Drosophila melanogaster has one of the best characterized antiviral immune responses among invertebrates. However, relatively few easily transmitted natural virus isolates are available, and so many Drosophila experiments have been performed using artificial infection routes and artificial host–virus combinations. These may not reflect natural infections, especially for subtle phenotypes such as gene expression. Here, to explore the laboratory virus community and to better understand how natural virus infections induce changes in gene expression, we have analysed seven publicly available D. melanogaster transcriptomic sequencing datasets that were originally sequenced for projects unrelated to virus infection. We have found ten known viruses—including five that have not been experimentally isolated—but no previously unknown viruses. Our analysis of host gene expression revealed that numerous genes were differentially expressed in flies that were naturally infected with a virus. For example, flies infected with nora virus showed patterns of gene expression consistent with intestinal vacuolization and possible host repair via the upd3 JAK/STAT pathway. We also found marked sex differences in virus-induced differential gene expression. Our results show that natural virus infection in laboratory Drosophila does indeed induce detectable changes in gene expression, suggesting that this may form an important background condition for experimental studies in the laboratory. Drosophila melanogaster is among the best characterized of any invertebrate, and antiviral responses in Drosophila are mediated by pathways that are conserved across many taxa [The antiviral immune response of any taxa . These iany taxa ,5,6,7,8.D. melanogaster infected with Drosophila C Virus (DCV) displays increased expression of genes such Spaetzle (Spz) and Drosomycin (Drs) that encode a cytokine and an antimicrobial peptide, respectively, and which are important in the toll pathway [listericin (CG9080) [Virus infection may be associated with dramatic changes in gene expression. First, infections trigger host signalling cascades that eventually alter the expression of host immune effector molecules. For example, wild-type  pathway . There i(CG9080) . Second,(CG9080) .Drosophila expression response to viral infection has come from transcriptome profiling studies e.g., [Much of what is known about the es e.g., ,12,13,14es e.g., ,16,17. Tes e.g., . The roues e.g., ,19. Remaes e.g., .Drosophila model. Such interactions can be highly host-specific [D. melanogaster, whereas it can suppress it in D. immigrans [Drosophila have used non-native viruses such as Flock House Virus , Sindbis, e.g., ), Inverts, e.g., ), and Crs, e.g., ).Drosophila has revealed over 120 naturally occurring fruit fly viruses spanning more than 25 families [Drosophila melanogaster viruses had available isolates; only the RNA viruses DCV, D. melanogaster Sigmavirus (DmelSV), Drosophila A virus (DAV), Drosophila X virus (DXV), nora virus, and the DNA virus, Drosophila Kallithea nudivirus virus [Despite the fact that metagenomic sequencing of wild families ,28,29,30us virus ,31,32,33us virus ,35, but Drosophila [Some of these viruses are known to occur in laboratory flies and cell culture. The most common viruses reported from laboratory fly stocks include DCV, nora virus, DAV, Newfield virus, DMelSV, and Thika virus . In addiosophila ,37,38,39osophila ,39,40,41D. melanogaster to quantify the prevalence of viruses in experimental studies, and to assess the impact of viruses on patterns of host gene expression. As these infections were incidental and unintended by the original authors, they reflect natural laboratory infection routes and host–virus combinations. The resulting changes in gene expression are suggestive of a reduction in host reproductive investment, and nora-virus-induced gut pathology and host repair. These results imply that background changes in gene expression due to viral infection may be relevant to laboratory experiments.Here, we survey seven publicly available adult transcriptome project datasets from laboratory D. melanogaster RNAseq ‘projects’ that each comprised at least 130 sequencing libraries, and downloaded them from the European Nucleotide Archive [We initially selected nine  Archive , Table 1 Archive ,44, the  Archive , the uti Archive , and the Archive . Two proD. melanogaster or to well-studied contaminating cellular organisms such as bacteria, fungi, trypanosomatids, and microsporidia  using Borted in . We thenorted in , and retorted in . Clusterorted in , retainiorted in  and all orted in . Contigsorted in . To mitiorted in , each viD. melanogaster genome  using tFEB2019,  and counFEB2019, . We veriTo test if there is an interaction between co-infecting viruses, we generated a contingency table of the total number of infected and uninfected libraries for each pair of viruses in each dataset. We then tested for statistical significance of association between the viruses using Fisher’s exact test in the R stats package .We quantified virus prevalence as the proportion of sequencing libraries within each published project for which the virus read number passed our presence/absence threshold . We then assumed binomial sampling to obtain confidence intervals for the proportion of affected libraries in each project. To explore the phylogenetic relationships between laboratory viruses and previously published virus sequences, we selected the sample within each project that had the highest virus read number for each virus. For galbut virus, which has two distinct strains, we selected the sample with the highest read number for each haplotype. We then performed phylogenetic analyses using the sequences from these samples and incorporating all the other sequences from each virus available in GenBank . For anaWe performed a differential gene expression analyses using ‘Dream’ , an R pap-value < 0.001 (to account for multiple testing) was used to indicate statistical significance. The genes with significant changes in expression from the differential expression analysis were subsequently analysed for gene ontology enrichment using VISEAGO [If the virus was present in only one sex in a mixed-sex project dataset, or the infected project datasets had only one sex, then the sex term and its interaction with virus were excluded. If a virus was present in only one dataset, then the dataset term was excluded from the model, and if the dataset had only one genotype, then the genotype term was also removed accordingly. Fly age was not incorporated into the models because these datasets are from published studies that do not report fly age. Cut-off log-fold changes, logFC > 0.5 and logFC < −0.5, were used to define increased and decreased expression, respectively, and a Benjamini–Hochberg adjusted  VISEAGO , which d VISEAGO .To compare the effects on the host by viral infection post hoc, we calculated a Pearson correlation matrix between the estimated changes in gene expression in response to viruses and their interactions with sex using the rcorr function in the Hmisc package  in R 6969.p-value = 2.2 × 10−16), consistent with a common source (Partitiviridae), Motts Mill virus (Solemoviridae), DAV (Permutotetraviridae), DCV (Dicistroviridae), nora virus , Dmel sigma virus (Rhabdoviridae), Bloomfield virus (Reoviridae), Craigie’s Hill virus (Nodaviridae), Thika virus , and Brandeis virus (cf. Negevirus). Out of a total of 2586 libraries across the seven datasets analysed, 625 (24%) were infected with only one virus and 104 (4%) were infected with two or more viruses. Using a p-value < 0.01 as a test of significance, we found that co-infecting viruses were independent of each other in the datasets used in this study, except for galbut virus and Motts Mill virus in PRJNA258012  and PRJNA281652 , DAV and Thika virus in PRJNA261333 , DAV and DCV in PRJNA281652 , and DAV and nora virus in PRJNA518903 . However, the number of coinfections was small; for example, in PRJNA258012, 49 (6.8%), and 171 (23.9%) libraries were infected with Motts Mill virus and galbut virus, respectively whilst one (0.1%) library was infected with both viruses. Similarly, in PRJNA281652, 45 (9.8%) and 79 (17.2%) libraires were infected with DCV and DAV, respectively whilst 17 (3.7%) libraries were infected with both viruses. In PRJNA518903, 10 (1.3%) and 31 (4%) libraires were infected with DAV and nora virus, respectively whilst four (0.5%) libraries were infected with both viruses. Finally, in PRJNA261333, 24 (6.1%) and 39 (9.8%) libraires were infected with DAV and Thika virus, respectively whilst seven (1.8%) libraries were infected with both viruses. Thus, the linear model should successfully disentangle the effects of the viruses, and any (unmodelled) interaction between viruses would only add to the residual variance.We examined a total of nine publicly available RNAseq projects, but two of these contained no viral reads and were excluded from further analyses . One pron source . We therSome samples displayed an extremely high virus titre . Most exDrosophila expression in response to these pathogens for the first time.Of these 10 viruses, nora virus had the highest prevalence, detectable in five projects, with a prevalence of up to 53%  is involved in synapse organisation [CG4676 and Apl are involved in protein transport and localisation [Ets21C, CG16995, and CG18649, were significantly upregulated  that is involved in mitosis [Of the 454 genes that displayed a logFC of more than 0.5 in response to Craigies Hill virus infection, only 4 were nominally significant , which is involved in gut epithelium repair via the JAK/STAT pathway was also significantly upregulated in response to nora virus infection. Numerous immune response genes were significantly upregulated in response to nora virus infection. These include Nazo, which encodes an antiviral effector protein that is expressed downstream of Sting and relish antiviral response [DptA and DptB, which both encode antimicrobial peptides regulated by the ImD pathway [Mtk, which encodes an antifungal peptide regulated by the ImD and Toll pathways [Of the 340 genes that showed a logFC of more than 0.5 in response to nora virus infection, 330 were significant malpha-BFM, which are involved in chitin binding and development [Of the 43 genes that showed a logFC of more than 0.5 in response to DAV, 12 were significant  between the DAV, galbut virus, and Motts Mill infection, although none were significant in all three  between the galbut:sex and Motts Mill virus:sex. That is, the effect of sex on virus infection induced similar changes in the expression of 209 genes in galbut-virus- and Motts-Mill-virus-infected flies, with four genes being nominally significant in both infections. These genes include three long, non-coding RNA, lncRNA:CR40465, lncRNA:CR44631, lncRNA:CR44289, and Yp2, which encodes the major yolk protein of eggs.To determine whether changes in gene expression in response to virus challenge were consistent across viruses, we compared the inferred changes between each of the viruses . The magll three . ChangesThere were no significantly enriched GO terms among the genes that changed expression in response to DmelSV, Bloomfield virus and DCV. Six GO terms related to cell proliferation were significantly enriched in Craigies-Hill-virus-infected flies . In geneDrosophila gene expression changes in response to virus infection either use non-native viruses or inject natural viruses into the host—which may not be a good reflection of what happens in nature. To understand how Drosophila gene expression changes in response to native virus infection, we analysed seven publicly available RNA-seq datasets that were serendipitously infected with ten different viruses.Most studies of how D. melanogaster suggested that the average global prevalences of nora virus and DAV is about 7% each [D. melanogaster collected from Athens . The prevalence of galbut virus in this study was 14%, which is much lower than has been reported in wild flies, where the prevalence of galbut is often over 50% [Drosophila to better understand host–virus interactions.The viruses found in this study were galbut virus, Motts Mill virus, DAV, DCV, nora virus, DmelSV, Bloomfield virus, Craigie’s Hill virus, Thika virus, and Brandeis virus. The most prevalent viruses in the datasets analysed were nora virus and DAV, with prevalences of up to 52% and 18%, respectively. This is higher than has been observed in wild flies; a study of wild  7% each , althougover 50% ,35. The Drosophila STING interacts with lipid-synthesizing enzymes such as acetyl-CoA carboxylase [Drosophila S2 cells [In response to DAV and nora virus infection, we found evidence of increased expression of immune genes involved in the antiviral STING pathway and changes in the expression of genes involved in lipid metabolism. Recent work has shown that flies mutant in STING display reduced lipid storage and downregulated expression of lipid metabolism genes . The autboxylase . It has S2 cells , which iCG15434 that is predicted to be involved in mitochondrial electron transport. The authors also found significant upregulation of chorion protein genes [Femcoat and Cp18, which were not differentially expressed in our study. In contrast to our findings, a study on Drosophila’s response to nora virus infection reported a downregulation of antimicrobial peptide genes regulated by the IMD pathway, such as DptA [Tep4 and Drs [As in previous studies, we also detected a significant downregulation of genes involved in mitochondrial function in response to nora virus infection. For example, using DNA microarray,  reportedin genes  such as  as DptA , which w and Drs , which wCp18 and downregulation of ribosomal protein genes [PGRP-LC and domeless knockout flies, suggesting the recruitment of IMD and JAK/STAT pathways respectively in DMelSV infection [Although we detected no significant changes in expression in response to DMelSV, it has been reported that DMelSV infection increases the rates of expression of chorion protein genes such as in genes . The autin genes . Howevernfection .Tret1-2, was significantly upregulated in response to DCV infection. This is in contrast with published work that showed that systemic DCV infection decreases the expression of many genes involved in trehalose metabolism, such as Tret1-1, CG5177, and Tps1 [Spz [vir-1 [In our study, only the gene encoding a trehalose transporter, and Tps1 . Using Dand Tps1 . DCV infps1 [Spz  and vir-z [vir-1  that aretipE in response to Motts Mill infection, which may have induced changes in the function of the metal ion channels. Temperature-induced paralytic E (tipE) encodes a protein with a cysteine-stabilized αβ scaffold (CSαβ). Drosophila peptides with CSαβ scaffold are implicated in the regulation of voltage-gated sodium ion channels [The changes in expression we have observed may be indicative of virus-induced pathologies in the host. For example, GO analysis showed that changes in the expression of metal ion transport was significantly enriched in the flies infected with Motts Mill virus. Perhaps, virus infection may have induced the deregulation of metal ion transport in flies infected with Motts Mill, leading to their change in expression. Conversely, there was an increase in the expression of channels .Drosomycin-like genes in the intestine [upd3 and the chitin metabolism gene, CG32302, were significantly upregulated in Drosophila infected with nora virus [Sox21a were also upregulated in response Thika virus infection in our study, which is in contrast with published work. For example, differential gene expression analysis on Drosophila midgut cells detected a significant downregulation of Socs36E, which is upregulated following gut septic injury [Jon65Ai, and the lysozyme gene, LysE [We observed an increase in the expression of genes involved in chitin biogenesis and binding, wound healing, and differentiation in the midgut stem cell, respectively. Perhaps, in response to nora-virus-induced damage of the gut epithelium , the hosntestine . This isra virus ,133. Genc injury  and encoc injury . Althougc injury . The autne, LysE .Drosophila fly culture, and that they can induce detectable changes in host gene expression. It may therefore be useful to consider their effects in experimental studies, especially those that generate transcriptomic data.Overall, our work confirms that viruses are often naturally present in laboratory


In the pathogenesis of influenza virus infection, lymphocyte apoptosis as a part of the infection and/or the immune response to the virus can be somewhat puzzling. The percentage of human T lymphocytes within the peripheral blood mononuclear cell population that becomes apoptotic greatly exceeds the percentage that are infected after exposure to the virus, consistent with substantial apoptosis of bystander T lymphocytes. Studies reveal an important role of viral neuraminidase expression by co-cultured monocyte/macrophages in induction of apoptosis, including that of uninfected bystander lymphocytes. Despite these observations, it is a reasonable perspective to recognize that the development of lymphocyte apoptosis during the response to infection does not preclude a successful immune response and recovery of the infected host in the great majority of cases. Further investigation is clearly warranted to understand its role in the pathogenesis of influenza virus infection for human subjects. Influenza A virus (IAV) is a major cause of respiratory infections in the United States and worldwide, with the most severe cases occurring in the very young and in elderly individuals ,2,3. AltThere have been four influenza pandemics in the last 103 years, in 1918, 1957, 1968 and 2009. Viral strains with pandemic potential continue to be isolated from avian species [Circulating lymphopenia is a clinical feature of influenza infections caused by seasonal influenza ,11, aviaA greater understanding of the pathogenesis of IAV infection is needed, including events in the leukocyte response to the challenge that are to a degree puzzling, such as lymphocyte apoptosis as a part of the infection and/or the immune response to the virus. A perspective regarding the existence and extent of lymphocyte apoptosis, especially that of uninfected bystander cells, is offered for consideration.Studies have been undertaken in our laboratories to delineate the extent and complexity, and analyze possible mechanisms, behind one of the potentially major contributors to IAV-induced leukopenia, namely the induction of apoptosis of human lymphocytes by the virus. Analysis of lymphocyte subpopulations after exposure of PBMC to IAV showed that a portion of CD3+, CD4+, CD8+, and CD19+ T lymphocytes became apoptotic, measured using a TUNEL assay  . In thesFurther studies confirmed that the percentage of cells that are infected by IAV is less than the percent of apoptotic cells after exposure . DepletiAdditional studies showed that, whereas exposure of PBMC to infectious IAV induced lymphocyte apoptosis, exposure to inactivated IAV did not result in apoptosis beyond that of sham-exposed cells . This inStudies by other investigators indicate that interferons (IFN) can greatly sensitize certain tissue culture cells such as murine fibroblasts, to apoptosis in response to IAV . IFNs maStudies of in vivo human IAV-induced apoptosis are apparently limited to postmortem examinations in cases of severe lethal infection. As noted above, lungs of patients infected by highly pathogenic avian influenza (HPAI) virus, such as the H5N1 virus, as well as patients with lethal human IAV infection, contain infiltrating apoptotic leukocytes .IAV infection-induced apoptosis of lymphocytes can be considered an important aspect of IAV pathogenesis since lymphocytes play critical roles in defense against and recovery from IAV infections. Divergent concepts regarding the ultimate role(s) of apoptosis during IAV infection have been raised mainly by studies examining cells other than leukocytes and using non-human models such as mice . This maStudies of pro-apoptotic and anti-apoptotic members of the cellular Bcl-2 protein family have been reported to show that the cell and IAV struggle to control apoptosis, with appropriately timed apoptosis of Madin–Darby canine kidney (MDCK) cells and mouse fibroblasts being important for the replication of IAV . Wurzer On the other hand, IAV-infected mouse and fruit fly cells, the latter host lacking adaptive immunity, were shown to undergo apoptosis and subsequent engulfment by macrophages and neutrophils, representing an apparent antiviral innate immune response mechanism that is conserved among multicellular organisms, according to Nainu et al. . Highly Thus, death receptor-mediated apoptosis represents a complex mechanism used by both the virus and the host to differing degrees depending upon the identity of the challenging virus ,44. The An alternative approach to the treatment of viral diseases has been proposed by some authors, namely, therapeutic strategies to enhance death receptor-mediated apoptotic clearance of virus-infected cells . Such anPerhaps the clearest indication that exposure to another RNA respiratory virus may not exhibit the lymphocyte apoptosis induced by IAV is provided by concomitant analyses of autologous human PBMC exposed to IAV or to respiratory syncytial virus (RSV) . The PBMStudies of other viruses can be cited relevant to the role of monocytes/macrophages in induction of lymphocyte apoptosis after exposure to a virus as described above. Thus, for example, macrophages infected by bovine viral diarrhea virus have been reported to induce lymphocyte apoptosis , and an It is very interesting and even provocative that monocytes/macrophages, long considered to have a central role in antiviral defense, such as by production of antiviral factors such as interferon  and by sRespiratory epithelial cells, including those of the alveoli, are the desired IAV target and are supportive of IAV replication ,57. FeatOpportunities do exist for monocyte/macrophage expression of IAV neuraminidase and induction of lymphocyte apoptosis as described in Apoptosis of IAV-infected cells makes them susceptible to phagocytosis , and thiInfluenza A virus infections are a continued threat to humans  and addrIn examining a process such as human lymphocyte apoptosis in the setting of IAV infection, it is reasonable and important to consider a teleological argument regarding the leukocyte responses. The vast majority of individuals survive the infection and are endowed with a substantial degree of resistance to re-challenge. They are usually infected again with a virus that has drifted or shifted away from their ability to recognize it immunologically and, even then, individuals are likely to survive the recurring challenge. The overall response to IAV supports survival of the human species. In the end, it is possible that lymphocyte cell death through apoptosis is part of an ultimately effective defense against the virus rather than an adverse event during infection that the host can survive. It remains to be determined what benefit is provided the host or the virus by the substantial bystander lymphocyte apoptosis that has been well documented, as well as the roles of monocytes/macrophages cited above.


Chlorovirus, family Phycodnaviridae) at the level of DNA replication are (i) the host cell has a DNA G+C content of 66%, while the virus is 40%; and (ii) the initial quantity of DNA in the haploid host cell is approximately 50 fg, yet the virus will make approximately 350 fg of DNA within hours of infection to produce approximately 1000 virions per cell. Thus, the quality and quantity of DNA (and RNA) would seem to restrict replication efficiency, with the looming problem of viral DNA synthesis beginning in only 60–90 min. Our analysis includes (i) genomics and functional annotation to determine gene augmentation and complementation of the nucleotide biosynthesis pathway by the virus, (ii) transcriptional profiling of these genes, and (iii) metabolomics of nucleotide intermediates. The studies indicate that PBCV-1 reprograms the pyrimidine biosynthesis pathway to rebalance the intracellular nucleotide pools both qualitatively and quantitatively, prior to viral DNA amplification, and reflects the genomes of the progeny virus, providing a successful road to virus infection.Viruses face many challenges on their road to successful replication, and they meet those challenges by reprogramming the intracellular environment. Two major issues challenging Paramecium bursaria chlorella virus 1 (PBCV-1, genus  The lifestyle of viruses includes the reprogramming of cell-based metabolic pathways to achieve efficient replication, assembly, maturation and escape. Many viruses achieve high levels of biomass production in the short time-periods of the infection cycle, and this seems to match up biosynthetic pathways with an appropriate substrate. However, very little is known about how giant viruses accomplish metabolic adjustments and optimizations.Chlorovirus  that infect certain eukaryotic green algae having mutualistic lifestyles as endosymbionts of various protists and metazoans  are highly conserved in many dsDNA viruses and are 100% conserved in the chloroviruses. The PBCV-1 RNR is a virion-associated protein, and the two genes are coordinately expressed early. The enzyme converts ribonucleoside diphosphate to deoxyribonucleotides A. With rpbcv-1_a416r) (E. C. 2.7.4.14) is conserved in the chloroviruses but not entirely; notably, most viruses in the subclade NC64A-I aspartate to carbamyl [U-14C]aspartate [pbcv-1_a169r) is not conserved in the chloroviruses but is found in those members of the C. variabilis NC64A-II subclade viruses only. This protein matches best to one of the two C. variabilis NC64A homologs .A gateway for pyrimidine biosynthesis de novo is through the complex and sensitive multi-enzyme complex referred to as CAD: carbamoyl-phosphate synthetase 2/aspartate transcarbamylase/dihyroorotase. This enzyme complex conducts the first three steps toward pyrimidine biosynthesis de novo and is sensitive to nucleotide pool concentrations, both positively and negatively C. The ce and UTP . Though  and UTP  and conf PBCV-1_A9R and prspartate . The virpbcv-1_a596r) is a highly conserved gene within the chloroviruses, as described above. The chlorovirus dCMP/dCTP deaminases are sensitive to nucleotide pool changes. The viral dCTP deaminase complements the cell (E. C. 3.5.4.13), while the viral dCMP deaminase augments the cell (E. C. 3.5.4.12), and both of the viral activities are negatively regulated by increasing levels of dTTP  of the purine biosynthesis pathway produces the same product from the pyrimidine biosynthesis pathway with uridine monophosphate synthetase: 5-phospho-alpha-D-ribose 1-diphosphate produced from the transfer of pyrophosphate from ATP to ribose 5-phosphate (PRPP). PRPP is one of the four key regulatory modulators of the purine biosynthesis pathway and is an essential component of the purine salvage pathway, as well as the synthesis of purines de novo. Phosphoribosylpyrophate synthetase is not the committed step in purine biosynthesis but is negatively affected by GDP, and at a distinct allosteric site is inhibited by ADP. The enzyme is activated by phosphate. Thus, the virus-encoded enzyme appears to be a portal between the pyrimidine and purine biosynthetic pathways, producing PRPP.Genomic annotations predict pyrimidine fluxes to be directed toward enhanced dTTP synthesis and less toward dCTP synthesis . This suFrom genomic and transcriptomic studies, we hypothesized that the nucleotide pools adjust to a state that favors the synthesis of viral nucleic acids, which is approximately 40% G+C, by the onset of viral DNA synthesis. A targeted liquid chromatography—tandem mass spectrometry (LC-MS/MS) metabolomic approach was developed to focus on the pyrimidine biosynthesis pathway intermediates during the early phase of infection (0–90 min p.i.). All flux patterns of the measured metabolites were complex, with either second- or third-order kinetics at the time of evaluation .Additionally, the analysis indicates that the pyrimidine biosynthesis pathway is up-regulated, generally, where most metabolites increase several-fold during the early phase up to 60 min p.i.  was approximately 4.5. This ratio is biased, seemingly to support a high G+C content of the organism (66%). As the infection progresses, there is a slow but steady increase in most of the pyrimidine intermediates, including both dCTP and dTTP, but at varying rates  most metabolites decline in abundance, then rebound . Of partng rates . By 40 m6 zmol/cell  were somewhat less affected by the infection progress . ATP andt = 60 = 125 zmol/cell:185 zmol/cell) was 0.67, where the abundances were maximal. This ratio is biased to support a high A+T content of viral DNA that is just beginning to be synthesized at this point in the infection and supports the initial hypothesis. In general, the pyrimidine intermediates were maximal at 40–60 min p.i., after which the abundances declined somewhat  of the virus matched that of the host, there would be a need to increase nucleotide biosynthetic rates but not shift away from the host ratios. This type of increase in nucleotide pools might be achieved with the augmentation of rate-limiting enzymes by viral homolog addition. However, if the nucleotide pool demand by the virus significantly differs from the host ratios, pathway fluxes would need to be (i) altered by post-translational modification of the host enzymes, or (ii) complementation with viral enzymes, or (iii) or both.3 particles per cell, resulting in a multi-fold increase in the total DNA of the cell. The hosts tend to have significantly higher G+C content in their nucleic acids than do the chloroviruses. We addressed the question of how does the infected cell adjust to an increased demand for nucleotide precursors to fulfill the need for viral RNA and DNA synthesis, as well as the crossover to a different balance of G+C content by the dominate virus replication machinery.Chloroviruses are large dsDNA viruses that replicate on the order of hours, with burst sizes up to 10Chlorovirus, PBCV-1, (ii) transcriptional profile data of a PBCV-1 infection, and (iii) analyte fluxes of the nucleotide biosynthesis pathways during the early phase of virus infection using a targeted metabolomics analysis. PBCV-1 genes either augmented or complemented the pyrimidine biosynthesis pathway, primarily, but also appeared to alter synthesis by the addition or subtraction of regulatory functions, such as the virus-encoded ATCase that lacks regulatory subunits (1/2 = 581 min) and 5′nucleotidase , suggesting some need for these enzymatic functions in the late phase of infection. This degradation of host pyrimidine biosynthesis mRNAs was accelerated relative to the majority of host mRNAs (mean t1/2 approximately 80 min). Notable exceptions with relatively long half-life values were mRNAs for protein synthesis , presumably to support virus replication [We hypothesized that chlorovirus-encoded protein functions alter the nucleotide biosynthesis pathway to reset the nucleotide balance while boosting the overall pool sizes, and that this alteration is pointed at the pyrimidine biosynthesis pathway primarily. This hypothesis was supported by the evaluation of the (i) genomic data of the type member of the genus subunits . The onssubunits . Most, blication . How degMost of the genes involved in the nucleotide biosynthesis process are conserved across a diverse set of chloroviruses, with notable exceptions . Within Pseudomonas aeruginosa and the associated myovirus phage PAK_P3, with burst sizes at 103 pfu/cell on the order of 10′s of min p.i. [In several respects, chlorovirus biology reflects aspects of certain bacteriophage infections. One case of interest is min p.i. . This inChlorovirus PBCV-1 infection reprograms the pyrimidine biosynthesis pathway during the early phase of infection to reflect the change in the host genome 66% G+C content to the 40% G+C content of the virus genome. We suspect this approach and these findings are generally useful for understanding how obligate intracellular symbionts control the environment of the host cell for their own benefit. This approach also allows for hypothesis development in terms of drug targeting of critical points in the replication pathways of many parasitic agents of interest that depend on intracellular metabolism.In summary, the in silico analysis and the experimental results support the hypothesis that


ELPKGR/GLF, which is a low-pathogenicity molecular characteristic. The XL virus had similar mammalian adaptations to human-originated H7N9 viruses, such as the polymerase basic protein 2 (PB2) Glu-to-Lys mutation at position 627 (E627K) mutation, but differed from avian-originated H7N9 viruses. The XL virus showed a higher SA-α2,6-Gal receptor-binding affinity and better mammalian cell replication than the avian H7N9 virus. Moreover, the XL virus had weak pathogenicity in chickens, with an intravenous pathogenicity index of 0.01, and intermediate virulence in mice, with a median lethal dose of 4.8. The XL virus replicated well and caused clear infiltration of inflammatory cells and increased inflammatory cytokines in the lungs of mice. Our data constitute the first evidence that the low-pathogenicity H7N9 influenza virus can infect camels and therefore poses a high risk to public health.The H7N9 subtype of influenza virus can infect birds and humans, causing great losses in the poultry industry and threatening public health worldwide. However, H7N9 infection in other mammals has not been reported yet. In the present study, one H7N9 subtype influenza virus, A/camel/Inner Mongolia/XL/2020 (XL), was isolated from the nasal swabs of camels in Inner Mongolia, China, in 2020. Sequence analyses revealed that the hemagglutinin cleavage site of the XL virus was IMPORTANCE H5 subtype avian influenza viruses can cause serious diseases in poultry and wild birds. On rare occasions, viruses can cause cross-species transmission to mammalian species, including humans, pigs, horses, canines, seals, and minks. The H7N9 subtype of the influenza virus can also infect both birds and humans. However, viral infection in other mammalian species has not been reported yet. In this study, we found that the H7N9 virus could infect camels. Notably, the H7N9 virus from camels had mammalian adaption molecular markers, including altered receptor-binding activity on the hemagglutinin protein and an E627K mutation on the polymerase basic protein 2 protein. Our findings indicated that the potential risk of camel-origin H7N9 virus to public health is of great concern. Influenza A viruses (IAVs) are eight-segmented, single-stranded negative-sense RNA viruses. IAVs can be classified into 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes based on the HA and NA proteins present 13. IAVs cThe H7N9 subtype of influenza virus emerged in China in early 2013 and caused five waves of human infection in 2013 to 2017 . Since 28–Anseriformes  and Charadriiformes (gulls and terns)  , 12. Thed terns) , 14.Dromedary or one-humped camels are exclusively domesticated species in arid areas, as both beasts of burden and production. Middle East respiratory syndrome (MERS) , brucell19–In this study, nasal swabs were collected from seven camels with respiratory symptoms (cough and runny nose) in 2020 in Inner Mongolia, China. One H7N9 virus was isolated from the nasal swabs, and its genome and biological characteristics were analyzed.50) and 50% tissue culture infective dose (TCID50) of the XL virus were determined to be 109.2/mL and 107.17/mL, respectively . Reverse transcription-PCR (RT-PCR) detection of the nasal samples confirmed negative nucleic acid test results for MERS and coronavirus disease 2019 and positive nucleic acid test results for the influenza virus. Furthermore, three of the seven samples were HA positive after isolation from chicken embryonated eggs. After three purifications using the plaque assay, one influenza virus was isolated. The PCR results and sequence analysis confirmed that the influenza virus was H7N9 and was A/camel/Inner Mongolia/XL/2020 (XL) (GISAID accession number EPI_ISL_12335304). No mutations were found in the genomic sequences of the purified virus compared with that of the PCR products from the original samples. The 50% effective infectious dose , 1,333 avian-origin H7N9 viruses, a human-origin virus from 2013 A/Anhui/1/2013 (AH), and 1,330 human-origin H7N9 viruses . The ami HA gene .Phylogenetic analysis showed that the HA gene of the XL virus belonged to the Yangtze River Delta branch of the Eurasian lineage . SimilarThe NA genes of XL, AH, and JD viruses belonged to the RD-5-like lineage . The nucAll the internal genes of the XL virus belonged to the Eurasian lineage . The PB2, PB1, NP, and M gene nucleotide sequences were most closely related to those of A/chicken/Zhejiang/SIC40/2015(H9N2), at 99.82, 99.40, 100, and 100% identity, respectively. The nucleotide sequence of the PA gene of the XL virus was most closely related to that of A/chicken/Jiangsu/XZ252/2015 (H7N9), with 99.81% identity. The nucleotide sequence of the NS gene of the XL virus was most closely related to that of A/chicken/Ganzhou/GZ79/2016 (H7N9), with 100% identity.Because A549 cells have relatively high levels of SA-α2,6-Gal receptors, whereas MDCK, DF1, and chicken embryo fibroblast (CEF) cells have similar levels of SA-α2,6-Gal and SA-α2,3-Gal receptors, these avian-origin CEF or DF-1 and mammalian-origin A549 or MDCK cells were selected for determination of viral replication curves, and avian-derived JD virus and human-derived H1N1 IAV A/California/04/2009 (CA) virus were selected for comparison. The titer of the XL virus was nearly the same as that of the CA virus in CEF, DF-1, MDCK, and A549 cells. However, the titer of XL virus was significantly lower than that of JD within 72 h postinfection in both CEF and DF-1 cells . The titTo test the receptor-binding activity of the XL virus, a solid-phase binding assay was performed to evaluate receptor-binding preferences. The XL and JD viruses could bind to both SA-α2,3-Gal and SA-α2,6-Gal receptors, whereas the CA virus could bind only to SA-α2,6-Gal receptors. The XL virus showed a higher affinity to SA-α2,6-Gal receptors but a lower affinity to SA-α2,3-Gal receptors, in contrast to the JD virus , indicatTo test the stability of the prefusion state of HA, the viruses were treated under low pH , and residual infectivity was measured using a hemagglutination assay. The results showed that the hemagglutination titer of the XL virus was similar to that of JD and CA viruses when exposed to different pH values . In addi50 of the XL virus was significantly higher than that of the JD or CA viruses at 1 and 3 days after incubation at 37°C  was used to determine the pathogenicity of XL virus in chickens. All infected chickens survived during the 10-day observation period , and the2 to 106 EID50 of the virus  of the XL virus was 104.83, indicating moderate virulence in mice.To measure the virulence of the XL virus in mice, 6-week-old BALB/c mice were injected intranasally at a dose of 10he virus  and moni6 EID50 of XL virus. The XL virus replicated efficiently in the lungs on days 3 and 5 postinfection with an EID50 of 105 and 104.5, respectively. XL viral replication was lower in the brain on days 3 and 5 postinfection with an EID50 of 101.5 and 102.42, respectively. However, XL viral replication was lower in the kidney and liver, and no virus was detected in the spleen and heart  to glutamic acid (E) at position 92 of the NS1 protein, which is required for viral virulence in mammalian species, especially swine , was notPolymerase activity of influenza viruses is pivotal for viral cross-species transmission. However, polymerase activity of AIVs is poor in mammals . Multipl50 determination, there were no clear histologic lesions or detectable IAV antigens in the brain, indicating that the XL virus only causes limited replication in the brain.Similarly, the camel-origin low-pathogenicity H7N9 virus was virulent in mice, suggesting that the XL virus with the E627K mutation in the PB2 protein has a high potential risk of mammalian infection. The XL virus was found to replicate in the lungs with alveolar hyperemia and inflammatory cell infiltration. Further immunohistochemistry tests indicated that the virus was widely distributed in the lung tissue bronchial and bronchiolar epithelial cells. Although low viral replication of the XL virus was detected in the brain of the infected mice based on EIDSeveral studies have shown that viruses with HA activation at lower pH are required for efficient airborne transmissibility and infection among mammals . HA actiThe thermal stability of the virus at physiological temperatures was also analyzed. The thermal stability of the XL virus at 37°C was significantly higher than that of the JD and CA viruses, whereas no difference was found in the thermal stability at 42°C, indicating a higher adaption ability of the XL virus in mammals. Further polymerase activity analysis revealed that the XL and CA viruses had higher polymerase activity in mammalian cells but lower polymerase activity in avian cells than the JD virus. These data indicated that the XL virus can efficiently replicate in mammals.https://www.tephinet.org/learning/fead/investigation-of-a-family-cluster-of-h7n9-avian-influenza-in-inner-mongolia-china-2017). Although vaccination with H5/H7 bivalent inactivated vaccines significantly reduced the potential risk of H7N9 infection in domestic poultry and humans .Challenge studies in chickens and mice were performed strictly following the Guidelines of Laboratory Animal Welfare and Ethics approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXKSU-2007-0005). When mice met the criteria of losing 25% or more of their initial body weight during the study, they were scored dead and euthanized under excess isoflurane anesthesia according to institutional guidelines.2. AIVs were propagated in 10-day-old SPF chicken embryos .The H7N9 AIV A/chicken/Huadong/JD/2017 strain was isolated in our previous study . H1N1 IAFresh nasal swab samples from seven camels were collected in January 2020 in Inner Mongolia. The samples were placed in phosphate-buffered saline  supplemented with 100 U/μL of penicillin and 100 U/μL of streptomycin and immediately shipped to the laboratory for further analysis. Treated samples were inoculated into 10-day-old SPF chicken embryos at 37°C for 72 h. After chilling at 4°C, allantoic fluids were harvested and analyzed for the presence of influenza virus using a hemagglutinin assay. As previously described, the HA and NA subtypes of HA-positive allantoic fluid were verified and identified using quantitative PCR (qPCR) . The virhttps://dongzhang0725.github.io/). The reference sequences were retrieved from the Global Initiative on Sharing Avian Influenza Data , and the sequence data were deposited in GISAID (accession number EPI_ISL_12335304).Viral RNA from both the original samples and purified viruses was extracted using TRIzol reagent . Extracted viral RNA was then reverse transcribed to complementary DNAs (cDNAs) using a cDNA synthesis kit , according to the manufacturer’s instructions. The synthesized cDNAs were amplified using PCR with universal primers  and sequ2. The cell culture supernatants were collected every 12 h. At 72 h postinfection, the mean tissue culture infective dose (TCID50) of the virus was determined for all samples.Monolayer MDCK, A549, DF1, and CEF cells were used to detect viral growth. Briefly, the cells were infected with the virus at a multiplicity of infection (MOI) of 0.01 in DMEM for 1 h. The cells were then washed to remove unbound viruses, and fresh DMEM with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (0.5 μg/mL) was added and incubated at 37°C with 5% CO8) were aliquoted into nine 60-μL vials, exposed to 56°C, and quickly cooled to 4°C after 0, 5, 10, 15, 30, 60, 90, 120, or 150 min of incubation  were mixed with equal volumes (100 μL) of 100 mM acetate buffer (pH 4.0 or 5.0), 100 mM phosphate buffer (pH 6.0), or neutral phosphate buffer (pH 7.0). After a 10-min incubation at 37°C, the titers of all samples were determined using a hemagglutination assay (The viruses (HA = 2on assay . The datRenilla plasmid (pTK-RL). After 24 h, luciferase activity was determined using the dual luciferase assay system kit , following the manufacturer’s instructions. Polymerase activity was calculated as relative luciferase activity, which is presented as the ratio of firefly luciferase activity to Renilla luciferase activity. The results are presented as means ± standard deviation (SD) from three independent experiments.The polymerase activity of the viruses was detected using a dual-luciferase assay. 293T or DF1 cells cultured on a 12-well plate were cotransfected with 200 ng/well of pCAGGS encoding PB1, PB2, PA, and NP genes from XL, JD, and CA viruses and a firefly luciferase reporter plasmid (p-Luci) or an empty plasmid, with 20 ng/well of an internal control Ten 6-week-old SPF white leghorn chickens were purchased from the Lihua Corporation, China. The chickens were injected intravenously with 0.1 mL of 1:10 diluted virus and monitored daily for clinical signs of disease for 10 days. The IVPI was calculated according to Office International des Epizooties recommendations.2, 103, 104, 105, or 106 EID50. PBS was used as a control. The mice were weighed individually and monitored for signs of illness and mortality daily for 2 weeks.The infection dose was determined using 30 6-week-old female BALB/c mice. Five mice per group were infected intranasally with isolated AIV at doses of 106 EID50 of the virus and six were intranasally dripped with PBS as the control. Three mice were euthanized on days 3 and 5 postinfection, and the lungs, brain, kidneys, spleen, heart, and liver were collected for viral titration and pathology studies. For virus titration, organs were collected on days 3 and 5 postinfection, and clarified homogenates were titrated for virus infectivity in eggs at an initial dilution of 1:10 (lungs), 1:2 (other tissues), or undiluted if negative at the lowest dilution. For the pathology study, the lung and brain tissues were fixed in formalin and subjected to paraffin sectioning with hematoxylin and eosin (H&E) staining. For immunohistochemical analysis, lung and brain tissue slides were rehydrated and incubated in 3% hydrogen peroxide blocking buffer for 25 min at 25°C before being blocked in 3% bovine serum albumin for 1 h at room temperature. The IAV antigen was detected with a mouse polyclonal antibody against IAV nucleoprotein and horseradish peroxidase-conjugated goat anti-mouse IgG antibody and visualized using diaminobenzidine (DAB) peroxidase as the substrate.Virus titration and pathology studies were performed using 12 6-week-old female BALB/c mice. Six mice were infected intranasally with 10AAGCCAGAGCTGTGCAGATGAGTA-3′; R, 5′-TGTCCTGCAGCCACTGGTTC-3′), IL-8 , IL-1β , TNF-α , and GAPDH .The expression of inflammatory cytokines in the tissues was determined using qPCR, with the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as an internal standard. The primer sequences used for amplification were as follows: IL-6 , and the sequence data of A/camel/Inner Mongolia/XL/2020 (XL) were deposited in GISAID .All data generated or analyzed during this study are included in the published article . The reference influenza viruses’ sequences were retrieved from the Global Initiative on Sharing Avian Influenza Data (GISAID;


Plant virus nanoparticles (PVNPs) have garnered considerable interest as a promising nanotechnology approach to combat cancer. Owing to their biocompatibility, stability, and adjustable surface functionality, PVNPs hold tremendous potential for both therapeutic and imaging applications. The versatility of PVNPs is evident from their ability to be tailored to transport a range of therapeutic agents, including chemotherapy drugs, siRNA, and immunomodulators, thereby facilitating targeted delivery to the tumor microenvironment (TME). Furthermore, PVNPs may be customized with targeting ligands to selectively bind to cancer cell receptors, reducing off-target effects. Additionally, PVNPs possess immunogenic properties and can be engineered to exhibit tumor-associated antigens, thereby stimulating anti-tumor immune responses. In conclusion, the potential of PVNPs as a versatile platform for fighting cancer is immense, and further research is required to fully explore their potential and translate them into clinical applications. Nanomedicine, a burgeoning area of multidisciplinary research, has exhibited enormous potential for transformation into a highly innovative development . DespiteNanomedicines encompass a broad range of nanomaterials, showcasing a particle size that spans from 1 nm to more than 400 nm, and represent a highly diversified array of materials. These materials may consist entirely of metal, as exemplified by gold and silver nanoparticles , or may Plant VNPs have been examined as an exclusive category of nanocarriers for biomedical applications, as documented in previous research . In addiPlant VNPs exhibit either a rod shape (tobacco mosaic virus (TMV) and potato virus X (PVX)) or an icosahedral shape (cowpea mosaic virus (CPMV)). These differing shapes elicit distinct nanoparticle responses in vivo. Tobacco mosaic virus is capable of assembling into VLPs without necessitating drug payload conjugation on the nanoparticle surface or within its inner channel, albeit to a limited degree. In contrast, potato virus X requires its RNA genome for self-assembly and can only carry a payload on its outer surface. Cowpea mosaic virus, on the other hand, can self-assemble into empty virus-like particles in the absence of its RNA genome and can therefore contain a payload both internally and externally within its protein shell.Despite being non-infectious in humans, the multivalent, repetitive coat protein assemblies of the VNPs and VLPs function as pathogen-associated molecular patterns (PAMPs), serving as danger signals to the immune system . VNPs adFor instance, TMV, PVX, and papaya mosaic virus VNPs are capable of eliciting robust cellular immune responses against fused antigenic epitopes . The potHence, VNPs function as exemplary epitope delivery vehicles for antigens and adjuvants of great applicability in vaccine and immunotherapeutic formulations . The preIn this review, we present a multitude of examples to analyze the manner in which the structural composition of plant viruses contributes to their efficacious utilization in the realm of cancer diagnostics and therapy. Furthermore, we delve into the origins of the implementation of plant virus architecture as nanoparticles in medical applications, along with the prospects of their propitious employment as pioneering cancer immunotherapies.For over a century, the history of the tobacco mosaic virus (TMV) has been significant and noteworthy. It all started when Beijerinck identified the mosaic disease of tobacco as a fluid that is capable of spreading, which was later referred to as a “virus” in modern language . TMV gotTMV represents the Tobamovirus genus and is part of the alphavirus-like supergroup. The virions of TMV are rod-shaped, measuring 300 × 18 nm, and have a hollow center that contains 95% capsid protein (CP) and 5% RNA. The capsid protein (CP) subunits interact with 3 nts in a helical pattern around the RNA. The virions remain stable for many decades and their ability to infect sap remains unchanged even after being heated to 90 °C .TMV’s application as a prototype system has been driving the field of virology research even in modern times. TMV was the first virus to go through chemical purification , detectiThe genome of TMV, consisting of a single positive-sense RNA molecule, spans 6395 nucleotides (nts) and encompasses four open reading frames (ORF) . The 5′-TMV is presently being employed extensively as a tool for biological research and biotechnology applications . VariousPVX is a plant virus that belongs to the Potexvirus genus of the Alphaflexiviridae family. It is regarded as the type member and is one of the oldest known viruses to infect potato plants, with scientific publications dating back to . This viPVX is a type of plant virus whose virions are made up of flexuous filaments that measure about 460–480 nm in length, 13 nm in diameter, and with a helical pitch of 3.4 nm. The genome of PVX is around 6.4 kb long and has both a 5′ cap and a 3′ poly(A) terminus. It consists of five open reading frames, with four considered essential for cell-to-cell and systemic movement, including one that encodes the viral replicase. Infected cells tend to have X-bodies, or cellular inclusions located near the nucleus, commonly observed in their vicinity .PVX is recognized as one of the top 10 plant viruses in molecular plant pathology due to its significant contributions as a model for studying plant–virus interactions. It has played a crucial role in advancing plant biology, including the development of antiviral gene silencing mechanisms, creating viral vectors for foreign gene expression, using green fluorescent protein (GFP) to track plant virus infection, discovering the function of viral movement proteins in transport through plasmodesmata, and cloning the Rx gene in potato through map-based cloning. The Rx gene is considered the first antiviral R gene isolated from potato ,36,37.PVX has greatly contributed to the initial characterization of the homology-dependent post-transcriptional gene silencing (PTGS) pathway, which is responsible for destroying viral RNAs. The PTGS mechanism targets highly structured regions of viral genomes or replication intermediates that can be cleaved by plant-specific Dicer enzymes, resulting in the formation of siRNAs . These studies were crucial and involved using PVX as an expression vector carrying cDNAs that encode foreign sequences such as green fluorescent protein (GFP), enabling the monitoring of siRNA production and mRNA turnover ,39.Improved PVX-based vectors are considered suitable for expressing any desired gene in PVX host plants. These vectors are favorable for protein overexpression, investigation of protein functions in plants, or utilization in virus-induced gene silencing mechanisms .The CPMV belongs to the Comovirus genus and is the representative member of this group. The Comovirus genus features a total of 13 other members that are part of the larger Comoviridae family . The famNicotiana benthamiana has proved to be an important experimental host. The virus is transmitted by various beetles that have biting mouthparts, and it has been reported in Africa, the Philippines, and Iran, but not in North and South America  bombesin-(7–14) can target the gastrin-releasing peptide receptor that is over-expressed in human prostate cancers. The phenomenon of tumor homing was observed through the utilization of human prostate tumor xenografts on the chicken chorioallantoic membrane model, through intravital imaging techniques .The filamentous PVX can be engineered to display green fluorescent protein (GFP) or mCherry as probes for optical imaging in human cancer cells and within a preclinical mouse model . Plant v3+, Zr4+, or Gd3+), or fluorescent dyes  and subjected to 808 nm irradiation [These unique structural and chemical properties of PVNPs make them highly suitable for combining therapeutic and diagnostic agents’ potentialities for in vivo applications . Metal–padiation . Gd-loadadiation . The capadiation .Engineered TMV-MOF  hybrid nanoparticles augmented the retention of these VNPs in murine models . ParticlIn yet another study, TMV VNPs were modified to target VCAM-1, the vascular cell adhesion molecule, and these particles were loaded with Gd-dodecane tetraacetic acid (Gd-DOTA). This led to highly sensitive recognition and visualization of atherosclerotic plaques in ApoE-/- mice, employing low doses of contrast agent and this resulted in enhanced relaxivity and moderate tumbling of the Gd-DOTA-TMV carrier with improved signal-to-noise ratio. Moreover, these coupling complexes showed greater imaging sensitivity, thus affording a 40-fold decrease in Gd dose compared to the standard clinical doses .Recent studies have shown that some PVNPs alone are immunogenic and highly effective as a monotherapy . Toward + and CD8+ T cell responses and promotes systemic tumor-specific cytotoxic CD8+ T cell activity [PVNP ISV is a treatment for solid tumors that involves several mechanistic processes. Firstly, the PVNP is transported into the tumor and assimilated by immune cells. Secondly, these activated innate immune cells release cytokines and chemokines to attract more immune cells to combat the tumor. Thirdly, T-lymphocytes become activated and are lured to the tumor to combat tumor cells with their cognate antigens. This results in tumor lysis. Finally, the activated T-lymphocytes travel throughout the body attacking metastatic tumors . For exaactivity .In the context of immunotherapy, PVNPs can also act as a nanocarrier for tumor antigens, or immune adjuvants for improving immunotherapeutic efficacy of vaccines ,105. In The integration of PVNP-based monotherapy and any therapy that induces tumor cell lysis and release of tumor-associated antigens (TAAs) might be one of the possible strategies to overcome complexity and tumor heterogeneity . The comWhen utilizing PVNPs, there are numerous challenges and limitations to consider. Firstly, in vivo biological barriers such as interactions with serum, immune cells, or antibodies can impact the use of native or functionalized PVNPs in clinical settings. Secondly, anti-PVNP antibodies can alter the way immune cells interact with PVNPs, potentially leading to their elimination before reaching the target site. Thirdly, the formation of protein corona (PC) can impede the development of PVNPs for in vivo applications. Fourthly, since PVNPs often target non-immune cells or tissues, clearance by the immune system should be minimized. Fifthly, expressing certain peptides genetically as part of coat proteins may impair capsid formation, which is necessary for effective infection and PVNP generation in plants. In the context of in situ vaccinations, there is also concern about viral escape from the site of injection, which could result in adverse side effects, in addition to the concern regarding virotherapy and the presence of neutralizing antibodies. Undoubtedly, the presence of existing circulating antibodies can increase the risk of adverse effects.Although PVNPs are safe for animals, some regulatory issues need to be considered. PVNPs require high throughput manufacturing, dedicated facilities, and adaptations to plant molecular farming. Further research on regulatory pathways, immune system effects, and novel administration methods, such as chimeras or semi-synthetic nanoparticles, is needed. PVNP production processes must adhere to manufacturing standards and quality control measures to ensure safety and consistency, as determined by regulatory agencies.PVNPs offer significant potential for various therapeutic applications, including but not limited to vaccines, imaging agents, cancer therapies, and drug delivery. The ongoing research endeavors to optimize VNP design, functionalization strategies, and targeting approaches to enhance their therapeutic efficacy. The capacity to modify viruses is constantly expanding, allowing for new therapeutic strategies. By incorporating bioactivatable aspects into the design of innovative viral reagents, researchers can create more flexible and effective treatment options. Directed evolution using mutagenesis strategies can be used to assemble virus libraries that meet specific objectives. Bioinformatics analysis can align different viral capsid genes/protein sequences or protein structures, leading to the generation of novel viruses with unique properties. Computer technologies like machine learning and mathematical modeling can also aid in the development of PVNPs for clinical cancer immunotherapy. In addition, collaborative interdisciplinary partnerships and investments in research infrastructure are crucial in stimulating innovation, expediting scientific progress, and revealing novel possibilities for VNP utilization. Nanoengineering research on VNPs is advancing their design, fabrication, and manufacturing, with various formulations and approaches on the cusp of clinical impact. The structural versatility of VNPs also presents new clinical opportunities across various dose forms. Given the incremental progress of VNP technology in the clinic, investigating pharmaceutical formulation technology may prove instrumental in improving prospects for further translational development. At the preclinical stage, conducting comprehensive research on the formulation of viral nanoparticles (VNP) in various dosage forms would facilitate the modification of VNP’s shelf life stability and compatibility with clinically significant routes of administration. This would in turn enhance the efficacy and safety of the product, while also providing a forecast for future costs and patient convenience.Conventional cancer treatment involves chemotherapeutic drug delivery through the circulatory system, causing dispersion and premature drug release before the tumor site. Drug dosage must be significantly increased to ensure efficacy at the target site. This approach may cause unwanted damage to cells and tissues. Precision cancer medicine aims to improve drug efficacy by targeting nanoparticles, demonstrating clinical potential. Plant VNPs are promising due to their self-assembling architecture and easy production. PVNPs offer quality control for uniform particle shape and size, unlike synthetic nanomaterials. Plant VNPs offer biocompatibility, efficacy, low cost, easy manufacturing, and optimal cargo loading capacity. Plant VNPs offer biodegradability, genetic engineering customization, and superior modification efficacy over synthetic nanomaterials, which rely on chemical synthesis and are persistent in the body. The fact that PVNPs can be used as theranostics for cancer detection and treatment could be a game changer for health outcomes in low- and middle-income countries. In these parts of the world, health burdens will increase and strain what little infrastructure is currently available for escalating elderly populations. The potential of plant VNPs could mitigate future health challenges for populations around the planet, particularly in the Global South. It is for this reason that plant VLPs must continue to be under exploration as a plausible solution for future health challenges.


Arabidopsis thaliana natural accessions and T‐DNA mutants revealed the presence of two virus‐like RNA sequences which showed the typical two‐segmented genome characteristics of a comovirus.Transcriptome studies of Illumina RNA‐Seq datasets of different A. thaliana plants cultivated under standard laboratory conditions. Hence it was named Arabidopsis latent virus 1 (ArLV1). Virus infectivity in A. thaliana plants was confirmed by quantitative reverse transcription polymerase chain reaction, transmission electron microscopy and mechanical inoculation. Arabidopsis latent virus 1 can also mechanically infect Nicotiana benthamiana, causing distinct mosaic symptoms.This comovirus did not induce any visible symptoms in infected A. thaliana RNA‐Seq repositories, including nearly 6500 Sequence Read Archives (SRAs) in the NCBI SRA database, revealed the presence of ArLV1 in 25% of all archived natural A. thaliana accessions and in 8.5% of all analyzed SRAs. Arabidopsis latent virus 1 could also be detected in A. thaliana plants collected from the wild.A bioinformatics investigation of A. thaliana plants. This has probably led to a worldwide distribution in the model plant A. thaliana with as yet unknown effects on plant performance in a substantial number of studies.Arabidopsis latent virus 1 is highly seed‐transmissible with up to 40% incidence on the progeny derived from infected  237: 1071–1073.Sáez & Pagán, See also the Commentary on this article by  Arabidopsis thaliana as model plant. Although community‐serving A. thaliana stock centers, such as the Arabidopsis Biological Resource Centre (ABRC) , tested positive for ArLV1. We identified different isolates of the virus across the NCBI Sequence Read Archives (SRAs) and investigated disease symptoms, infectivity, plant growth and effects on the A. thaliana transcriptome and on abiotic stress resilience. Regular screening for the presence of this widely present – but unnoted – virus and further investigation of its possible effects is highly relevant for the plant science community working with A. thaliana.Here, we describe a comovirus; Arabidopsis latent virus 1 (ArLV1), which we encountered in et al.  using standard settings and the resulting contigs were blasted against the NCBI RefSeq database. Contigs showing a clear identity to different comoviruses were retained for further analysis. As per A. thaliana genotype , we assembled unique contigs, of which the virus isolate in ALL1‐3 from the Wageningen dataset was submitted to GenBank under accession nos. MH899120.1 (RNA1) and MH899121.1 (RNA2), respectively. This Wageningen isolate is later referred to as ArLV1_A, the Col‐0 isolate from Utrecht as ArLV1_B. To identify the phylogenetic position of this virus within the genus Comovirus, RefSeq comovirus sequences were downloaded from NCBI and the amino acid sequence of the conserved Pro‐Pol region from RNA1 was aligned using Mafft v.7.475 with the auto option  were downloaded from the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra) and searched for the presence of ArLV1 RNA1 and RNA2 sequences with the Blastn_vdb program from the NCBI SRA toolkit v.2.9.0 .Illumina‐generated enomic Workbench v.20) against the ArLV1 sequences (MH899120.1 (RNA1) and MH899121.1 (RNA2), respectively) with options ‘low coverage definition threshold = 3’ and ‘insert N‐ambiguity symbol’. The ML phylogenies were reconstructed using the conserved Pro‐Pol region from RNA1  using IQ‐Tree v.2.0.3 with ModelFinder and 1000 ultrafast bootstrap replicates  for 36 accessions with available latitude and longitude coordinates, was made with the R package ggplot2. Isolation by distance was assessed using the Mantel test implemented in the R package ade4, where distance was calculated as Euclidean distance from the coordinates using the R package sp and genetic distances were estimated using IQ‐Tree.From the NCBI SRA output, accessions containing ArLV1 were selected based on the presence of at least 500 reads of RNA2, representing full coverage. From these, one SRA each from 38 randomly chosen accessions was selected, from which the consensus nucleotide sequences of RNA1 and RNA2 were retrieved by reference mapping  in an inoculation buffer . For inoculation, leaves of A. thaliana plants in leaf stage 8–10, or Nicotiana benthamiana plants in leaf stage 5–8 were lightly dusted with carborundum powder, and inoculum was applied by gentle rubbing. Plants were rinsed with water 10 min after inoculation. Virus symptoms were assessed visually at 7–10 d post‐inoculation (DPI). Presence of virus in inoculated plants was checked by SYBR Green quantitative reverse transcription polymerase chain reaction (RT‐qPCR) and transmission electron microscopy (TEM) with a JEM1400 Plus  using a leaf dip assay according to standard protocols . This analysis clearly identified the virus as a distinct comovirus  up to 88.2% (isolate ArLV1_A), and accession Pent‐1 (CS76209) up to 83.0%. We also identified an extremely high abundance of similar RNA1 and RNA2 reads from the same comovirus in some, but not all, RNA‐sequenced samples of accession Col‐0 at Utrecht University, with a mapping percentage of up to 90.08% of viral reads (isolate ArLV1_B) .An analysis of RNA‐Seq transcriptome datasets from several natural h et al. , hereaftrus Fig. . Samples_B) Fig. . The occA. thaliana plants, grown from infected seed batches, to N. benthamiana, known for its susceptibility to many plant viruses   Fig. . Taken tLV1 Figs , S3, indA. thaliana datasets other than the Wageningen and Utrecht datasets, we initiated a search in publicly available A. thaliana RNA‐Seq datasets. Out of a total of 6477 RNA‐Seq datasets analyzed, 547 (8.45%) contained at least 500 reads mapping to RNA1 and 500 reads mapping to RNA2 of ArLV1, indicating a full coverage of the viral genome. Altogether, ArLV1 was detected in 176 out of 711 accessions (24.75%) in the public datasets analyzed. Arabidopsis latent virus 1 RNAs could not be detected through reference mapping in a randomly selected set of 35 SRAs from the related species Arabidopsis lyrata. Therefore, the precise host range of ArLV1 remains to be determined. To assess the genetic diversity of ArLV1, nucleotide sequences from the Pro‐Pol regions of RNA1 and from the full open reading frame of RNA2 of four isolates from different A. thaliana ecotypes from Wageningen and Utrecht and 38 different NCBI‐derived datasets, each with a full coverage of RNA1 and RNA2, were used for phylogenetic analyses . RNA1 showed evidence of isolation by distance , but not RNA2 , leaving it inconclusive as to whether limited geographical dispersal played a role in diversification. To find out if ArLV1 was also present in wild A. thaliana populations, we sampled 27 different plants in Arnhem, Wageningen and Woerden (the Netherlands), where the presence of ArLV1 outside of the laboratory environment was confirmed in independent RT‐PCR assays for eight out of 27 plants   affected the area of the pectin mucilage layer, ArLV1 infection of the parent plant did not affect the size of the mucilage layer (P = 0.147)  Fig. . Likewising Fig. , suggestet al., et al., et al., et al., et al., The advent of high‐throughput sequencing technologies has greatly contributed to the understanding of the concept of holobionts . We showed the infectivity of this newly discovered virus for A. thaliana and N. benthamiana and its high transmissibility to A. thaliana progeny via seeds from ArLV1‐infected plants. The TEM studies confirmed typical comovirus particles in infected A. thaliana plants and a RT‐qPCR test was developed that allows detection of the virus in plant material.In this study, Illumina‐derived RNA‐Seq datasets from A. thaliana SRAs that were tested, 8.45% contained evidence of an ArLV1 infection, accounting for near 25% of the ‘natural’ A. thaliana accessions and some mutant lines in the dataset. This indicates that ArLV1 is present in the A. thaliana stocks of laboratories worldwide. We also confirmed its presence in wild A. thaliana populations, both in the Netherlands and in Spain. A total of 38 A. thaliana accessions were selected for a phylogenetic analysis of ArLV1 sequences, including the widely used accession Col‐0. The phylogenetic analysis divided these virus sequences into three clades with different geographical distributions, where occasional recombination and re‐assortment also occur. This, in addition to its apparent latent nature, its occurrence in plants directly collected from the wild and a large number of laboratory stocks of A. thaliana accessions collected from many geographical regions, clearly suggests that ArLV1 is a virus that has been naturally associated with A. thaliana for a long period of time.From the nearly 6500 public A. thaliana accessions in independent studies in both Wageningen and Utrecht show that an ArLV1 infection in A. thaliana can result in > 90% of virus‐specific reads. This is an indication that ArLV1 can potentially reach very high titers in infected plants. Interestingly, these large differences in ArLV1‐specific read numbers  were observed in datasets from both Wageningen and Utrecht, between individual plants from the same accession grown and processed in the same experiment. In addition, RT‐qPCR results for individual plants grown under the same conditions varied between ΔCt values of −20 and −6. The two laboratories in Wageningen and Utrecht used different plant growth conditions  should be directed to the Click here for additional data file.


In 2013, the Shanghai Hospital Development Center issued a policy to advocate public hospitals to report their information about costs on diseases. The objective was to evaluate the impact of interhospital disclosure of costs on diseases on medical costs and compare costs per case following information disclosure between hospitals of different rankings.The study uses the hospital-level performance report issued by Shanghai Hospital Development Center in the fourth quarter of 2013, which covers quarterly aggregated hospital-level discharge data from 14 tertiary public hospitals participating in thyroid malignant tumors and colorectal malignant tumors information disclosure from the first quarter of 2012 to the third quarter of 2020. An interrupted time series model with segmented regression analysis is employed to examine changes in quarterly trends with respect to costs per case and length of stay before and after information disclosure. We identified high- and low-cost hospitals by ranking them on a costs per case basis per disease group.This research identified significant differences in cost changes for thyroid malignant tumors and colorectal malignant tumors between hospitals after disclosing information. A hospital’s discharge costs per case for thyroid malignant tumors increased significantly among top-cost hospitals , while decreased for thyroid and colorectal malignant tumors among low-cost hospitals .Our findings indicate that information disclosure of costs on diseases results in changes in discharge costs per case. And low-cost hospitals continued to maintain their leading edge, whereas the high-cost hospitals changed their position in the industry by reducing discharge costs per case after information disclosure.The online version contains supplementary material available at 10.1186/s12913-023-09510-8. In China, public hospitals, which provide over 80% of overall inpatient and outpatient services, play the most important role in service delivery . HoweverUnder the fee-for-service payment system, hospitals have the incentive to increase the volume of services and to choose treatments with a greater profit margin . The ShaThe rationale behind information disclosure is compelling . InterhoIn addition, a few studies have been conducted to evaluate the impacts of information disclosure on healthcare providers and patients and these studies reported conflicting results –18. For Therefore, the principal objective of this study was to evaluate the impact of interhospital disclosure of costs on diseases based on hospital-level performance reporting on medical costs and compare costs per case following information disclosure between hospitals of different rankings. Additionally, in order to further explore the potential reasons for the change of costs, we also analyzed the drug costs and consumable costs. With the results of our study, we aim to provide an insight into the role of performance reporting in the healthcare industry.This study uses interrupted time series with segmented regression analysis to evaluate the impact of information disclosure through hospital-level performance reporting. The intervention under study is the implementation of an internal policy in January 2013 that was passed at the Shanghai Hospital Development Center advocating hospitals to report information about costs on diseases. The Shanghai Hospital Development Center, as a municipal government bureau, is responsible for the investment, construction, operation, management and assessment of municipal public hospitals. In October 2013, the Shanghai Hospital Development Center released the first report to hospitals, covering medical cost information from January to August. From the fourth quarter of 2013, Shanghai Hospital Development Center began to publish quarterly reports for interhospital use. Hence, in this quarterly data-based study, the pre-intervention period included the first quarter of 2012 to the third quarter of 2013, whereas the post-intervention period included the fourth quarter of 2013 to the third quarter of 2020. This change was applied across 33 tertiary public hospitals in Shanghai with the exclusion of one hospital with missing data and four newly built hospitals in the suburban area.Our data source included hospital-level costs from the first quarter of 2012 to the third quarter of 2020, collected from the hospital-level performance report issued by Shanghai Hospital Development Center, which was established in 2005. Before the disclosure of costs on diseases information of public hospitals in the fourth quarter of 2013, the report only included quarterly data at the hospital level, but did not include data for each disease. Therefore, we assume that before the disclosure of costs on diseases, the trend of expenses of each disease is consistent with the trend of total costs in each hospital. Accordingly, we considered the cost of each disease from the fourth quarter of 2013, based on the quarterly rate of change in the total costs, and estimated the costs per case for each disease before the disclosure of costs on diseases. Municipal-level public hospitals are established by the municipal government unit and are usually larger hospitals. The contents disclosed in the hospital-level performance report include the following information: the hospital name, medical costs , admission and discharge-related information per case, disease-related information , and hospital characteristics   for analysis. Subsequently, we separately analyzed the impact of information disclosure on discharge costs per case, drug costs per case, consumable costs per case, and length of stay across two diseases, as well as compared discharge costs per case, drug costs per case, consumable costs per case, and length of stay separately for each disease following information disclosure between hospitals of different rankings for the same disease. Hospitals were stratified according to quartiles of average costs per case in the fourth quarter of 2013, where Q1 indicated hospitals with the lowest costs and Q4 indicated hospitals with the highest costs  , number of beds, proportion of cases with targeted disease, and fixed effects of seasons to assess the robustness of the model. All analyses were performed using Stata 16.0 with the ce level .Table For both thyroid and colorectal malignant tumors, there were decreasing but insignificant step change in discharge costs per case  when information was disclosed. Ascending change in trend of discharge costs per case after information disclosure were observed in thyroid malignant tumors . In contrast, discharge costs per case in colorectal malignant tumors presented significant decrease in trend of discharge costs per case  after information disclosure . However, both thyroid  and colorectal malignant tumors  presented significant descending changes in trend of drug costs per case , as well as significant decrease in both step change  and change in trend  among Q4 hospitals. For colorectal malignant tumors, all 4 subgroups experienced significant descending change in trend of drug costs per case, among which Q4 hospitals demonstrated the largest decrease  , Q4 hospitals presented immediate decrease for colorectal malignant tumors , while Q2 and Q3 hospitals experienced insignificant step change. Ascending change in trend of consumable costs per case were observed in both thyroid and colorectal malignant tumors across the four subgroups  and colorectal malignant tumors . Increase in trend of length of stay were observed in both diseases but only significant in thyroid malignant tumors   among cases with colorectal malignant tumors (supplementary Table The sensitivity analysis in which we adjusted season fixed effects, hospital type, number of beds and proportion of cases with targeted disease gave similar but more precise results compared with the primary analysis. The findings produced smaller step changes, and the directions of pre-disclosure and post-disclosure trends were consistent with those observed in the main analysis. However, more statistical significance in changes was found compared with in the initial analysis Table .In this study, disclosure of information among public hospitals in Shanghai had effect on costs per case in each hospital. For malignant thyroid and colorectal tumors, the discharge costs per case presented an insignificant downward trend immediately upon information disclosure, but a significant different trend change subsequently. Meanwhile, the results showed that information disclosure resulted in a significant increase in the quarterly trend of discharge costs per case among low-cost hospitals, whereas a slight decrease in the discharge cost trend was observed among high-cost hospitals.It is important to note that significant declines in both drug expenditures and length of stay were detected. In this study, the reduction of discharge costs per case may be related to reduction in unnecessary drug usage or excessive hospitalization. Moreover, the reduction in the consumable costs per case is not yet demonstrated. In addition, in the fourth quarter of 2013, the main policy that affected the health cost was the disclosure of costs on diseases, and there was no other policy that directly affected the costs. Therefore, the time displacement of the introduction of different reform measures, to some extent, eliminates the interference of other policy factors on information disclosure of this study.Our results provide evidence that information disclosure did indeed generate effects partially consistent with the intended purposes. The findings suggest that the cost change was primarily driven by disease information, which was presented in the form of a series of cost and efficiency indicators. For public reporting what matters most is that hospital managers are shown to meet acceptable performance standards . For exaIn addition, similar information reported high- and low-cost hospitals by rank-ordering them based on costs. This can have a powerful effect on reputation, as high-cost hospitals can easily imitate the disclosure behavior of low-cost hospitals, as we observed in this study. The findings indicated that making interhospital information public stimulated medical cost reduction in low-cost hospitals. Concern for public image appeared to be a key motivator for hospitals’ medical cost change efforts , 27. In It is also worth noting that the findings in this study were entirely driven by supply-side responses to performance information. Performance information generally has decision value, and as stated by Narayanan and Davila : “Most fTo the best of our knowledge, this is the first study to evaluate the effect of information disclosure on medical costs among medical organizations. We believe that our findings will not only have a strong positive impact on medical cost change efforts but it will also enable the formation of an effective information disclosure form, which in turn could help improve and enhance the supervision and governance among medical institutions in Shanghai. Moreover, this form of information disclosure, once fully established, can give provider-specific performance information within the industry. Further, the decision making by hospital can be better informed. Finally, our study can serve as useful background research for further large-scale, multicenter studies and for research after comprehensive medical information disclosure.Our study does have limitations that need to be acknowledged. First, in our analysis, we examined changes in costs per case over approximately 27 quarters after the disclosure of disease information. Further high-quality studies should focus on evaluating changes in efficiency and quality of services provided that we were unable to observe with the current data. Further, more importantly, the degree to which the observed increased quality and efficiency improvement efforts yield actual improvements in outcomes should be observed. Second, we could not examine other effects of hospital-level changes that may be important, such as, different personal preferences of executives at the hospital level, different cultural backgrounds of hospitals, and different strategies used to confirm the development goals. Examination of these issues was beyond the scope of the current study. These will be critical topics for future research. Third, although this research cannot draw a firm causal link between information disclosure and outcomes in light of a non-experimental design, an interrupted time series design was used to study time trends in an adjusted analysis for important prognostic factors and for exploring the impact of information disclosure on outcomes. We did not identify significant changes in healthcare-related policies in fourth quarter of 2013, which reduces the concern that we may be misattributing changes in outcomes to factors other than the intervention in question. Finally, because of the limitation of sample selection, the results of our study can only represent the cities with comparable sample GDP levels. Future studies are needed to disentangle the impact of these policies.In conclusion, information disclosure resulted in a significant increase in the quarterly trend in discharge costs per case among low-cost hospitals, whereas a slight reduction in the costs among high-cost hospitals was seen. Our findings support the continued disclosure of medical information, improvements in the disclosure form, and enlargement of the public reporting systems in China, given its potential for triggering changes in healthcare costs. In future studies, evaluating the types of information or presentation methods that are effective in making medical decisions and ultimately providing care will help verify our findings.Below is the link to the electronic supplementary material.Supplementary Material 1


Pseudomonas aeruginosa PAO1 as a model microorganism. The NO treatment involved the injection of NONOates (NO donor compounds) into mature biofilms, while QQ was implemented by immobilizing QQ bacteria (Escherichia coli TOP10-AiiO or Rhodococcus sp. BH4) in alginate or polyvinyl alcohol/alginate beads to preserve the QQ activity. When QQ beads were applied together with (Z)-1-[N-(3-aminopropyl)-N-(n-propyl) amino]diazen-1-ium-1,2-diolate (PAPA NONOate), they achieved a 39.0% to 71.3% reduction in biofilm formation, which was substantially higher compared to their individual applications (16.0% to 54.4%). These findings highlight the significant potential of combining QQ and NO technologies for effective biofilm control across a variety of processes that require enhanced biofilm inhibition.Membrane biofouling is an inevitable challenge in membrane-based water treatment systems such as membrane bioreactors. Recent studies have shown that biological approaches based on bacterial signaling can effectively control biofilm formation. Quorum quenching (QQ) is known to inhibit biofilm growth by disrupting quorum sensing (QS) signaling, while nitric oxide (NO) signaling helps to disperse biofilms. In this study, batch biofilm experiments were conducted to investigate the impact of simultaneously applying NO signaling and QQ for biofilm control using  Membrane bioreactors (MBRs) combining biological treatment and membrane filtration processes allow for the efficient removal of suspended solids, organic matter, nutrients (such as nitrogen and phosphorus), and even micropollutants ,2. Moreo1R2N−(NO−)−N=O, where R1 and R2 represent alkyl groups, in both batch biofilm test and continuous water treatment systems diazen-1-ium-1,2-diolate , (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate , and (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate  were purchased from Cayman Chemical . Stock solutions of the NO donors were prepared by dissolving them in 10 mM NaOH to prevent NO release before the experiment.Various NONOates, which can release NO spontaneously at ambient temperatures, were used as NO donors. (Z)-1-[E. coli TOP10-AiiO or Rhodococcus sp. BH4) at an OD600 of 3. The alginate beads were prepared based on a previously reported method in which a 1% (w/v) alginate–bacterial cell mixture was dropped into a 4% (w/v) calcium chloride solution [w/v) PVA  and 1% (w/v) alginate–bacterial cell mixture was dropped onto a 7% (w/v) boric acid and 4% (w/v) calcium chloride solution, followed by incubation for 1 h for the first cross-linking process [Two kinds of hydrogel beads, alginate and polyvinyl alcohol (PVA)/alginate, were used to immobilize QQ bacteria . The bacterial solutions (1 mL) were placed in the wells of a 24-well microtiter plate and incubated for 6 h at 30 °C with shaking (180 rpm). After 6 h of culture, 10 mM NaOH (Control) or the prepared NO donor stock solution was added to each well to obtain final concentrations of 50 and 100 μM. The plate was incubated with shaking for an additional 30 min after the injection of the NO donor.The impact of NO and/or QQ on the biofilm formation of ocedures . The modP. aeruginosa PAO1 as described above, transwells were installed in the 24-well plate; then, a QQ and a control bead (immobilizing E. coli TOP10-Empty) were placed into the corresponding transwell.To test the individual or combined effects of QQ, transwells  were used to prevent the QQ beads from directly contacting the 24-well plates . Followiw/v) CV for 20 min and washed two times with phosphate-buffered saline buffer. The wells were then destained with 99.9% ethanol, and the quantity of biofilm was determined by measuring the absorbance at a wavelength of 550 nm using a microtiter plate reader .The biofilms formed on the well surface were quantified using crystal violet (CV) assays. The biofilm remaining in the wells was stained using 0.1%  by the combination of QQ and NO, the biofilm reduction (%) by QQ, and the biofilm reduction (%) by NO, respectively.The expected combined effect of biofilm reduction was estimated using the following :Rexp = 1AHLs were quantified using a luminescence-based bioassay method that has been described in a previous study . The samQQ activity tests were conducted to confirm the AHL degradation capability of the QQ beads. Twenty milliliters of 200 nM OHHL were placed into a conical tube, along with 50 QQ beads. The mixture was cultured at 30 °C and 100 rpm, and 500-μL samples were collected at reaction times of 0, 1, 10, 30, 60, 120, and 180 min. These samples were centrifuged at 13,500 rpm for 3 min, and 200 μL of the supernatant was treated at 95 °C for 2 min before storing it at −20 °C. Then, the residual OHHL concentrations at each time point were measured using the AHL bioassay method described in t [The rate coefficient of QQ was expressed as a pseudo-first-order reaction according to the degradation rate of the OHHL at time t :(2)d[AHLE. coli TOP10-AiiO or Rhodococcus sp. BH4 strains was measured as shown in E. coli TOP10-AiiO or Rhodococcus sp. BH4 was tested in suspension, both of which completely degraded OHHL within several minutes [−1) compared to BH4 alginate beads (0.95 h−1), resulting in the complete degradation of 200 nM OHHL in 120 and 180 min, respectively. While there was a difference in the AHL degradation rate between the two beads, both beads effectively degraded AHL. Although the rate of AHL degradation may decrease when microbial cells are confined within hydrogel beads compared to when they are in a suspended state, the protection of QQ bacteria can provide a safer environment for continuous process applications [The QQ activity of alginate beads entrapping  minutes ,25. Thisications .E. coli TOP10-Empty). The addition of the BH4 alginate bead also yielded a substantial biofilm reduction (30.7%) compared to the vacant bead . In any case, the results confirmed that quenching AHL using alginate beads with immobilized E. coli TOP10-AiiO or Rhodococcus sp. BH4 can mitigate the biofilm formation of P. aeruginosa PAO1.To evaluate the efficacy of biofilm reduction by the QQ beads, a biofilm test was conducted using a microwell plate setup with a transwell, as shown in To assess the NO-induced dispersal of biofilm cells, we selected three specific NO donors: PAPA NONOate, Spermine NONOate, and DPTA NONOate. These compounds have half-lives in water ranging from several minutes to a few hours, which are neither too short nor too long . Thus, tThe effectiveness of different types and concentrations of NO donors in mitigating biofilm formation was assessed by quantifying the percentage of biofilm reduction following a 30 min incubation period with NO donors. The results showed that both 50 μM and 100 μM PAPA NONOate additions led to substantial reductions  in biofilm formation . This waAfter confirming the biofilm reduction effects of the individual QQ and NO treatments, we investigated their combined inhibitory effect on biofilm formation. As shown in The BH4 alginate beads also exhibited a similar trend when combined with AiiO alginate beads and PAPA NONOate beads. When a BH4 alginate bead and 50 μM PAPA NONOate were applied separately, they showed 23.1% and 42.0% reductions in biofilm formation, respectively b. When tP. aeruginosa PAO1 is known to produce more than two types of AHLs, including N-butanoyl-homoserine lactone and N-3-oxo-dodecanoyl-homoserine lactone. The biosensor A136 used in this experiment can respond to various types of AHLs; therefore, bioassays using this biosensor cannot determine which AHLs were generated and degraded. However, the luminescence values obtained from the sample can be represented as OHHL equivalent values using the standard curve established with OHHL [ith OHHL .P. aeruginosa PAO1 within the transwell microplate setup were effectively degraded by the AiiO bead. The injection of PAPA NONOate did not have any impact on either the production or degradation of the AHLs. In the case of the BH4 alginate bead, 68.2% of the AHLs were found to be degraded compared to the well where a vacant bead was injected (In the absence of a PAPA NONOate injection (control), an 85.3% reduction in AHL levels was observed upon the injection of an AiiO alginate bead compared to the well with an Empty alginate bead a. This cP. aeruginosa PAO1 utilizing an extracellular signal molecule called Pseudomonas quinolone signal (PQS) in addition to AHL. PQS influences the synthesis of QS-dependent extracellular products such as pyocyanin and elastase, which are associated with biofilm formation [This discrepancy could be attributed to ormation . Moreoveormation .P. aeruginosa PAO1, thus inhibiting biofilm formation, although the reduction rate was limited (16.9–23.1%). However, when NO was applied together with QQ beads, the reduction rate increased to 44.6–59%, indicating that the combination of the two techniques can achieve more effective biofilm reduction.In summary, the QQ beads effectively degraded the AHLs produced by Despite their high biocompatibility and ease of preparation, alginate beads have the disadvantages of low physical strength and stability ,33,34. T−1, respectively. Compared to the alginate beads, these are somewhat reduced values, and the key trend observed is an unavoidable trade-off that occurs as the physical strength of the bead increases.As expected, the addition of PVA resulted in beads with higher physical strength and stability, but their AHL degradation ability showed a slight decrease . Over 12As shown in This study investigated the combination of QQ and NO signaling for effective biofilm control. Biofilm tests were performed using a microplate equipped with a transwell that confirmed that biofilm reduction occurred when the QQ bacteria-immobilized beads and NO donors were applied separately. When these two approaches were combined, they led to a more significant biofilm reduction compared to the outcomes of their individual applications, thus demonstrating the effectiveness of their combination. By comparing the results with the expected combined effect calculated mathematically, no synergistic or antagonistic effects were observed in their combination. Although this research study has limitations in terms of its use of a monospecies microorganism for a batch-type biofilm test, the results highlight the promising prospects of combining these two technologies for more efficient biofilm inhibition in various processes requiring biofilm control, including membrane filtration processes. By exploring these possibilities and confirming their effectiveness in future laboratory- or pilot-scale water treatment processes, we can assess their practical applicability in full-scale processes.