 min, the homogenate was centrifuged at 13,000 × g for 5 min at 4°C. The supernatant was separated in SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk and incubated with the polyclonal antiserum of the BYSMV N (1:3000), P6 (1:3000), RFP (1:2000), GFP (1:3000), GST (1:5000), His (1:5000), LsCUL1 (1:1000), Actin (1:3000), or LsTIM (1:1000) antibodies at 37°C for 1 h. Goat anti-rabbit or anti-mouse IgG horseradish peroxidase conjugate  were used as secondary antibodies. Finally, membranes were incubated with NcmECL Ultra chemiluminescent substrate  and analyzed using Azure Sapphire RGBNIR .To generate anti-LsCUL1 and anti-LsTIM antibodies, the cDNA fragments corresponding to the 53. Feeding behaviors of SPBHs on barley were monitored continuously for 8 h using an 8-channel DC-EPG device  in an electrically grounded Faraday cage. After a 4 h pre-starvation period, dorsum of healthy and infected planthoppers was connected to a gold wire (2-cm long and 12.5-µm in diameter) through a water-soluble silver conductive paint . The other end of the wire was connected to the Giga-8 DC-EPG amplifier through the EPG probe. Then the planthoppers were carefully moved to barley plants. The hard copper wire of plant electrode was inserted into soil. The data sets were recorded and analyzed by Stylet+ software .EPG assays were performed as described previouslyI16A were individually cloned into the pGADT7 vector as described previously49, and the LsCSN5 ORF was introduced into the pGBKT7 vector. Plasmids expressing AD-P6/BK-LsCSN5, AD-P6I16A/BK-LsCSN5, and negative controls were transformed into AH109 or Y187 yeast cells and grown in Trp/Leu double-deficiency yeast plates. The co-transformed cells were diluted and dropped on -Trp/-Leu double-deficiency and -Trp/-Leu/-His/-Ade/3-AT quadruple-deficiency yeast plates with 5 mM 3-amino-1,2,4-triazole (3-AT) and incubated at 30°C for 3–5 d.To verify the P6–LsCSN5 interaction, the ORFs of P6 and P6I16A were engineered into the pGEX-KG vector for expressing GST-P6 and GST-P6I16A, respectively. Fusion proteins were expressed in the E. coli strain BL21 under induction of 0.1 mM Isopropyl-D1-Thiogalactopyranoside at 18°C for 16 h. In GST pull-down assays, GST, GST-P6, or GST-P6I16A proteins were incubated with LsCSN5-6×His or DmCSN5-6×His proteins in 500 µL binding buffer  with 30 µL glutathione Sepharose4B beads  at 4°C for 2 h. After centrifugation at 800 × g for 1 min, beads were washed five times with series concentrations of washing buffer , boiled in SDS buffer for immunoblotting analyses with anti-GST (1:5000) and anti-His (1:5000) antibodies.The ORFs of LsCSN5 and DmCSN5 were cloned into the pET-30a (+) vector for expressing LsCSN5-6×His and DmCSN5-6×His proteins, respectively. The ORFs of P6 and P669. To pull down the P6 protein from viruliferous SBPHs, total proteins were extracted from 4th-instar viruliferous planthoppers using RIPA Lysis Buffer (Strong) supplemented with PMSF. Approximately 10% of the total protein fraction served as input samples. Then, the extracted total protein samples were incubated with LsCSN5-6×His was at 4°C for 3 h, and then added Ni-NTA agarose for 2 h. The GFP-6×His protein was used as a negative control. Finally, the beads were washed and boiled for immunoblotting analyses.Co-IP analysis was performed as described recentlyLsCSN5  were amplified using primers containing T7 RNA polymerase promoter at the two termini as shown in Supplementary Table dsGFP served as a negative control. Approximately 40 ng dsRNA (2.0 µg/µL) was injected into the thorax of SBPHs.The specific fragments of To detect SBPH honeydew secretion, barley plants (one plant per cup) were covered with a transparent plastic cup and placed over a filter paper. Ten SBPHs were placed into the cups for feeding. After 2 d, the filter papers were dried in an oven for 30 min, and then soaked with 0.1% (w/v) ninhydrin in acetone solution. After 30 min at 65°C, the amino acid contents of honeydew were stained until violet or purple spots appeared. The areas of stained honeydew were analyzed by Image J software.t test was used to compare the significance of only two groups. Statistical analyses of RT-qPCR of clock-related gene mRNAs in different SBPHs among Zeitgeber times were performed using two-way ANOVA followed by Tukey’s post hoc test, P < 0.05. Asterisks indicate statistical significance .Statistical analyses were performed using Graphpad Prism 8. Two-tailed Student’s https://www.ncbi.nlm.nih.gov/) with the following accession numbers: Lstimeless (MG356485.1); Lsperiod (MG356486.1); DmCSN5 (NM_058094.5); LsActin (AY192151); Dmtimeless (AB059649.1); Dmperiod (NM_080317.2); Dmclk (NM_079240.3); Dmcyc (NM_079444.3); Dmtubulin (M14643.1); Lsclk (OR514639); Lscyc (OR514641); Lscsn5 (OR514640); LsCUL1 (OR514638).The accession numbers of described genes in this study can be found in GenBank database (Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileSource Data


The most important variables for predicting the contact time were pH, temperature, and virus type. The estimated contact time for 3 log inactivation of RNA virus was <2 h at pH 12, which was achieved by adding 1.8 and 3.1% slaked lime to wastewater and faeces, respectively. The contact time decreased exponentially with the pH of the sludge and wastewater. In contrast, the pH of the sludge and wastewater increased linearly with the slaked lime dosage. Lime treatment is a promising measure where long-term containment in latrine is not feasible in densely populated areas, as 1 day is sufficient to inactivate viruses. The relationship we have identified between required contact time and lime dosage is useful for practitioners in determining appropriate treatment conditions of human waste.Water, sanitation, and hygiene provisions are essential during emergencies to prevent infectious disease outbreaks caused by improper human excreta management in settlements for people affected by natural disasters and conflicts. Human excreta disinfection is required when long-term containment in latrines is not feasible on-site. Alkali additives, including lime, are effective disinfectants for wastewater and faecal sludge containing large amounts of solid and dissolved organic matter. The aim of this study was to determine the minimum dose and contact time of alkali additives for treating virus-containing human excreta in emergency situations. We used literature data collected by searching Google Scholar and Web of Science. The date of the last search for each study was 31th May 2023. Only peer-reviewed articles that included disinfection practices in combination with quantitative data for the physicochemical data of a matrix and viral decay were selected for data extraction. Two reviewers independently collected data from each study. We extracted datasets from 14 studies that reported quantitative information about their disinfection tests, including viral decay over time, matrix types, and physicochemical properties. Three machine learning algorithms were applied to the collected dataset to determine the time required to achieve specified levels of virus inactivation under different environmental conditions. The best model was used to estimate the contact time to achieve a 3-log People otavirus . Infectiotavirus . Maintaiotavirus . Proper A common emergency measure is disposing in sewers; however, sewer system and treatment facilities are heavily damaged by earthquakes, stopping functioning for a long period . In the 2) and burnt (CaO) lime. Lime has been used for treating high-organic-loading waste because it raises the sludge pH to >10, which is sufficient to inactivate viruses and bacteria (Entamoeba histolytica and Giardia lamblia (3) that can diffuse through the complex wall of protozoan oocysts  of microorganisms as a function of variables related to the matrix and environmental conditions . Machine2.What is the contact time with alkali additives for treating a virus-containing matrix?” Google Scholar and Web of Science were used for collecting relevant articles published between 1950 and May 2023. The keywords used were:  AND (virus) AND (inactivation OR disinfection) AND (biosolids OR sludge). The date of the last search for each study was 31th May 2023. Only peer-reviewed articles that included disinfection practices in combination with quantitative data for the physicochemical data of a matrix and viral decay were selected for data extraction. A single reviewer decided whether a study met the inclusion criteria for the review. The inclusion criteria for the papers were as follows: (1) published in English, (2) peer-reviewed, (3) not a review paper, (4) used alkali additives as disinfectants, and (5) containing the time-course change of virus infectivity and quantitative information on the physicochemical parameters of the matrices. Two reviewers independently extracted data from each study. Data extraction from one figure was performed using WebPlotDigitizer version 4.2 , as described in a previous study , light GBM, and Automatic Relevance Determination (ARD) . The model features were the physical and chemical properties of the matrix during the treatment, which are individual measurable properties or characteristics of a phenomenon. We split a dataset into train and test sets, and used resampling methods to handle the uncertainty in the representativeness of our dataset and estimated the performance of a modelling procedure on dataset not used in that procedure. Of the complete dataset, 80% was used to train the model and the remaining 20% to evaluate its prediction accuracy. The values of the hyperparameters were determined via a 5-fold cross validation. We conducted this set of data allocations and cross-validation 10 times. The mean square values (MSE) for predicting the test (MSEtest) and training (MSEtrain) data were calculated. We also estimated the ratio of MSEtest to MSEtrain, indicating the degree of overfitting to the training data. The best model was selected based on the mean value of MSE and the ratio of MSEtest to MSEtrain in the 10-time calculation.9 copies/mL and 106 copies/mL, respectively, by RT-qPCR  , virus type (phage or virus), matrix type , suspended solid contents , pH, temperatures, and logarithmic initial virus concentration. MSEtest and MSEtrain by three machine learning models were shown in The linear relationships were identified between the logarithmic times for inactivation and the pH of the matrix ( < 0.05) ; howeverp < 0.05, nonzero slope t-test). Normal Q-Q plot was shown in Five additional studies were included to estimate the additive dosage required to achieve an alkaline pH . The pH The estimated T99.9 for RNA virus in different alkaline pH, temperatures, and matrices are presented in In the developed model, 52 estimates were shorter than the observed data (<10 h), indicating the possible underestimation of contact time using the developed models. We carefully examined mispredicted data to avoid misleading treatment conditions . More co4.This study aimed to determine the alkali dosage and contact time required for virus inactivation in faecal sludge and wastewater with high solid loading. Since alkaline treatment is widely used for sludge treatment, we identified several reviews aimed at evaluating the pathogen inactivation efficacy of alkaline treatment . HoweveThe pH of the matrix was the predominant factor causing an exponential increase in the contact time. A previous study also applied a logarithmic scale for T99 against the pH of different organisms, including phages and mammalian viruses . This stLime treatment is a promising measure where prolonged human waste storage is inappropriate in densely populated areas, as 1 day is sufficient to achieve a 3-log virus reduction. When matrix pH <12 because of limited alkaline additives available, the required contact time with lime should be increased considerably: T99.9 for faeces was 4.0 h at pH 11 and 20.7 h at pH 10 (25°C). Therefore, we recommend precise pH monitoring of the matrix soon after adding disinfectants.Our results indicated that slaked lime and burnt lime increased the pH of the matrix with a lower mass than ash. A previous systematic review also reported that ash does not appear to have the same level of disinfection efficacy as lime for the treatment of faecal sludge . The reqWe estimated the treatment times for RNA viruses because many waterborne viruses in excreta are RNA viruses, including Picornaviridae, Caliciviridae, Hepeviridae, Reoviridae, and Astroviridae . They arn = 5). More treatment data on mammalian DNA viruses are required to discuss the applicability of DNA phage as an appropriate surrogate in alkaline treatment of sanitation-relevant matrices. The prediction accuracy for LRVs of DNA phages was lower than for other surrogate types, indicating that the prediction of DNA phages was less reliable. This could be due to our assumption that ignored the variable tolerance between four different phages included in our dataset . Incorporating the species differences as a feature or developing individual models may improve the prediction accuracy, while more data from experimental conditions would be needed to reduce the uncertainty due to incomplete coverage of the domain in modelling.The extent of misprediction was exceptionally large for DNA phages, indicating that the time for inactivating DNA phage should be relatively longer . The wat10 inactivation was shorter than for ammonia treatment. T99 ranged from 1 day to 800 days at 0.1–600 mM NH3, while it was within 1 day at pH 9–12  in faeces or when additional risk barriers  are introduced in the faecal-oral route. Anaerobic treatments including anaerobic baffled reactor, anaerobic filter, and biogas reactor are applicable onsite, while more treatments are used as semi-centralized treatment   .5.We conducted a systematic review to determine the contact time with alkali additives for treating a virus-containing matrix. Fourteen studies reported quantitative information on their disinfection tests, including the time-series decay of viruses, types of matrices, and physicochemical properties. We obtained synthesised datasets of the required contact time associate to pH, temperature, matrix type, virus type, and initial concentration of virus. The size of datasets applied to further analysis was 136 for T90, 105 for T99, 77 for T99.9, and 57 for T99.99. The pH of the matrix and contact time with lime were determined using machine learning algorithms. The slaked lime quantity required to achieve pH 12 was 1.8 and 3.1% on the wet matter basis of wastewater and faeces, respectively. When matrix pH <12 because of limited alkaline additives available, the required contact time with lime should be increased considerably: T99.9 for faeces was 4.0 h at pH 11 and 20.7 h at pH 10 (25°C). Thus, lime treatment is a promising measure where prolonged human waste storage is inappropriate in densely populated areas, as 1 day is sufficient to inactivate viruses. Nevertheless, further studies are necessary to investigate the contact time for >3 log inactivation of RNA virus.The original contributions presented in the study are included in the article/WO: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing – original draft. DS: Data curation, Supervision, Writing – review & editing.


Physostegia virginiana is an important ornamental and cut-flower plant in China. Its commonly used method of clonal propagation leads to virus accumulation in this plant. However, which viruses can infect the Physostegia virginiana plant remains to be illuminated. In this work, five viral pathogens in a Physostegia virginiana plant with virus-like symptoms of yellow, shriveled, and curled leaves were identified using RNA-seq, bioinformatics, and molecular biological techniques. These techniques allowed us to identify five viruses comprising one known alfalfa mosaic virus (AMV) and four novel viruses. The novel viruses include a virus belonging to the genus Fabavirus, temporarily named Physostegia virginiana crinkle-associated virus 1 (PVCaV1); two viruses belonging to the genus Caulimovirus, temporarily named Physostegia virginiana caulimovirus 1 and 2 (PVCV1 and PVCV2); and a virus belonging to the genus Fijivirus, temporarily named Physostegia virginiana fijivirus (PVFV). The genome sequences of PVCaV1, PVCV1, and PVCV2, and the partial genome sequence of PVFV were identified. Genome organizations and genetic evolutionary relationships of all four novel viruses were analyzed. PVCaV1 has a relatively close evolutionary relationship with five analyzed fabiviruses. PVCV1 and PVCV2 have separately a closest evolutionary relationship with lamium leaf distortion-associated virus (LLDAV) and figwort mosaic virus (FMV), and PVFV has a close evolutionary relationship with the five analyzed fijiviruses. Additionally, PVCaV1 can infect Nicotiana benthamiana plants via friction inoculation. The findings enrich our understanding of Physostegia virginiana viruses and contribute to the prevention and control of Physostegia virginiana viral diseases. Physostegia virginiana, also known as false dragon head flower, belongs to the Physostegia genus of the Lamiaceae family and is a perennial herbaceous plant. Physostegia virginiana is an important ornamental plant in many public gardens and a cut-flower plant in China because of its beautiful and colorful flower; it was also one of the eight flowers presented to winners at the 2008 Beijing Olympic Games . P. P13]. PSecoviridae were downloaded from the GenBank database for phylogenetic tree construction , DNA-binding protein (DNAb), CP, polymerase polyprotein (PP), and inclusion body protein (IB)  A,B 34]..Caulimovthan 80% , and basThe genome of PVCV1 began with the conserved tRNAMet sequence (5′-TGGTATCAGAGCC-3′), which served as a primer binding site and was complementary to the plant consensus sequence of the tRNAMet binding site. BLASTn search showed that the genome of PVCV1 had the highest homology of 48.55% with lamium leaf distortion associated virus  . ORF1 ofIn addition, the genome of PVCV1 contains a large intergenic region of 767 bp between ORF6 and ORF1, and a small intergenic region of 175 bp between ORF6 and ORF5. An RNA polyadenylation signal  and a negative-sense strand primer-binding site sequence  are present in the large intergenic region, while a TATA box  is present in the small intergenic region, as published previously ,43.https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 3 August 2022). BLASTp alignment showed that PVCV2 MP shared the highest aa identity of 57.84% with that of FMV  with that of figwort mosaic virus (FMV) . ORF1 oft of FMV . This MPt of FMV C. ORF2 ous genus . CDD anaus genus C. Previo.48% withus genus C. PVCV2 us genus . ORF4 ofus genus . Like PVus genus B,C. ORF5us genus . Like PVus genus C. ORF6 ous genus C and shaus genus .In addition, the genome of PVCV2 contained a large intergenic region of 857 bp between ORF6 and ORF1 and a small intergenic region of 31 bp between ORF6 and ORF5. An RNA polyadenylation signal  and a negative-sense strand primer-binding site sequence  were present in the large intergenic region, as published previously .Caulimovirus, as well as 14 representative members from the family Caulimoviridae in the GenBank database  with the P2 of maize rough dwarf virus (MRDV) (ORF1 of PVFV RNA1 was predicted to encode a RdRp of 168.5 kDa (hereafter designated P1) that shared the highest aa sequence identity (45.90%) with the P1 of southern rice black-streaked dwarf virus (SRBSDV) . Accordis (MRDV) . PVFV RNs (MRDV) . A putats (MRDV) . ORF5 ofs (MRDV) . The obts (MRDV) . PVFV RNs (MRDV) . The obts (MRDV) .Spinareoviridae, and tobacco mosaic virus (TMV) of the genus Tobamovirus from the NCBI database ; two new viruses belonging to the genus Caulimovirus, provisionally designated Physostegia virginiana caulimovirus 1 and 2 (PVCV1 and PVCV2); and a new virus belonging to the genus Fijivirus, provisionally designated Physostegia virginiana fijivirus (PVFV). The full-length genome sequences and genome organizations of PVCaV1, PVCV1, and PVCV2 and most genome sequences and genome organization of the nine RNA segments of PVFV were identified. This is the first time that fabavirus, caulimovirus, and fijivirus were identified in Physostegia virginiana plants. The results of the phylogenetic tree and viral protein aa identity analyses show that PVCaV1 is clustered in the branch of fabaviruses and has a close evolutionary relationship with BBWV1, BBWV2, CuMMV, LMMV, and GeMV (Physostegia virginiana viruses and contribute to effective strategies for the prevention and control of the viral diseases of Physostegia virginiana.In this work, we used RNA-seq, bioinformatics, and molecular biological techniques to investigate the viral pathogens of a and GeMV ; Table 1and GeMV . PVFV isand GeMV ; Table 4N. benthamiana plants through friction inoculation, but it cannot cause obvious virus-like symptoms in the infected N. benthamiana plants (Physostegia virginiana plant needs to be further studied.Among these five identified viruses, only PVCaV1 can infect a plants A. TherefPhysostegia virginiana plants include sowing, ramet, and cottage, whereas the ramet and cottage are the main propagation methods of Physostegia virginiana plants because of their low seed setting rate and fewer seeds. The frequently used clonal propagation method may lead to the accumulation of a variety of viruses in Physostegia virginiana plants. Thus, we believe that the phenomenon of multiple virus coinfection in Physostegia virginiana plants is closely related to its horticultural propagation mode. In the future, it is necessary to strengthen the virus quarantine, keep virus-free plants for reproduction, and control transmission vectors such as aphids and leafhoppers.The propagation methods of Caulimovirus genus can integrate their partial genome sequence into the genomes of their hosts [Physostegia virginiana plants remains to be fully explored using Southern blot hybridization and other molecular experiments. Furthermore, the full-length genome including RNA 6 and the genomic structure of PVFV need to be further determined, and the possible synergistic or antagonistic effects between these viruses need to be further elucidated. In addition, a virus field survey needs to be performed to clarify the prevalence of the five identified viruses in Physostegia virginiana plants in China and their host ranges, and the potential threats of these four new viruses to crops need to be assessed.Whether PVCaV1, PVCV1, PVCV2, and PVFV are transmitted by insect vectors in the field needs to be further verified. However, according to the published reports about viruses in the same genus ,46,47, wir hosts . Whether


Viruses produce a variety of illnesses, which may also cause acute respiratory syndrome. All viral infections, including COVID‐19, are associated with the strength of the immune system. Till now, traditional medicine or vaccines for most viral diseases have not been effective. Antiviral and immune‐boosting diets may provide defense against viral diseases by lowering the risk of infection and assisting rapid recovery. The purpose of this review was to gather, analyze, and present data based on scientific evidence in order to provide an overview of the mechanistic insights of antiviral bioactive metabolites. We have covered a wide range of food with antiviral properties in this review, along with their potential mechanism of action against viral infections. Additionally, the opportunities and challenges of using antiviral food have been critically reviewed. Bioactive plant compounds, not only help in maintaining the body's normal physiological mechanism and good health but are also essential for improving the body's immunity and therefore can be effective against viral diseases. These agents fight viral diseases either by incorporating the body's defense mechanism or by enhancing the cell's immune system. Regular intake of antiviral foods may prevent future pandemic and consumption of these antiviral agents with traditional medicine may reduce the severity of viral diseases. Therefore, the synergistic effect of antiviral foods and medication needs to be investigated. Regular intake of antiviral foods may prevent future pandemic and the use of antiviral food with traditional medicine may reduce the severity of viral diseases. The most hazardous viral infections include Ebola, Influenza, AIDS (acquired immunodeficiency syndrome), Dengue fever, and SARS (severe acute respiratory syndrome). Each year, for example, influenza causes roughly 3 million occurrences of severe illness and 300,000–500,000 deaths. Every year, the number of people diagnosed with viral infections rises rapidly due to point mutation, recombination, and reassortment mechanism of the viral genome, population growth, urbanization, the increasing ease of international travel of human and virus‐infected animals or arthropods, trans‐species transmission of the viral agent, monocultures of genetically identical individuals, drug resistance, deforestation, global warming, intensive farming practices, and degradation of the environment.Escherichia coli O157:H7, hantavirus, dengue fever, west Nile virus, and the zika virus  ‐like 2 (Nrf2) is the most powerful antioxidant and is able to inhibit the Angiotensin II Type I Receptor (AT1R) axis in particular  obtained in Kimchi demonstrated remedial activity against the H1N1 influenza virus. Park et al. (Lactiplantibacillus plantarum (DK119) also collected from Kimchi increases mouse resistance against the H1N1 influenza virus. Furthermore, Kimchi's Lactobacillus plantarum Ln1 strains have recently been found to have immune‐stimulating properties  and Bifidobacterium species (B. longum) have been demonstrated to have anti‐rotavirus effects in several investigations , CD8+, immunoglobulin (Ig)G+ and IgA+ B cells, T cells, neutrophils, as well as cytokines ‐2, IL‐12, and interferon gamma‐γ) are some of the mechanisms of action of kefir  is a glycoprotein that naturally occurs in various mucosal secretions. It has the ability to bind with iron, is non‐toxic, and has been found to possess immunomodulatory and anti‐inflammatory properties. LF is crucial in the initial line of defense against microbial infections  plant is well‐known for its medicinal properties. Antiviral qualities are conferred by the presence of the active plant component carvacrol, which protects against viral infection. Oregano oil has protective activity against several viruses such as the HSV type 1, rotavirus, and respiratory virus, according to several researches  are its powerful antiviral, anti‐inflammatory, antioxidant, and antibacterial capabilities  is a licorice‐flavored herb that triggers immunity, reduces body's inflammation, and fights viral infection. Trans‐anethole, one of the active compounds in fennel seeds, is known for fighting against herpes viruses  leaves are mainly composed of menthol, menthone, neomenthol, and iso‐menthone, with strong anti‐inflammatory, antibacterial, antiviral, scolicidal, immunomodulatory, antitumor, neuroprotective, antifatigue, and antioxidant activities , is a weed that can be used as a tonic, anti‐inflammatory, antioxidant, and diuretic in the medication of a variety of disorders, including pneumonia, sore teeth, stomach discomfort, tonsillitis, wing worms, pain, inflammation, fever, and tumors  plant has antiviral activity against a variety of viruses, including the coronavirus SARS‐CoV‐1, HSV type 1, HSV type 2, Hemorrhagic Viral Rhobdavirus Septicaemia (VHS), HIV, Varicella‐Zoster virus, Influenza virus, Cytomegalovirus, poliovirus, and Human papillomavirus  is one of the polyphenolic substances whose antiviral activity is based on their antioxidant properties, as well as their ability to inhibit enzymes, break cell membranes, block viral binding and penetration into cells, and activate the host cell's self‐defense systems  experiments  possess a powerful antiviral agent whose every day consumption can be a great way to improve one's immune system  is a prominent medicinal plant found throughout the southern Asian continent  and its aqueous extracts might be useful in preventing foodborne virus contamination, especially for those foods that are commonly not processed sufficiently before consumption  contains a huge number of bioactive compounds filled with medicinal qualities. It contains active elements like curcumin a phytochemical that possesses strong antioxidant, anti‐inflammatory, and antiviral activities. Regular intake of turmeric milk can trigger immunity response and help combat viral infections  is a nutritious herbaceous plant that has been utilized in the treatment of respiratory diseases, diabetes, hypertension, inflammation, and influenza since ancient times  is a common culinary ingredient that has been quite useful as a home remedy for millennia in different countries. Cinnamaldehyde, cinnamyl alcohol, coumarin, cinnamic acid, and eugenol are the main components of cinnamon bark , has been utilized as a traditional cure in various cultures from ancient times. Licorice root is regarded as a life‐enhancing substance, and several researches has revealed that it has a wide range of pharmacological properties  includes a large number of bioactive chemicals that have been demonstrated to be beneficial to human health, including antiviral, anti‐inflammatory, antipyretic, and immunological effects. The principal phytochemicals found in peppers are alkaloids, terpenes, flavonoids, steroids, etc. , vesicular stomatitis Indiana virus (an enteric virus), and human parainfluenza virus (a respiratory infection causing virus)  is a medicinal and nutritious plant with a wide range of applications. All components of the tree i.e., bark, root, leaf, seeds, pod, gum, fruit, and flowers are rich in vitamins, antioxidants, minerals, proteins, nitrile glycosides, and phenolics, but the leaves are the most nourishing , and polypore mushrooms efficiently fought off the flu‐causing influenza viruses, according to Lipipun et al. , ganoderans, ganopoly, and protein LZ 8 are immune modulators found in cauliflower mushrooms and Reishi, and Lingzhi mushrooms, respectively.By restricting virus adsorption onto cells, inhibiting viral nucleotide synthesis enzymes, and boosting cellular immunity, several mushrooms, including 2.5Functional foods are now generally recognized for the prevention and treatment of major non‐communicable diseases (NCDs), particularly those characterized by inflammatory and oxidative stress‐related disorders like diabetes and cardiovascular disease. A variety of oily fish, fruits, vegetables, nuts, olive oil, and legumes are all regarded as functional foods since they include natural levels of nutraceuticals such as terpenoids, polyphenols, flavonoids, sterols, pigments, alkaloids, and unsaturated fatty acids. It has been suggested that bioactive peptides, which are inherently present in fruits and vegetables are developed as nutraceuticals according to their amino acid chain length, molecular weight, or peptide composition, which can cause a broad spectrum of physiological responses linked to the gastrointestinal, cardiovascular, neurological, and other hormonal functions of the human body , the Nordic diet, and its integration with other lifestyle strategies. All necessary nutraceuticals with disease‐preventive, anti‐inflammatory and antioxidant properties may be found in fermented foods, dairy products, teas, tree nuts, and olive oil. Olive oil contains monounsaturated fatty acids (MUFAs), such as oleic acid. Tree nuts like walnuts contain polyunsaturated fatty acids (PUFAs), notably alpha‐linolenic acid. Docosahexaenoic and eicosapentaenoic acids are present in fish oil. Fruits and vegetables contain significant levels of flavonoids, polyphenols, and antioxidants. The prevalence of such functional foods and their constituent parts vary by region, but their cardiometabolic preventive effects in lowering key NCDs and fatality risks are widespread. By enhancing and protecting the immune system and having antiviral defensive actions, the challenge is to transform these functional properties into the protection of new CDs like COVID‐19  as well. Trans‐ and cis‐isomeric versions of resveratrol are available. Resveratrol was shown to significantly limit MERS‐CoV replication in vitro by inhibiting RNA synthesis, as well as having additional pleiotropic effects. Studies have shown that resveratrol and indomethacin are useful supplements for SARS‐CoV‐2 ; visualization ; writing – original draft (lead); writing – review and editing . Md. Nahidul Islam: Conceptualization (lead); project administration (lead); writing – original draft (lead); writing – review and editing . Md. Wahiduzzaman: Visualization (lead); writing – review and editing (supporting). Mahabub Alam: Writing – review and editing . Akib Atique Khan Dhrubo: Writing – review and editing .The authors declare no conflict of interest.No ethical approval was required for this article since no human or animal study was Conducted.


The paper presents virophages, which, like their host, giant viruses, are “new” infectious agents whose role in nature, including mammalian health, is important. Virophages, along with their protozoan and algal hosts, are found in fresh inland waters and oceanic and marine waters, including thermal waters and deep-sea vents, as well as in soil, plants, and in humans and animals (ruminants). Representing “superparasitism”, almost all of the 39 described virophages (except Zamilon) interact negatively with giant viruses by affecting their replication and morphogenesis and their “adaptive immunity”. This causes them to become regulators and, at the same time, defenders of the host of giant viruses protozoa and algae, which are organisms that determine the homeostasis of the aquatic environment. They are classified in the family Lavidaviridae with two genus . However, in 2023, a proposal was presented that they should form the class Maveriviricetes, with four orders and seven families. Their specific structure, including their microsatellite (SSR-Simple Sequence Repeats) and the CVV  system described with them, as well as their function, makes them, together with the biological features of giant viruses, form the basis for discussing the existence of a fourth domain in addition to Bacteria, Archaea, and Eukaryota. The paper also presents the hypothetical possibility of using them as a vector for vaccine antigens. Virophages, like their “host” giant viruses, are new infectious agents in the viral world. Their role in nature, mainly in the aquatic environment, but also in the context of human and livestock health, is essential ,4,5,6,7.Lavidaviridae [Maveriviricetes, with four orders and seven families. Like giant viruses and “classical” viruses, virophages are incapable of independent replication, and their process occurs in the viral particle factory of the giant viruses in which they parasitize. They can form provirophages by integrating their genomes with these viruses and host cells [Their destructive effect on giant viruses occurs through their genetic “action”, e.g., tRNA, which influences the replication and morphogenesis of giant viruses, as well as their “adaptive immunity”, making them regulators and, at the same time, defenders of giant virus hosts, which are protozoa and algal organisms that primarily condition and shape the homeostasis of the aquatic environment ,6,7,9,10aviridae ,13,14,15st cells ,13,14,15st cells ,16,17,18A. polyphaga, A. castellani) and flagellates (Cafeteria roenbergensis—now C. burkhardei) or the giant virus, virophage, retrotransposon system in the unicellular eukaryote Bigelovatella natans [Additionally, associated with the discovery of virophages is research into a three-part system termed CVV (cell–virus–virophage), which is formed by giant virus host cells, a giant virus, and a virophage, although an analogous relationship occurring between giant viruses, virophages, and transposons has also been described ,9,18,19.a natans ,21,22,23a natans ,25,26.An analysis of virophage genomes has shown that their dsDNA is 10–42.3 kbp long. Their genomes contain 16-to-34 genes which encode 12–39 predicted proteins that are mostly differentially expressed ,18. All On the other hand, the tRNAs present in virophages may serve to complement the host codon or help virophages utilize amino acids during their replication . It has Among the 39 virophages described thus far , only 22Mamavirus ACMV (Acanthamoeba (A) castellani mamavirus) resembles the giant Mimivirus AMPV (Acanthamoeba polyphaga mimivirus), which belongs to the genus Mimivirus, family Mimiviridae, and of which ACMV has been found in water from the Bradford cooling tower. There, small virions have been detected. They have been named after the Earth’s first satellite, the virophage Sputnik, meaning “travelling companion” [During observations in 2008 by transmission electron microscopy of a viral particle factory of a giant virus, mpanion” ,26,42,43Sputnikovirus and the family Lavidaviridae [This virophage was initially classified into satellite viruses ,26,44,45aviridae ,15,28. Taviridae ,24,26,42aviridae ,26,44,46This MCP protein assembles pseudohexameric and pentameric capsomeres to form the outer shell of the capsid of this virophage . It has APMV giant virus [APMV giant virus. One is a homologue of the integrase of archeon viruses, and the other four are a predicted primase-helicase, an ATP-packing ATP-ase (homologue in bacteriophages and eukaryotic viruses), a distant homologue of the bacterial insertion sequence of the DNA-binding subunit of transposase, and the Zn ribbon protein [The organization of the Sputnik genome is typical of viral genomes, namely a tight arrangement but little overlap of the genes. The genome virophage contains 21 genes encoding proteins ranging in size from 88 to 779 amino acids, with only slight overlap ,26,44,48nt virus ,26. Withnt virus . In cont protein ,26. Thre protein ,26, exce protein ,26.Furthermore, g06 and g07 encode a protein containing a highly conserved collagen triple helix motif, while g13 encodes a protein consisting of two domains involved in viral DNA replication ,26. It hAdditionally, g04 encodes a Zn ribbon protein but lacks highly conserved homologues ,26, whilMimiviridae is the giant Mamavirus ACMV, which infects the amoebae A. castellanii [Mimivirus APMV, of the same genus and family but living on the amoeba A. polyphaga [Mamavirus ACMV filaments are essential elements [Mamavirus ACMV, as its replication has not been detected during coinfection with its naked form [ACMV giant virus results in the formation of its defective virions, by which its replication efficiency is reduced by up to 70%. Such an effect on ACMV giant viruses determines its protection against amoebae [For the Sputnik virophage, the dominant host among viruses of Lineages A, B, and C of the family tellanii ,42,48,49olyphaga ,21,49. Iolyphaga . These folyphaga . The threlements , as the ked form . The Spuked form ,33,46,50 amoebae ,42,46,50 amoebae .Lentillevirus of the family Mimiviridae that parasitized the amoeba A. polyphaga, which was isolated from a contact lens solution belonging to a 17-year-old patient with keratitis [Sputnikovirus, family Lavidaviridae [Mimiviridae family of Lineages A, B, and C, causing their destruction, thus showing a protective effect against their amoeba hosts cell [Lentillevirus, a provirophage integrated with it was also found [Lentillevirus and the amoeba A. polyphaga, which is an example of a tripartite CVV system, with a novel transpovirion forming the CVV system and consisting of the transpovirion, the virophage, and the giant virus parasitizing the amoeba [Virophage Sputnik 2 was described in 2012 in a giant virus of the genus eratitis ,26,43,49aviridae ,43,49. Taviridae ,25,26. Iaviridae ,25,26. Tsts cell . With thso found . It is te amoeba ,42.Mimiviridae family C-lineage giant viruses. Although, as with the Guarani virophage, it has been shown that this virophage can be “free” of the giant virus [Mimiviridae family—Lineages A, B, and C, including APMV giant viruses parasitizing the amoebae A. polyphaga [Mimivirdae family, mainly of Lineage C, although also of Lineages A and B, causing the formation of abnormal virions of these giant viruses and reducing their infectivity and lytic capacity against their hosts cells, which are amoebae [Sputnikovirus, the family Lavidaviridae. However, it differs from them by less than 10 base pairs, although all these virophages have a low G+C content (approximately 30%), which is also typical of giant viruses of the family Mimiviridae [This virophage was described in 2013  in a soint virus . This stolyphaga ,17,25,26olyphaga ,24,43. Tolyphaga ,17,25,26olyphaga . Similarolyphaga ,25,26. Tolyphaga ,26. This amoebae ,26,48. Liviridae ,15,28.Mimivirus argentum, on which it parasitizes and whose host is probably the amoeba A. castellani [Mimiviridae family Lineages A, B, and C [Gp2, Gp5, Gp11, Gp12, and Gp16. In comparison, the remaining 75% of the genome encodes proteins containing DNA-binding domains, proteins related to its morphogenesis, and proteins containing triple helix repeats, among others [This virophage was described in 2022. Its name is derived from the giant virus stellani . It is aB, and C  and is cB, and C . Its genB, and C . It has g others .Cafeteria (C) roenbergensis virus), genus Cafeteriavirus, family Mimiviridae infecting the unicellular phototrophic marine flagellates C. roenbergensis [C. roenbergensis cells with CroV giant virus co-occurs, the expression of Mavirus virophage genes is activated, leading to the replication and synthesis of its virions from proviruses [C. roenbergensis flagellate cells transporting these provirophages not being directly protected against giant virus-CroV infection. However, when infection with this virus occurs and Mavirus provirophages are released in subsequent coinfections, they inhibit giant virus-CroV replication and protect the C. roenbergensis flagellates on which these viruses parasitize [C. roenbergensis worms by proviruses against the CroV giant virus appears to take place in an altruistic model, as some cells are sacrificed to protect others [C. roenbergensis has also been demonstrated, providing the Mavirus virophage with the opportunity to exist as a provirophage. In contrast, these flagellate populations benefit from the Mavirus virophage infecting CroV giant virus [The Mavirus virophage was obtained in 2010 from the coastal waters of Texas, USA, from the giant virus, CroV  of the Phycodnaviridae family, it was shown that as many as 7408 bp of the OLV virophage encodes g17–22 proteins, similar in 32–65% to sequences in the OLPV-1 and OLPV-2 regions of the Phycodnaviridae family giant viruses [Phycodnaviridae family and infecting unnamed phototrophic marine algae, the OLV virophage influences their growth and abundance, thus playing a pivotal role in regulating the microbial network of organic lake waters [Furthermore, by studying the OLV virophage, six genes are linked to genes of giant viruses of the nfection ,29,37. T viruses . This vi viruses . By infeA. castellanii infected with Samba giant virus, genus Mimivirus, family Mimiviridae Lineage A [The RNV virophage was the first virophage discovered in Brazil in 2011 in the waters of the Negro River in Amazonia and was found in the amoeba ineage A ,25,30. TA. castellanii amoebae, causing its defective shape and abnormal capsid and reducing the abundance of this giant virus in amoebae by more than 80% [It is also indicated that the sequence of the MCP protein gene of the virophage RNV is also partly identical to the gene encoding the MCP protein of the virophage Sputnik ,30. Thisthan 80% .Phaeocystis globosa virus), genus Prymneovirus, family Phycodnaviridae, infecting algae of the genus Phaeocystis residing in the Dutch coastal waters of the North Sea [Phaeocystis globosa—14T (PgV-14T), together with the giant virus Phaeocystis globose, are probably new virophages that have been named PLV “Gezel–14T”, which are different from all known virophages but also have a destructive effect on specific giant viruses [The PGVV virophage was obtained in 2013 from the giant virus PgV–16T , was obtained from the waters of artificial Dishui Lake in Shanghai, China; it has also been found in other freshwater bodies ,34,40,56er algae . Metagenaviridae ,34,51,52aviridae .Phycodnaviridae, parasitizing unnamed freshwater algae [g19), and DNA helicase/primase/polymerase (g23) [Phycodnaviruses , which are known to infect unicellular green algae [Phycodnaviridae is evidence that it replicates in these viruses [This virophage was identified in 2016 in the surface waters of Qinghai Lake in the mountains of Tibet, which is rich in a planktonic microbial community ,35. Thisse (g23) ,35. In aen algae ,35, and  viruses .Mimivirus, family Mimiviridae, which parasitizes the amoeba Saccamoeba lacustris, which lives in sycamore trees [comedere—to eat, devour [This virophage was found in 2018 in the giant virus KSLT-5, probably belonging to the genus re trees . This vire trees . When in, devour .Phycodnaviridae, which infects the freshwater alga Chrysochromulina parva, living in the waters of Lake Tai in China and Lake Erie in the United States [These three virophages  were described in 2019 along with the CpV–BQ2 giant virus from the family d States ,36. InitIn contrast, the CpV–PLV Moe virophage was shown to be very similar to the CpV–PLV curly virophage and, despite having a genome of 21,750 bp with only 30.1% G+C content, encodes 23 genes . MeanwhiChlorella virophage, CVV–SW01, residing in the giant Chlorella virus XW01 (CV-XW01) of the family Mimiviridae, which parasitizes algae of the genus Chlorella, was obtained from the waters of Lake Dishui [Bigelovatella natans [In 2022, a e Dishui . Describe Dishui ; this haa natans ,22,23. TCafeteria or the first species of a new genus closely related to it. It should be added that despite the close relationship between the two giant viruses, CV–XW01 and CroV, their virophages are poorly related. Given these facts, it is suggested that the interaction of the virophage CVV–SW01, the giant virus CV–XW01, and the alga Chlorella sp. is likely to be different from the interaction of the virophage Mavirus—giant virus CroV—the flagellate C. roenbergensis (now C. burkhardaei) [Furthermore, as many as 82 genes of this virophage’s CV–XW01 giant virus host show homology with the CroV giant virus, which is most closely related . It shouhardaei) . Notablyhardaei) . It has hardaei) .The virophages YSLV 5, YSLV 6 and YSLV 7 were identified by metagenomic analyses in 2013 in water samples from Yellowstone Lake, USA, without identifying a giant virus for them and their host cell; they share genetic homology with the virophage Zamilon ,37. The The Virophage Zamilon 2 was described in 2015 as homologous to the virophage Zamilon, which was recorded in the metagenome of a bioreactor of poplar wood material (sawdust) in New York State, USA, already in 2012 ,17,37,43Tectiviridae family and archaea viruses [These virophages were already recorded in 2009 and 2012 but were only described in 2017, presenting them as 25 new “non-breeding” virophages, including 17, presumably, complete genomes . They we viruses . It shou viruses .Phycodnaviridae infecting unspecified freshwater algae, although possibly also on giant viruses of the genus Mimivirus infecting amoebae [Phaeocystis [Mimivirus giant viruses [Virophages DSLV 2–8 were described in 2018 as seven complete genomes already recorded in 2016 in the freshwater of Dishui Lake, Shanghai, China, which reside on various giant viruses of the family  amoebae ,40. Thei viruses .Mimivirus, for which no host cell has been identified [Mimivirus giant viruses [Klosneuviruses (LCMiAC01 and LCMiAC02) do not contain equivalent LCV virophage promoters, it has been suggested that it is possible that their host cell could be not only Mimivirus giant viruses but also Pitoviruses, for which, as for Mimiviruses, no host cell has been identified [Researching the metagenome of marine sediment from the Loki Castle hydrothermal vent area—named after the Norse God of Fire located on the Mid-Atlantic Ridge in the Arctic Ocean—two Sputnik-like virophages, LCV 1 and LCV 2, were described in 2019 without specifying their genome and shape and capsid symmetry . A phyloentified . In addientified . It is i viruses ,16,25. IMimiviridae family from the A, B, and C lineages [Tupanovirus [ACMV giant virus in the amoeba A. castellanii, it was shown that its long replication cycle is linked to its delayed replication associated with the utilization of the late promoter of this giant virus, which starts at the latest stage and extends to the final stages of its morphogenesis process [Mimiviridae family Lineages A, B, and C, affects their replication and infectivity, resulting in a decrease in their abundance by up to 90% and consequently increasing the survival of the amoebae and protists on which these giant viruses parasitize [This virophage was described in 2019 in freshwater samples collected in the Pampulha lagoon in Belo Horizonte, Brazil, and, like the virophage Sputnik 3, it may be “free” of its specific giant virus ,17,25. Tlineages , during anovirus . Evaluat process ,14,25. T process . Its genrasitize .This virophage was recorded in 2019 in Tunisia but has not been fully described [In the form of 32 genomes, these virophages were described in 2021 in the waters of the oligotrophic alpine lake Gossenköllesee in 2021 in Austria, although they had already been recorded in 2017 and 2018 . These vMimivirus giant viruses on humans has been demonstrated, as they have been registered, among others, as an infectious agent in patients with CAP (community-acquired pneumonia), and antibodies to collagen encoded by these viruses are involved in human rheumatoid arthritis [The effect of virophages on the human body is not yet known. However, the effect of rthritis ,20,43,48rthritis ,43 that rthritis . In thesrthritis ,43 at thMoreover, the replication and expression of the virophage genome leads to reverse transcription of viral and virophage RNA ,43, prev


Host-virus interactions are complex cross-talk between the virus and its host cell. These interactions are not limited to causing disease or changing the physiology of host cells but they also influence viruses. Studies showed bidirectional changes in viruses and their host which co-evolved together , 2. The The presence of virus in the host body does not always cause diseases. A majority of viruses present in the host body are commensal . Host boThere are several pieces of evidence showed that the virus and its host can benefit each other. A study with tropical panic grass, fungal endophyte, and fungal virus showed a three-way symbiosis. Where virus-infected fungus confers heat tolerance to both, tropical panic grass and fungal endophyte. Virus-free fungi were unable to confer heat tolerance, but heat tolerance was restored after the virus is reintroduction to fungi . SimilarListeria monocytogenes and Yersinia pestis by activating the host immune response  or small interfering RNAs (siRNAs) which silence the viral gene in subsequent infection , 37, 38.the host , 40. Anothe host . Cellulathe host . Studiesthe host . Howeverthe host .Translocation of virus particles to the host cell is the first step in virus infection. This translocation is initiated by the interaction of distinct molecules present on the outer layer of the virus with specific receptors on the host cell . This inThe outcome of viral disease is greatly influenced by viral factors such as virulence and host factors like susceptibility. Broadly, damage in the host during virus infection can be divided into two, (A) direct damage by the virus when virus replicates in the host cell and cause damage in cells either by hijacking cellular machinery, changing cellular physiology, or damaging cellular components by its structural or non-structural proteins –63. (B) Host cells have several inherent antiviral factors such as antiviral proteins which control virus replication. A few of the antiviral proteins are APOBEC3G , ZAP , SAMHD1 (SAM domain and HD domain-containing protein 1), 2′-5′-Oligoadenylate Synthetase (OAS), Mx Proteins and Tetherin, which is also known as BST-2  –75.APOBEC3G exhibits its antiviral effect by inducing hypermutation in viral genomes. APOBEC3G catalyzes the deamination of cytosine (dC) to uracil (dU) in single-stranded DNA, leading to non-functional viral DNA and prevents virus replication . While ZSAMHD1, another cellular protein exhibits its antiviral effect by hydrolyzing nucleotide triphosphates (dNTPs), dNTPs are needed by the virus to synthesize its genome while the lower concentration of dNTP in cells suppress the virus replication . However2′-5′-Oligoadenylate Synthetase (OAS), activates RNase L, an endoribonuclease that cleaves single-stranded viral RNAs and suppresses its replication , while MHost-virus interaction studies provide valuable information about viruses, and their molecular characteristics which facilitate them to mutate, change their virulence, and shift host range. Additionally, these studies help in understanding the effect of virus infection on host cells, cell organelles, and the physiological/metabolic activity of the cells. These studies also help in identifying the cellular molecules which could have antiviral properties. These molecules could be used to suppress the broad range of pathogenic viruses.MR: Writing—original draft, Writing—review and editing. NT: Writing—original draft, Writing—review and editing.


Oncolytic viruses have many anticancer strategies. Genetically modified viruses may more effectively infect cancer cells than normal cellsNumerous preclinical studies have shown the treatment’s safety, and evidence of both a local and systemic antitumor effect has emerged. Using CF33, it would be able to enhance the clinical advantages and quality of life for patients with malignancies that are now untreatable with standard therapy methods. Phase 1 clinical studies will use CF33-hNIS, a new chimeric orthopox virus, either as monotherapy or in combination with pembrolizumab to evaluate the safety and effectiveness of the treatment regimens and the immunological alterations in the tumor microenvironmentGene therapy Talimogene Laherparepvec (T-VEC) overexpresses granulocyte macrophage colony-stimulating factor by using an attenuated herpes simplex virus 1 virus, which is then used in dendritic cell-mediated antigen presentation studies. Patients with advanced, nonresectable melanoma who received intratumoral granulocyte macrophage colony-stimulating factor alone had a lower rate of long-term response compared to those who received T-VEC+ T-cell infiltration and interferon-gamma signaling and by upregulating PD-L1 in the microenvironmentOncolytic virus injection may enhance the effects of checkpoint inhibitors by enhancing CD8In addition to the encouraging findings from the preclinical studies, we are very excited to explore the possibilities presented by the oncolytic virotherapy platform comprised of VAXINIA and T-VEC.This case report has been reported in line with the Case Report (CARE) guidelines.Not applicable.None.R.A.: conceptualization, data curation, writing – original draft preparation, writing – reviewing and editing. B.R.S., P.P., and A.R.: data curation, writing – original draft preparation, writing – reviewing and editing. T.B.E.: writing – reviewing and editing, visualization, supervision. All the authors confirm the concept, design, data collection and analysis and interpretation, and writing are our own. The authors confirm there are no other contributors.The authors declare that they have no financial conflict of interest with regard to the content of this report.None.https://orcid.org/0000-0003-3188-2272. E-mail: talhabmb@bgctub.ac.bdTalha Bin Emran, PhD, Associate Professor, Department of Pharmacy, BGC Trust University Bangladesh, Chittagong 4381, Bangladesh. Tel: +880 303 356 193, Fax: +880 312 550 224. Not commissioned, externally peer reviewed.