Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions–PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS. The central nervous system (CNS) is one of the most common extra-respiratory tract sites of infection for influenza A viruses. In ferrets—an animal model used to study the pathogenesis of influenza—highly pathogenic avian influenza H5N1 virus can enter the CNS via the olfactory nerve, resulting in the development of a severe meningo-encephalitis. In the present work, we evaluated the evolutionary dynamics of the virus populations entering and spreading throughout the CNS. We show that once inside the CNS, H5N1 viruses can acquire mutations that increase the polymerase activity in vitro. In vivo, the virus bearing these mutations retained its capacity to infect the CNS but showed reduced spread to other anatomical sites. Analysis of virus populations revealed that infection from the nasal turbinate to the olfactory bulb did not present a genetic bottleneck, suggesting a diffusive passage of viruses from the nasal cavity to the CNS. Inside the CNS, specifically in the brainstem, we found signs of positive selection. These findings support the idea that H5N1 viruses can invade the CNS efficiently via the olfactory nerve, and have the potential to adapt to the CNS. Central nervous system (CNS) disease is the most common and potentially fatal extra-respiratory tract complication of influenza A virus infection ,2. The aOnce inside the CNS, virus populations encounter cells of neuronal and glial origin that differ phenotypically from cells of the respiratory tract. Viral adaptation to these cells could occur if conditions are conducive to positive selection. A major prerequisite of positive selection is genetic variation. It is therefore relevant to evaluate whether a genetic bottleneck constricts the diversity of viral populations accessing the CNS, which would reduce the potential for adaptation. Whether or not it is subject to a bottleneck, the diversity of the seed population can however be augmented through de novo mutation, resulting from the error-prone replication of the viral genome by the influenza virus RNA-dependent RNA polymerase. The potential for positive selection to lead to adaptation is then further reliant on the extent to which selection and drift are active in the virus population . Selectiin vitro and in vivo, of the virus with the CNS-associated mutations (H5N1-CNS virus) and compared it with the wild type virus (H5N1-WT). We found that the H5N1-CNS virus demonstrated higher polymerase activity than the wild type virus in vitro but did not show increased replication in the CNS in vivo. Analysis of viral variant dynamics within the infected ferrets revealed that viral dissemination into the CNS via the olfactory nerve was not subject to stochastic loss of diversity through genetic bottlenecks. Importantly, the H5N1-CNS virus also showed signs of positive selection in the brainstem, suggesting that H5 HPAI viruses are able to evolve under positive selection within the CNS.In the present study, we evaluated an H5N1 HPAI virus that acquired three substitutions–PB1 E177G and A652T and NP I119M - in the CNS of an experimentally inoculated ferret in order to evaluate the adaptive potential of H5N1 virus to the CNS. This ferret developed a severe meningo-encephalitis associated with virus spread throughout the CNS, including the olfactory bulb, cerebrum, cerebellum, and spinal cord  TCID50/mL for H5N1-WT group and 105.7 [0.8] TCID50/mL for H5N1-CNS group) [2.8 [0.9] TCID50/mL) and for H5N1-CNS at 5 dpi (103.1 [0.8] TCID50/mL). At 1 dpi, viral titers in nose swabs from ferrets inoculated with H5N1-WT virus (103.8 [0.5] TCID50/mL) were significantly higher compared to ferrets inoculated with H5N1-CNS virus (102.1 [0.5] TCID50/mL)  of 10S group) , S4 DataID50/mL) , S4 DataMacroscopic examination revealed a moderate-to-poor physical condition in two and one ferret from the H5N1-WT and H5N1-CNS groups, respectively at 6 dpi and good physical condition of all other ferrets at 3 and 6 dpi . In tissIn tissues of the CNS, virus was detected in the olfactory bulb from both H5N1-WT and H5N1-CNS virus inoculated ferrets at 3 and 6 dpi with significantly higher titers in the olfactory bulb of H5N1-WT virus inoculated ferrets compared to ferrets inoculated with H5N1-CNS virus , S5 DataWe also examined the presence of virus in the heart, liver, spleen, adrenal gland, kidney, pancreas, jejunum, blood, serum and plasma. Virus was sporadically detected in the liver, spleen, adrenal, kidney, pancreas, and jejunum of ferrets inoculated with H5N1-WT, but not in ferrets inoculated with H5N1-CNS .Together, these data suggest that H5N1-CNS virus retained the capacity to invade and spread throughout the CNS, although virus titers were lower in the olfactory bulb, lower respiratory tract and extra-respiratory organs compare to ferrets inoculated with H5N1-WT.In the respiratory tract, microscopic examination revealed mild-to-moderate rhinitis and necrosis in the epithelium of the nasal mucosa in all ferrets , Fig 4. in vivo.To assess the stability of the CNS-mutations in vivo, and to ensure that the H5N1-WT virus did not acquire mutations at these sites during the experiment, viruses from the respiratory tract and CNS were sequenced using the Illumina platform. The original sequences at the sites of the three CNS-associated mutations were maintained in all ferrets Tables  Tables, 4 TCID50/g, which included one ferret at 3 dpi and three ferrets at 6 dpi. Contrary to expectation for a stringent bottleneck, we found increased diversity in the olfactory bulb compared to the nasal turbinates in all four animals  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Sabra L. KleinAssociate EditorPLOS PathogensAna Fernandez-SesmaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************The concerns raised by Reviewer 2, in particular, should be carefully considered including how to address the concern that the CNS-associated virus mutations do not cause the same phenotype in ferrets. The biological relevance of the virus mutations must be addressed.Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Siegers et al have performed detailed experiments to explore the mechanisms by which the HPAI H5N1 virus evolves in the mammalian CNS. The authors demonstrate that an HPAI H5N1 virus can enter the CNS via the olfactory nerve without a genetic bottleneck. And the authors conclude that viral dynamics within the CNS are conducive to positive selection, which gives the potential for improvement of viral replicative capacity and may lead to more severe disease.Reviewer #2: Professor van Riel and colleagues report findings from in vitro and in vivo studies that examined the contribution of specific amino acid residues of an H5N1 influenza A virus strain to CNS invasion and neurological disease. Cell culture studies revealed that the CNS-associated mutations enhanced the in vitro viral replication phenotype. Follow-up analysis with the ferret model of influenza and an H5N1 influenza virus strain harboring these putative CNS-associated mutations revealed an attenuated viral phenotype, including restricted tissue distribution. The results suggest the consequence of positive selection on viral phenotypes and potential evolution of influenza viruses.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1.Lines 104-111. Please explain why the change in the surface potential from negative to neutral in the apo form and from moderately negative to moderately positive charge in the promotor-bound form influence interactions with potential (host) binging factors?2. Lines 133-137. Both the single substitution PB1-652T and the combined substitutions PB1-652T/177G with NP-119M can result in a significant increase of polymerase activity in A549 cells. However, the combination of PB1-652T with NP-119M maintained wild-type levels of polymerase activity. Please give some reasonable explanation for these phenotypes that seem contradictory.Reviewer #2: Genetic analysis of the viral population within the nasal cavity did not reveal a potential bottleneck or selection of virus with CNS-associated mutations. These results suggest the possibility that viral quasispecies contribute to the observed CNS invasive phenotype, neurological disease, and broad tissue distribution, which was lost by selection of a specific viral strain with the CNS-associated mutations. Another limitation of the study is that this phenotype was observed for one infected ferret. It is unclear how the host-specific response of this one animal contributed to the observed phenotype and if the selective forces are reproducible in shaping evolution of influenza viruses.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1.It is better to specify which amino acids were site-directed mutated to generate the H5N1-CNS virus in Materials and methods or anywhere proper.2.Line 125. Where are the large positively charged RNA-biding grove? Please highlight it in the supplementary figure 5.3. Line 127. “different ‘human’ cell types” is suggested for more specific description.4.Fig 5. Whether the difference of genetic diversity is significant statistically?Line 205.5.Line 205. The study aimed to explore the CNS-associated mutations of an H5N1 HPAIV, did not involve reassortment events among different viruses, so “variant” may be appropriate than “genotypes”.6.Lines 222, 223. Please provide more information to explain what the sentence “it is highly unlikely for common variants penetrated the CNS from the nasal turbinate” .7.Line 278. what’s means of “stochastic fixation”？8. Statistical method in Fig. 2. Whether one-way ANOVA should be used instead of two-way ANOVA?Reviewer #2: not-applicable**********what does this mean?). 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Read more information on sharing protocols at  10 Jan 2023AttachmentRebuttal letter.docxSubmitted filename: Click here for additional data file. 16 Feb 2023Dear Dr van Riel,We are pleased to inform you that your manuscript 'Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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KleinAcademic EditorPLOS PathogensAna Fernandez-SesmaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************************************************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The manuscript modification meets the requirement.Reviewer #2: This in vivo study examined the replication phenotype and tissue distribution of H5N1 highly pathogenic avian influenza virus in the ferret model of influenza. The authors note that the findings are limited to one animal but the data indicate the impact of genetic evolution for virus replication and viral pathogenesis. Overall, the authors deserve high marks for execution and scholarship.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: NoReviewer #2: Not applicable.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: NoReviewer #2: Not applicable.**********what does this mean?). 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Plant viruses, as obligate intracellular parasites, rely exclusively on host machinery to complete their life cycle. Whether a virus is pathogenic or not depends on the balance between the mechanisms used by both plants and viruses during the intense encounter. Antiviral defence mechanisms in plants can be of two types, i.e., natural resistance and engineered resistance. Innate immunity, RNA silencing, translational repression, autophagy-mediated degradation, and resistance to virus movement are the possible natural defence mechanisms against viruses in plants, whereas engineered resistance includes pathogen-derived resistance along with gene editing technologies. The incorporation of various resistance genes through breeding programmes, along with gene editing tools such as CRISPR/Cas technologies, holds great promise in developing virus-resistant plants. In this review, different resistance mechanisms against viruses in plants along with reported resistance genes in major vegetable crops are discussed. Plant viruses cause significant yield loss in the cultivation of many agricultural and horticultural crops around the world. Conventional methods of virus control have been used for a long time, including crop rotation, other types of cultivation, removal of infected plant debris, and chemical control of insect vectors. Since these methods have very little impact in the mitigation of virus diseases, numerous inventive methods to manage viral infections in plants have been developed as a result of a growing understanding of the molecular genetics of plant viruses as well as of the hosts’ defence mechanisms. The primary defence mechanisms against viruses in plants include innate immunity, RNA silencing, translational repression, dominant resistance genes, and autophagy-mediated degradation . The risThe virus infection cycle within the plants can be narrated as described by Calil and Fontes . VirusesThis infection process is targeted by several host defence systems which help the host to counter the virus attack. There are several points in the infection cycle which can be targeted by host defences such as the uncoating of the virus capsid, translation events, encapsidation, and cell-to-cell movement .This can be divided into two types: Natural resistance to plant viruses and Engineered resistance to plant viruses.Possible natural resistance mechanisms against viruses include innate antiviral immunity, translation repression, small RNA-mediated antiviral defence, dominant viral resistance genes, resistance to virus movement, autophagy, and cross protection.1)). PTI is the initial mechanism by which plants detect the microbes at the cell membrane through detection of conserved PAMPs by extracellular pattern recognition receptors (PRRs) [Arabidopsis [Arabidopsis showed PTI-like responses due to the coat proteins of tobacco mosaic virus and potato virus X, respectively [There are two layers of plant immune responses against microbial pathogens, i.e., PAMP (pathogen-associated molecular pattern) triggered immunity (PTI) and Effector-triggered immunity (ETI) (s (PRRs) . PRRs bes (PRRs) . Activats (PRRs) . Althougs (PRRs) . Howeverbidopsis . In anotectively ,9.Pathogens in response to PTI introduce particular proteins called effectors in plant cells to weaken PTI-mediated defence. In response to these effectors, host plants depend on specific intracellular receptors known as R gene proteins which cause direct or indirect identification of the pathogen effector molecules. These R gene products can block the effectors and activate the effector-triggered immunity (ETI) . R genes2)). The most thoroughly studied RIP with antiviral activity is the pokeweed antiviral protein (PAP) from Phytolacca americana. PAP slows down the spread of various plant viruses, including the cauliflower mosaic virus, potato virus X, and cucumber mosaic virus [Arabidopsis thaliana and Chenopodium foetidum [The initiation of translation is a critical step in protein synthesis that demands an array of eukaryotic initiation factors (eIFs). The translation of eukaryotic mRNA hinges on the association between the translation eukaryotic initiation factor 4E (eIF4E) and their 5′ m7 G cap structure, and the 3′ polyA tail’s interaction with the polyA-binding protein (PABP) also heightens the process. Although translation factors are not typically encoded by viruses, they have developed several techniques to hijack translation factors from their hosts, resulting in the promotion of viral RNA translation while compromising the translation of endogenous mRNAs. Mutated isoforms of the translation IFs, i.e., eIF4E and eIF4G have been found linked to many plant recessive resistance genes. These mutations typically prevent host factors from interacting with viral RNAs or proteins to suppress viral protein translation, an endogenous antiviral mechanism that has emerged recently . Recessiic virus . In the ic virus . Additiofoetidum .3b))Small RNAs (sRNA) are essential for the epigenetic and post-transcriptional control of gene expression in plants throughout growth, developmental, and biotic/abiotic stress responses. Recent research has highlighted the role of two distinct families of small RNAs, known as small interfering RNA (siRNA) and micro RNA (miRNA) with respect to biotic stress responses in plants. By regulating the gene expression of the modulators of host defence pathways, these sRNAs activate antiviral defence during virus infection. RNA silencing, commonly referred to as RNA interference (RNAi), is a conserved evolutionary method for regulating endogenous expressions of genes and preventing the entry of foreign nucleic acids such as viruses and transposons . The pla3a)). Recently, the roles of various miRNAs, such as miR168, miR528, miR319, and miR444 in rice antiviral immunity have been identified. By modifying jasmonic acid (JA) signalling, MiR319 has been reported to confer antiviral resistance to rice against the rice-ragged stunt virus and to wheat against the rice black-streaked dwarf virus. [Unlike siRNAs, miRNAs are endogenously produced non-coding short RNAs by RNA polymerase II from ssRNA precursors with a hairpin structure. . Along wf virus. .Arabidopsis, and it particularly provides resistance to a number of potyviruses, including TEV, lettuce mosaic virus (LMV), and plum pox virus by inhibiting their long-distance movement [Arabidopsis called Jacaline-type lectin required for potexvirus resistance 1 (JAX1) imparts broad spectrum resistance to potexviruses at the initial infection stage by suppressing the activity of viral RNA-dependent RNA polymerase (RdRp) [Musa paradisiaca, attaches to the TMV CP and inhibits virus infection in plants [A few dominant resistance genes that function independently of the traditional innate immune signalling pathway have also been discovered in the past ten years. These dominant resistance genes express proteins that cannot be incorporated into the so-called plant innate immunity because they differ structurally from conventional R proteins. The majority of these dominant resistance genes inhibit the action of viral proteins by interacting with them in a direct manner. These dominant resistance gene products are referred to as atypical dominant viral resistance proteins (ADVRPs). The majority of ADVRPs that have been identified so far are from a protein family called lectin. Restricted TEV movement (RTM) 1, a member of the lectin-protein family is found in movement . A lectie (RdRp) . BanLec-n plants .cum1 and cum2 mutations in Arabidopsis cause reduced multiplication of CMV, thus rendering its movement in adjacent cells [Pvr11 and Sbm1 were found to be mutated at a locus expressing eIF4E in pepper and pea, respectively [RTM1, RTM2, and RTM3 prevent systemic tobacco etch virus (TEV) movement between plant cells. These genes interact with the virus CP and are expressed in phloem sieve elements [Ny-1 gene confers potato virus Y (PVY) resistance in potatoes [For plant viruses to infect the entire plant system, they must spread from the initially infected cells to nearby ones. This must occur after the virus multiplication has begun within the cytoplasm and/or nucleus of a plant cell in a susceptible host. Host resistance to viral infection is evident when the virus only appears to affect one or a small number of cells but is unable to move past this initial focus of infection. At this point, resistance can be brought on by active host defence systems that swiftly thwart virus spread or by a breakdown in the connections between plant and viral components necessary for cell-to-cell movement. It is known that a variety of host gene alterations hinder the cell-to-cell movement of plant viruses. The nt cells . Pvr11 aectively ,31. eIF4elements . Similarelements . By indupotatoes .ATGs) get activated and initiate the formation of autophagosome, which ultimately fuse with the vacuole leading to the vacuolar degradation of autophagosome contents which also harbour viral particles (4)). As TMV aggregates at infection sites in Beclin-1 or ATG7 silenced plants, it has been hypothesised that autophagy contributes to plant defence against viruses. It is interesting to note that induction of autophagy extends beyond the TMV infection sites to uninfected neighbouring tissues, where it prevents cell death [ATG8 specifically interacts with the beta-satellite (βC1) of the CLCuMuV [ATG6, also known as Beclin1) to prevent viral replication [Arabidopsis. [Proteins and defective organelles are transferred to vacuoles or lysosomes for destruction through autophagy, which is an evolutionarily conserved intracellular degradation mechanism . Autophall death . Several CLCuMuV . Not onllication . Additioidopsis. .Cross protection has been utilised to manage viral diseases together with protein and RNA-mediated resistance. By infecting the host plant with a mild strain of the virus, the resistance is conferred effectively. The infection caused by attenuated strain makes the affected plant immune to further infections by a viral strain that is closely related to the inoculated virus. After the attenuated strain has been inoculated, it may trigger strain or sequence-specific resistance against the challenger virus. In a number of experiments, the effect of coat protein on cross-protection reactions has been examined. When systemically expressed using PVX as a virus vector, various TMV CP mutants with altered CP aggregation demonstrated that the CP mutants with high assembly capacity offered effective cross protection against TMV infections. TMV cross protection and CP-mediated resistance may therefore depend on CP’s capacity to prevent the challenging virus from uncoating its capsid . NumerouEngineered resistance against plant viruses can be divided into two parts: pathogen-derived resistance (PDR) and gene editing technologies.The majority of the functions played by plant viruses during the replication cycle are well-characterised since they have short genomes and few genes. Viruses are suitable targets for developing artificial resistance based on the concept of pathogen-derived resistance (PDR). Sanford and Johnston were the ones who originally introduced this idea . The mosThese are further divided into Engineering ZFN or TALEN-based resistance and CRISPR/Cas technology.Arabidopsis [Rep genes tobacco curly shoot virus (TbCSV) and tomato yellow leaf curl China virus (TYLCCNV) in tobacco plants, which showed a considerable suppression of viral genome replication [It only became possible a decade ago to manipulate the genetic material in many cellular organisms, thanks to a novel technique called genome editing. The first-generation genome editing technologies are zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) . TALENs bidopsis . This prbidopsis . The BSClication .A promising tool for plant genome modification is the CRISPR /CRISPR associated 9 (CRISPR/Cas9) system. The Cas9 nuclease and a single guide RNA (sgRNA) with a 20-nucleotide-long spacer sequence which guides the Cas protein to the DNA or RNA target make up the CRISPR/Cas machinery. Recently, various research has revealed the generation of virus-resistant plants with CRISPR/Cas9 technique having resistance durability . By disrStreptococcus pyrogenes  after it targeted the viral dsDNA for cleavage [Arabidopsis. It was reported that Arabidopsis transgenic plants involve the expression of several sgRNAs that target the CaMV CP coding sequences. Moreover, it was found that siRNAs with 21–24 nucleotides (nt) were produced from sgRNAs, most of which were mapped to 3’ end of the sgRNA backbone region and less frequently to the spacer region, which binds to the CaMV coat protein-coding sequences. This finding proved CRISPR/Cas9 effectiveness against double-stranded DNA viruses as well [DNA is the target molecule for the CRISPR/Cas9 technology derived from yrogenes A. In ordn plants ,60,61. Tcleavage . A similcleavage . CRISPR/ as well .Francisella novicida, a pathogenic bacterium, whose FnCas9 orthologue targets RNA has been exploited to neutralise various human and/or plant viruses. Numerous Cas protein variants, including the Cas9 from Francisella novicida (FnCas9) and Cas13a from Leptotrichia shahii (LshCas13a) or Leptotrichia wadei (LwaCas13a), have been reported to target RNA in vivo, which shows promising results against RNA viruses .Avr) gene products of the pathogen [Avr determinants is dependent on precise molecular interactions, virus resistance genes appear to have more specificity than one might anticipate [It was found that around 80% of the host-virus combinations have monogenic resistance, with the remaining 20% being either oligo- or polygenic . Genes tpathogen . Membersticipate . While aticipate . As viruticipate . In the Despite great progress over the past decade, many questions remain unanswered, such as the molecular mechanisms controlling incompatible virus-plant interactions, the existence of PAMPs from viruses and their associated PRRs, and the mechanisms controlling the mixed infection-assisted resistance loss in host plants. Increased understanding of the natural resistance mechanisms of plants against viruses has been attained through in-depth investigations on virus/plant interactions employing innovative technologies. Numerous antiviral defence strategies have also been identified by utilising the growing understanding of the molecular interactions between viruses and their host plants. The use of well characterised natural resistance genes that can be inserted by marker-assisted breeding techniques is very promising along with genome editing tools, especially CRISPR/Cas-mediated technologies which stand out for their simplicity, robustness, and versatility in developing virus-resistant plants.A promising approach to building genetic resistance to viruses in plants is to use host genes with antiviral activity. However, viruses are rapidly evolving and have a remarkable ability for mutation. Although significant progress has been made over the past few years in the understanding of the structure and function of the plant-virus resistance genes and in the deployment of these genes in various fields and horticultural crops, more work still needs to be carried out. Viral disease threats are emerging, particularly in developing countries, due to the expansion of monoculturing of specific crops for which the resistance resources are still unknown. Interpretation of the techniques which are significantly promising in virus resistance, and the different pathways by which these mechanisms can be deployed in resistance breeding are definitely going to be a centre of attraction for research in the coming future.


This study compared, for five distinct detergents, the CMC in ddH2O side-by-side with two biopharmaceutical process intermediates relevant to plasma-derived (Immunoglobulin) and cell-derived  products, respectively. Depending on the matrix, the CMC of detergents changed by a factor of up to ~4-fold. Further, the CMC in biotechnological matrices did not correlate with antiviral potency, as Triton X-100 (TX-100) and similar detergents had comparatively higher CMCs than polysorbate-based detergents, which are known to be less potent in terms of virus inactivation. Finally, it was demonstrated that TX-100 and similar detergents also have virus-inactivating properties if applied below the CMC. Thus, the presence of detergent micelles might not be an absolute prerequisite for the disruption of virus envelopes.For decades, the ability of detergents to solubilize biological membranes has been utilized in biotechnological manufacturing to disrupt the lipid envelope of potentially contaminating viruses and thus enhance the safety margins of plasma- and cell-derived drugs. This ability has been linked to detergent micelles, which are formed if the concentration of detergent molecules exceeds the critical micelle concentration (CMC). Traditionally, the CMC of detergents is determined in deionized water (ddH Further, while Gooran et al. have recently postulated an indispensable role of micelle formation, the corresponding experiments were based on modeling the interaction of detergents with the virus lipid envelope via a supported lipid bilayer platform; thus, they did not involve virus infectivity assays . Virus reduction factors were calculated in accordance with the EU Committee for Proprietary Medicinal Products guidance [MDBK cells (source: ATCC/CCL-22) were propagated in growth medium , 10% fetal calf serum, 2 mM L-Glutamine, 1 mM sodium pyruvate, 1x non-essential amino acids, 0.15% sodium bicarbonate, and 100 µg/mL gentamycin sulfate, and used to generate stocks of bovine viral diarrhea virus . BVDV is a model virus for Hepatitis C virus (HCV), for which the transmission to recipients of plasma-derived medicines has happened before the introduction of S/D treatment. Titration of BVDV infectivity was accomplished with a median tissue culture infectious dose (TCID2O) to reach the desired final concentration. Addition of detergent working solution was accomplished using a Hamilton syringe. Samples for virus titration were drawn at 1 to 2 min, 10 ± 1 min, 30 ± 1 min, and 59 ± 1 min after detergent addition.Twenty-five mL of filtered and diluted plasma protein matrix (see above) was tempered to 17 °C ± 1 °C and continuously mixed by a magnetic stirrer. Spiking with BVDV was performed at a ratio of 1:31, i.e., 0.8 mL of virus stock solution were added. Within 1 to 2 min after spiking, two samples for virus titration were drawn; the spike control (SC) was titrated immediately, while the hold control (HC) was incubated in the same cooling circuit as the spiked matrix and titrated at the end of the experiment. The spiked matrix was weighed to determine the necessary amount of a detergent working solution (prepared by 1:20 dilution in ddH


This cohort study assesses the association of high out-of-pocket costs and initiation of a sodium-glucose cotransporter 2 (SGLT2) inhibitor or glucagon-like peptide-1 receptor agonist (GLP-1 RA) among adults who are treated with metformin and have type 2 diabetes and established cardiovascular disease. What is the association between high out-of-pocket costs and initiation of a sodium-glucose cotransporter 2 (SGLT2) inhibitor or glucagon-like peptide-1 receptor agonist (GLP-1 RA) among adults with type 2 diabetes and established cardiovascular disease?This retrospective cohort study examined 80 807 adult patients followed for a median of 1080 days. Compared with patients in plans with the lowest quartile of out-of-pocket costs, patients in plans with the highest quartile of costs were less likely to initiate a GLP-1 RA or an SGLT2 inhibitor.These findings suggest that adults with type 2 diabetes in the highest quartile of OOP costs were 13% and 20% less likely to initiate a GLP-1 RA or SGLT2 inhibitor, respectively, when compared with those in the lowest quartile of OOP costs. The latest guidelines continue to recommend sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 receptor agonists (GLP-1 RAs) for patients with type 2 diabetes (T2D) and established cardiovascular disease (CVD). Despite this, overall use of these 2 drug classes has been suboptimal.To assess the association of high out-of-pocket (OOP) costs and the initiation of an SGLT2 inhibitor or GLP-1 RA among adults with T2D and established CVD who are treated with metformin-treated.This retrospective cohort study used 2017 to 2021 data from the Optum deidentified Clinformatics Data Mart Database. Each individual in the cohort was categorized into quartiles of OOP costs for a 1-month supply of SGLT2 inhibitor and GLP-1 RA based on their health plan assignment. Data were analyzed from April 2021 to October 2022.OOP cost for SGLT2 inhibitors and GLP-1 RA.The primary outcome was treatment intensification, defined as a new dispensing  of either an SGLT2 inhibitor or GLP-1 RA, among patients with T2D previously treated with metformin monotherapy. For each drug class separately, Cox proportional hazards models were used to adjust for demographic, clinical, plan, clinician, and laboratory characteristics to estimate the hazard ratios of treatment intensification comparing the highest vs the lowest quartile of OOP costs.Our cohort included 80 807 adult patients  with T2D and established CVD on metformin monotherapy. Patients were followed for a median (IQR) of 1080 days (528 to 1337). The mean (SD) of OOP costs in the highest vs lowest quartile was $118 [32] vs $25 [12] for GLP-1 RA, and $91 [25] vs $23 [9] for SGLT2 inhibitors. Compared with patients in plans with the lowest quartile (Q1) of OOP costs, patients in plans with the highest quartile (Q4) of costs were less likely to initiate a GLP-1 RA  or an SGLT2 inhibitor . The median (IQR) number of days to initiating a GLP-1 RA was 481 (207-820) days in Q1 and 556 (237-917) days in Q4 of OOP costs and 520 (193-876) days in Q1 vs 685 (309-1017) days in Q4 for SGLT2 inhibitors.In this cohort study of more than 80 000 older adults with T2D and established CVD covered by Medicare Advantage and commercial plans, those in the highest quartile of OOP cost were 13% and 20% less likely to initiate a GLP-1 RA or SGLT2 inhibitor, respectively, when compared with those in the lowest quartile of OOP costs. To address this evidence gap, we assessed the association of OOP costs and initiation of an SGLT2 inhibitor or GLP-1 RA using commercial claims data among adults with T2D and established CVD who are treated with metformin.High out-of-pocket (OOP) drug costs are identified as a factor that limit initiation of and adherence to prescribed glucose lowering therapies.STROBE) reporting guideline.This cohort study was approved by the University of Pittsburgh institutional review board. Informed consent was waived because this was not human participant research. This report follows Strengthening the Reporting of Observational Studies in Epidemiology (We conducted a retrospective cohort study using data from 2017 to 2021 from Optum deidentified Clinformatics Data Mart Database (CDM), a longitudinal database that sources deidentified administrative claims for members of commercial and Medicare Advantage plans from the largest health insurer in the US. This database has been used extensively in studies of prescription drug use, adherence and outcomes and includes patient enrollment, physician, facility, and pharmacy claims, and laboratory results.International Statistical Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) diagnosis and procedure codes and Current Procedural Terminology (CPT) codes during the preindex period16  members who had less than 365 days of continuous enrollment prior to their first metformin dispensing; (2) who did not have evidence of established CVD, based on validated The Optum CDM database includes detailed information on copays and deductibles for each adjudicated pharmacy claim. Our measure of OOP drug costs summed both copay and deductibles. However, OOP costs cannot be measured for patients who never filled a prescription for an SGLT2 inhibitor or GLP-1 RA. To overcome this limitation, we assigned OOP costs for all members in our cohort by using copay and deductible data from individuals in the Optum CDM database who were not part of our cohort but had the same health plan as our study participants . To do this, we first calculated the mean OOP cost within each plan year for all of Optum CDM members not part of our study cohort who had 1 or more fills for an SGLT2 inhibitor or GLP-1 RA (n = 445 873). We then assigned the mean OOP cost to members in our cohort. These costs represent the OOP costs faced by those in the same health insurance plan, allowing us to approximate OOP costs not biased by inclusion of only those who fill prescriptions in the cohort. We scaled all costs to a standard 30-day supply. We excluded members where an OOP cost could not be determined based on all available data (n = 3631) .Our primary outcome was treatment intensification, defined as a new dispensing  of either an SGLT2 inhibitor or GLP-1 RA among patients previously treated with metformin monotherapy. We do not use the term treatment intensification to refer to an increase in the dose of a drug.The primary exposure was OOP cost for SGLT2 inhibitors and GLP-1 RA. In each treatment group separately (SGLT2 inhibitor and GLP-1 RA), we divided members into quartiles based on their mean 30-day OOP cost.We measured all covariates in the 365 days before their first metformin dispensing during the study time period. Baseline characteristics that were considered to be clinically related to treatment intensification were selected a priori. These included sociodemographic , clinical comorbidities , insurance type , clinician type  of the metformin prescriber, and presence or absence of a hemoglobin A1c (HbA1c) result.Race was considered in this study because previous studies have demonstrated racial disparities in access to newer glucose lowering medications for Americans living with type 2 diabetes. Race and ethnicity was self-identified at enrollment, and categories included Asian, Black, Hispanic, White, and unknown/missing.Summary statistics were reported for the overall cohort as well as for the lowest and highest quartiles of OOP costs for each of SGLT2 inhibitor and GLP-1 RA drug classes. Continuous measures were presented as mean (SD) and categorical measures were presented as No. (%). To depict time until initiation of an SGLT2 inhibitor and GLP-1 RA, we presented cumulative incidence curves  for each drug class separately stratified across the 4 quartiles of OOP costs. For each drug class separately, we used Cox proportional hazards models adjusting for demographics, clinical comorbidities, insurance type, clinician, and laboratory characteristics to estimate the hazards of treatment intensification comparing the highest vs the lowest quartile of OOP costs. Follow-up began on the date of the index metformin dispensing and ended when a member either initiated a target drug of interest or reached the end of the study period . Patients were censored if they had a gap in enrollment (plus a grace period of 30 days) or if they disenrolled or died. We constructed 6 Cox models based on the independent variables included as factors of initiation. Model 1 included the quartile of OOP costs only. Model 2 included the quartile of OOP costs and demographic variables. Model 3 included all variables from Model 2 and clinical comorbidities. Model 4 included all variables from Model 3 and insurance type. Model 5 included all variables from Model 4 and included clinician type. Model 6 included all variables from Model 5 as well as the presence (vs absence) of an HbA1c laboratory result.To improve the rigor of our study, we conducted a falsification test using statin initiation as the outcome of interest. In this test, we wanted to examine whether OOP cost quartiles for SGLT2 inhibitors or GLP-1 RAs were significantly associated with statin initiation. The presence of a strong negative association would suggest the presence of confounding . All analyses were performed using R version 4.2.0  assuming a Type I error rate α = .05 and no multiplicity adjustments. Data were analyzed from April 2021 to October 2022.Our cohort included 80 807 adult patients  with T2D and established CVD on metformin monotherapy . Most paAmong those in the GLP-1 RA cohort, those in the highest quartile of OOP costs were slightly older (mean age 73.6 vs 71.3), more likely to be male (59.7% vs 50.2%), more likely to be White (70% vs 53.8%), and had a higher proportion with diabetic nephropathy (23.4% vs 19.5%), all when compared against those in the lowest quartile of OOP costs . In addiFor those in the SGLT2 inhibitor cohort, those in the highest quartile of OOP costs were similarly older , more likely to be male (11 932 [59.5%] vs 10 405 [51.4%]), more likely to be White (13 997 [69.8%] vs 10 943 [54.1%]), and had a higher proportion with diabetic nephropathy (4370 [21.8%] vs 3783 [18.7%]), all when compared against those in the lowest quartile of OOP costs . PatientCompared with those in the plans with the lowest quartile of OOP costs, individuals in the highest quartile were least likely to initiate a GLP-1 RA, with an unadjusted HR of 0.70  . In the Similarly, for SGLT2 inhibitors, those in the highest vs the lowest quartile of OOP costs had an unadjusted HR of 0.68  for SGLT2 inhibitor initiation . In the The median (IQR) number of days between the index metformin dispensing and initiation of a GLP-1 RA was 481 (207-820) days, 485 (210-874) days, 548 (234-889) days, and 556 (237-917) days for Q1, Q2, Q3, and Q4 of OOP costs, respectively . The medWe compared the hazards of statin initiation among the quartiles of OOP costs for GLP-1 RAs and SGLT2 inhibitors. Individuals in the highest quartile of OOP costs had similar (but slightly higher) likelihood of statin initiation compared with those in the lowest quartile of OOP costs   were least likely to start 1 of these medications.21 The copay cap does not have any effect on those with commercial insurance, the uninsured,22 or those receiving SGLT2 inhibitors or GLP-1 RAs, which are increasingly recommended as first line agents for patients with diabetes and preexisting cardiovascular or renal disease. However, the bill does redesign the Medicare Part D benefit and institutes a $2000 OOP cap on copayments, which would have a beneficial effect on beneficiaries who take these medications.21 Strengths of our study include its use of a large administrative claims database, large sample size, and focus on a specific population of patients with strong indications for use of newer glucose lowering medications .There has been recent policy action to address high prescription costs, especially among Medicare beneficiaries. Recently, the president signed the Inflation Reduction Act of 2022, which institutes a $35 copay cap for insulin among Medicare beneficiaries.This study had limitations. First, our results were drawn from 1 national health insurance company with most patients covered under Medicare Advantage. Therefore, our results may not be generalizable to patients covered by other commercial plans, Medicaid beneficiaries, or individuals with Medicare fee-for-service. Future studies should consider the impact of OOP costs on medication initiation among patients covered by these plans. Second, we did not have access to formulary data which precluded a precise calculation of each member’s exact OOP cost for SGLT2 inhibitors or GLP-1 RA. However, we did estimate OOP costs using data drawn from other Optum CDM members with the same unique plan identification number and year of index metformin dispensing as members in our cohort, which we believe addresses a shortcoming of trying to measure OOP costs for those who do not fill a medication. Third, Optum CDM claims did not include information on samples or prescriptions paid with cash or reimbursed through secondary payers, which may have led to outcome misclassification. Notably, our results would not likely be biased by differential use of manufacturer-sponsored drug coupons since the use of these types of discounts are not allowed in Medicare plans. Fourth, our results may not apply to all patients since SGLT2 inhibitors and GLP-1 RAs are increasingly being used as front-line agents in T2D, while our cohort entry criteria required prior use of metformin. Fifth, our results may be subject to residual confounding due to unmeasured factors, although our use of a statin initiation falsification test is a strength and suggests that bias related to who enrolls in higher OOP plans does not explain our findings on SGLT2 inhibitor and GLP-1 RA initiation. Important areas for future research include whether overall OOP costs are associated with the use of these therapies and whether the findings we describe here also apply to medication adherence.In this cohort study using data from more than 80 000 patients with T2D and established CVD, those with the greatest cost-sharing for SGLT2 inhibitors and GLP-1 RA had a significantly lower likelihood of treatment intensification to a drug class with proven cardiovascular benefits. The median duration of delay comparing the highest vs the lowest quartile of OOP costs ranged from 3 to 6 months. Future studies should examine whether these differences in prescribing translate into meaningful differences in outcomes that are meaningful to patients in clinical settings.


Most emerging virus infectious diseases in humans, such as H5N1 and H7N9 subtypes of avian influenza , Ebola vIn order to improve the prevention and control of zoonotic emerging viral infectious diseases, further research is necessary to identify and characterize unknown viruses in both domestic and wild animals. It is important to monitor the diversity, distribution, evolution, and potential risks of various viruses that have the potential to cause zoonosis. Additionally, it is crucial to clarify the pathogenesis of these viruses and develop effective strategies and measures for detecting, surveying, preventing, controlling, and eliminating zoonotic emerging viral infectious diseases. These studies are essential for risk analysis and policy-making in animal protection and public health, and are therefore significant areas of research in veterinary and medical sciences.Zoonotic Emerging Viral Infectious Diseases. This Editorial is to summarize the findings and significance of these six papers.Recently, Frontiers in Veterinary Science published six articles on the topic of Chen et al.). It suggests that strengthened control measures have helped to shift the outbreak from a rapidly increasing trend to a declining one. However, some American countries are still experiencing sustained community transmission of Mpox, and it is crucial to enhance control measures in these regions (The first article provides a summary of the epidemiology of monkeypox (Mpox), which has spread to over 100 countries since May 2022. Through a literature review, this article highlights the risks associated with this outbreak and proposes eight strategies for its elimination, primarily based on the summarized epidemiology . This virus can represent a new species in the genus of Protoparvovirus, which currently includes three species of carnivore parvoviruses, four species of primate parvoviruses, three species of rodent parvoviruses, two species of ungulate parvoviruses (including porcine or bovine parvoviruses), and one species of bat parvovirus (The second article presents evidence for the identification and genomic characterization of a new porcine parvovirus (PPV) in China, tentatively named PPV8 . BCoV is a major cause of infectious disease in cattle worldwide and can infect humans and multiple other species of mammals. The authors developed a multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combination assay, which targets a highly conserved region of the viral nucleocapsid (N) gene for BCoV detection. This MIRA-LFD assay is superior to classical real-time PCR assays as it does not require sophisticated instruments and the results can be visually observed. Furthermore, this method has the potential to be used for the on-site detection of other zoonotic viruses.The third article reports a novel approach for the on-site detection of bovine coronavirus (BCoV) infection in suspected farms that is rapid, reliable, and simple . The study also highlights the veterinarians' belief that SwIV is becoming a more significant health issue in swine. SwIV infection was the second most commonly confirmed disease in respiratory porcine tissue cases in some regions in the USA between 2010 and 2019 (The fourth article describes the varying perspectives among veterinarians in the USA on swine influenza virus (SwIV) infections in pigs . Group A rotaviruses of the family Reoviridae is one of the important intestinal pathogens causing diarrhea in piglets and humans. A human-porcine reassortment rotavirus was identified from outbreak of diarrhea in suckling piglets with 60.00% (324/540) morbidity and 20.99% (68/324) mortality in Guangdong Province of China in 2022. The virus strain likely emerged as the result of genetic reassortment between porcine and human rotaviruses, suggesting that the strain has a potential risk of cross-species transmission.The fifth article describes the emergence of a human-porcine reassortment porcine rotavirus A strain in China (Nasua narica) in Mexico . Although Mexico has successfully eliminated human rabies transmitted by dogs, the country still faces the challenge of wildlife transmitting the virus to humans and domestic animals. The study reports 13 cases of rabies in white-nosed coatis in Mexico between 1993 and 2022. The rabies virus strains from nine of the samples were antigenically and genetically characterized. Coatis have not been previously considered important vectors of rabies virus, but the significance of rabies virus infections in coatis should be carefully monitored in order to prevent human infections transmitted by this species.The six article focuses on rabies virus in white-nosed coatis (In summary, the six articles mentioned above make significant contributions to risk analysis and the selection of control strategies for zoonotic emerging viral infectious diseases. They also aid in identifying new viruses with zoonotic potential, developing new techniques for on-site detection of zoonotic viruses, and enhancing surveillance of zoonotic emerging viral infectious diseases. These articles underscore the importance and necessity of conducting studies on various aspects related to zoonotic emerging viral infectious diseases.J-MC and Y-FJ wrote this editorial. Z-JD and BW revised it. All authors approved the submitted version.


Agricultural production is hampered by disease, pests, and environmental stresses. To minimize yield loss, it is important to develop crop cultivars with resistance or tolerance to their respective biotic and abiotic constraints. Transformation techniques are not optimized for many species and desirable cultivars may not be amenable to genetic transformation, necessitating inferior cultivar usage and time-consuming introgression through backcrossing to the preferred variety. Overcoming these limitations will greatly facilitate the development of disease, insect, and abiotic stress tolerant crops. One such avenue for rapid crop improvement is the development of viral systems to deliver CRISPR/Cas-based genome editing technology to plants to generate targeted beneficial mutations. Viral delivery of genomic editing constructs can theoretically be applied to span the entire host range of the virus utilized, circumventing the challenges associated with traditional transformation and breeding techniques. Here we explore the types of viruses that have been optimized for CRISPR/Cas9 delivery, the phenotypic outcomes achieved in recent studies, and discuss the future potential of this rapidly advancing technology. It is estimated that plant disease induces global yields losses between 20 and 40% for rice, maize, wheat, potato, and soybean, five of the most important crops worldwide  of trans-species genes  virus host range and tissue specificity or exclusion ; 2) genomic cargo carrying capacity of the virus; 3) mode and efficiency of transmission , and 4) biosafety and biocontainment of the engineered viruses. The host range of a given virus tends to be restricted to either dicots or monocots, although some monocot viruses have been successfully propagated in dicotyledonous species, particularly The size limits of foreign inserts are often dictated by the physical structure of the virus. Typically, rod-shaped viruses can incorporate more foreign genomic material than small (ca. 30 nm) icosahedral viruses  or insect transmission  were determined to have editing efficiencies up to 48% . It was later revealed that PVX could be useful for delivering multiplexed gRNAs, and that edits were heritable when gRNAs included mobile FT modifications  to create a synergistically more robust infection   and pea early browning virus (PEBV), are currently being used as CRISPR/Cas9 delivery vectors. TRV, with a wide host range and an easily modifiable bipartite positive sense RNA genome, is a proven VIGS vector for N. benthamiana plants   through double stranded break/homologous recombination of the wild type CRTISO sequence delivered by the BeYDV-Cas9 construct (Dahan-Meir et al. Most of the CRISPR/Cas9 virus delivery systems to date rely on RNA viruses; however, there are a few DNA viruses – notably geminiviruses – that also have been developed for this purpose (Baltes et al. r et al.  developeN. benthamiana or Arabidopsis.Traditional transformation methods with CRISPR/Cas9 constructs were used to target a variety of plant phenotypes, including reduced lodging in elite rice cultivars, drought tolerance in Arabidopsis and maize, and pathogen resistance across numerous plant species (Hu et al. FT RNA sequences greatly increased the likelihood of heritable editing (Ellison et al. Given the success of the 40+ plant viruses engineered as VIGS or protein expression vectors, solving current limitations of viral CRISPR/Cas9 vectors will undoubtedly yield remarkable results in plant genome editing studies (Cody and Scholthof In addition to effective germline targeting in a given plant/virus system, production of a desired phenotype such as drought tolerance or pathogen resistance requires identification of appropriate gene targets. Due to the robustness of viral infection, a positive attribute of viral delivery of CRISPR/Cas9 constructs is that the phenotypic outcome of gene editing can be analyzed directly in infected tissue prior to seed set and plant selection, enabling timely refinement or modification of the gRNA sequences if needed for optimal results (Cody and Scholthof FT gRNA in N. benthamiana (Uranga et al. Broad application of virus delivery of CRISPR/Cas9 constructs will aid researchers much like VIGS systems have greatly advanced our understanding of plant gene function Fig. . AdditioAs highlighted in this review, many new CRISPR/Cas9 viral delivery systems are being developed and are ready for application in plant functional genomics studies. Ultimately, once biosafety and bioethics concerns have been addressed, it may eventually be possible to deploy these virus constructs via their natural insect vectors to rapidly save stressed crop plants in field applications. Thus, we anticipate these technologies will be adopted by many laboratories to advance their crop improvement studies for understanding gene function and developing disease/insect resistant and abiotic stress tolerant cultivars.


Viruses, since 2019, highlighting the increasing effort of the scientific community for the surveillance and further research of new emerging or re-emerging viruses. As organisms with the fastest evolutionary rates, viruses pose challenges for many research areas relevant to human health, agricultural practices, and the world economy. The SI 2022 reflects the insights of virologists around the globe about relevant and potentially threatening new viruses, especially those arising in the edge between rural and forest biomes. Indeed, the emergence of new virus species and/or isolates infecting wild animals from the interface between forest/rural/metropolitan areas and animals from growing farms were the most prevalent issues in this SI. New arteriviruses, hepinaviruses, coronaviruses, and astroviruses were identified in rodent, vole, and gosling species, respectively [The Special Issue “Emerging Viruses: Surveillance, Prevention, Evolution and Control” has been published annually by ectively ,4. Also,ectively ,6,7.Oecomys sp., unveiled a new arterivirus, classified in the Variarterivirinae clade. Using a similar approach, the same group also identified a new hepinidavirus-like in the marsupials Marmosa demerarae, opossums, adjacent to a rural municipality of Peixe-Boi, Pará State, Brazil [Henipavirus is a Paramyxoviridae genus that comprises some emerging viruses of serious public health concern. The novel virus called the Peixe-Boi virus (PBV) corroborates the circulation of henipa-like viruses far from Africa, Asia, and Australia. It was the first description of henipa-like viruses in marsupials worldwide, indicating the importance of including these animals in surveillance studies. The vulnerability of deforested environments such as those at the study site, where wild habitats overlap with rural areas, resembles the conditions in which Hendra henipavirus (HeV) and Nipah henipavirus (NiV) outbreaks arose previously [Human–animal interface in the Amazonian metropolitan area represents a constant opportunity for the emergence of new viruses. In an effort to identify viral communities in wild animals from this region, Da Paz and collaborators  conducte, Brazil . Henipaveviously . Betacoronavirus species designed as Grimsö virus was found in bank voles (Myodes glareolus) in Grimsö, Sweden [Puumala orthohantavirus and Francisella tularensis. The study analyzed vole communities for 3 years, and the Grimsö virus was detected during this time. The phylogenetic analysis of ORF1b, S, and N genes, as well as a partial RdRp gene, suggested a relatively broad geographic distribution of CoVs in bank voles in Europe, which is indicative of possible long-term host–virus association [In another effort, using next-generation sequencing, but primer-targeted to coronavirus spike, a new , Sweden . Bank voAvastrovirus 3 (AAstV-3), while GoAstV-1 belongs to Avastrovirus 1 (AAstV-1). In 2016, an outbreak of a novel gout disease affecting goslings emerged in China. A novel goose astrovirus (GoAstV) was associated with the disease, and successive outbreaks resulted in serious economic loss. In the study by Fu et al. , GoAstV Sghaier et al. reported the re-emergence and spread of the epizootic hemorrhagic disease virus strain 8 (EHDV-8), causing epizootic hemorrhagic disease in cattle in Tunisia in 2021 . The EHDCulex tritaeniorhynchus Giles (Diptera: Culicidae), indicating that it could be a vector of the virus.In October 2021, an outbreak of suspected Lumpy skin disease virus (LSDV) occurred on a mixed yak and cattle farms in Sichuan Province, China. The outbreak resulted in high morbidity and mortality rates in yaks. Li et al.  detectedTogaviridae family. Human transmission is mediated by Aedes spp. The virus was introduced in Brazil in 2014; however, there is still limited information about the genomic epidemiology of CHIKV from surveillance studies. Souza et al. [Three human-infecting viruses were discussed in this SI, including the genomic surveillance of Chikungunya virus (CHIKV) in a Brazilian state  and the a et al.  reportedIn a different approach, Lim and colleagues  developePhlebovirus, family Phenuiviridae) genome, they obtained around 80–90% of the genome covering of the virus from not enriched samples. The proposed amPCR method achieves sufficient sequence coverage for genotyping and other genomic epidemiological studies such as transmission dynamics.In an interesting paper, Juma et al.  reportedReoviridae, a double-stranded RNA genome with no envelope; foot-and-mouth disease virus (FMDV), family Picornaviridae, a single-stranded RNA genome with positive polarity; small ruminant morbillivirus, family Paramyxoviridae, a single-stranded RNA genome with negative polarity and lipid envelope; and lumpy skin disease virus (LSDV), family Poxviridae, a double-stranded DNA genome with lipid envelope. The general conclusion was that cards are safe if samples are appropriately applied. The report also described which card showed the best nucleic acid recovery.Finally, Keck and collaborators  tested aIn summary, the manuscripts featured in this Special Issue encompass studies on a wide range of viral species, offering invaluable insights into their origin, evolution, transmission patterns, and potential outcomes. These studies underscore the significance of comprehensive research not only to enhance our understanding of these novel viruses but also to enable effective strategies for their surveillance and control.