The precise location of the human female genital representation field in the primary somatosensory cortex (S1) is controversial and its capacity for use-associated structural variation as a function of sexual behavior remains unknown. We used a functional magnetic resonance imaging (fMRI)-compatible sensory-tactile stimulation paradigm to functionally map the location of the female genital representation field in 20 adult women. Neural response to tactile stimulation of the clitoral region (vs right hand) identified individually-diverse focal bilateral activations in dorsolateral areas of S1 (BA1–BA3) in alignment with anatomic location. We next used cortical surface analyses to assess structural thickness across the 10 individually most activated vertices per hemisphere for each woman. We show that frequency of sexual intercourse within 12 months is correlated with structural thickness of the individually-mapped left genital field. Our results provide a precise functional localization of the female genital field and provide support for use-associated structural variation of the human genital cortex.SIGNIFICANCE STATEMENT We provide a precise location of the human female genital field in the somatosensory cortex and, for the first time, provide evidence in support of structural variation of the human genital field in association with frequency of genital contact. Our study represents a significant methodological advance by individually mapping genital fields for structural analyses. On a secondary level, our results suggest that any study investigating changes in the human genital field must map the field individually to achieve sufficient precision. Our results pave the way for future research into the plasticity of the human genital cortex as a function of normal or adverse experience as well as changes in pathologic conditions, i.e., sexual dysfunction, sexual deviation, or sexual risk-taking behavior. The precise location of the female genital representation field in the primary somatosensory cortex (S1) is still a matter of contention . FurtherIn their first presentation of the somatosensory homunculus, The mode of stimulation used in functional mapping studies may contribute to heterogeneous results concerning the location of the genital field in humans. Specifically, electrical stimulation is not equivalent to sensory touch and elicits less focal responses . Self-deCommensurate with the fact that the precise location of the genital field remains controversial, there is no evidence regarding its capacity for structural change in association with use in humans. It is well established that the human brain has substantial capacity for plasticity as a function of experience . Use-depWe here combine the investigation of the location of the female genital field with the question of structural variation of this field as a function of sexual behavior, considering the important issue of individual variability: (1) we provide a precise localization the human female genital representation field by using a focal sensory-tactile nonarousing stimulation paradigm during functional MRI (fMRI) to contrast neural response of stimulation of the clitoral region versus the right hand. (2) We use individually-mapped genital fields based on the 10 most activated vertices per hemisphere for each woman and assess structural thickness in the individually-mapped field using cortical surface analysis. (3) We show that thickness of the individually-mapped genital field varies with the frequency of sexual intercourse in the past 12 months, compatible with use-associated plasticity.We recruited 25 adult healthy women aged 18–45 years. General exclusion criteria applied to select women were lifetime or current psychiatric disorders, exposure to childhood abuse or neglect , neurologic disorders, physical disease, central nervous system or urogenital surgery, psychotropic medication within six months, sexually transmitted disease, sexual disorders , past or current pregnancy, and current menstruation. Exclusionary conditions were assessed using clinician-administered interviews and standard questionnaires . Women wWomen underwent a standardized study visit at the Institute of Medical Psychology and the Berlin Center for Advanced Neuroimaging, both at Charité−Universitätsmedizin Berlin. During the visit, women underwent all study procedures, including interviews and questionnaires for demographics and exclusionary conditions. To localize the genital representation field in S1, women underwent (1) fMRI scanning during sensory-tactile stimulation of the clitoris versus dorsum of the right hand; (2) structural MRI to assess thickness of the individually mapped genital field; and (3) a detailed sexual history to assess frequency of sexual intercourse, i.e., genital sensory touch, in the past year and lifetime for the assessment of use-dependent plasticity of the individually mapped genital field. The study was approved by the institutional ethics committee and was conducted in accordance with the Declaration of Helsinki. Written informed consent of the participants was obtained.3 isotropic T1 anatomic scans were acquired in the sagittal plane using the magnetization-prepared rapid gradient echo sequence . Structural MRI acquisition took 2 × 6:03 min. fMRI scans were obtained using a T2*-weighted echoplanar image (EPI) pulse sequence . The functional imaging paradigm comprised four scanning blocks with a duration of 5:36 min, respectively.Structural MRI was performed using a 3.0 T Siemens Tim Trio MRI scanner  with a standard 12-channel head coil. Two 1-mmWe developed an MRI-compatible sensory-tactile stimulation paradigm that allows for administering a defined focal sensory stimulus to the clitoral region see . The stiThe paradigm was performed in an ABBA versus BAAB block design with stimulation of either the clitoral region (A) or the dorsum of the right hand (B) interspersed with 10-s periods of no stimulation. Each of the four runs started with a period of no stimulation and included a total of eight clitoral and eight dorsum manus stimulation phases. The order of these phases was fixed and counterbalanced between women. Synchronization of the trigger pulses from the MRI scanner and the timing of the stimulation was controlled using Presentation . During the sensory-tactile stimulation, subjects were asked to fixate a cross on a screen. One woman completed only three runs.Pleasantness and sexual arousal during clitoral stimulation were assessed after each run using a seven-point visual analog scale. Subjects were instructed to use a fiber-optic response box, indicating changes in pleasantness and sexual arousal. We then computed combined ratings on overall pleasantness and sexual arousal after the scan. We further inquired on the subjective appropriateness of the location of the clitoral membrane during the experiment as well as on sensations in other body parts during clitoral stimulation. There was no evidence for dislocation of the stimulation membrane in the sample.Statistical parametric mapping 12  was used to perform functional image analysis to localize the genital field in S1. Standard spatial preprocessing of functional images, including realignment and coregistration to T1 image, was separately performed for each of the four scanning blocks. Data were high-pass filtered with a default cutoff period of 128 s to correct for slow drift artifacts. There was no head motion above 3.0 mm and 3.0° of maximal translation and rotation in any direction throughout a scanning block.t tests for contrasts between the two regressors of the design matrix, resulting in individual patterns of neural activation in response to clitoral versus hand stimulation. We identified an activated region in SI for each participant at p < 0.001 without correction or p < 0.05 with family-wise error (FWE) correction for multiple comparisons. Individual neural activations were overlaid onto coregistered anatomic scans and saved as individual regions of interest (ROIs) for the left and right hemisphere, respectively. Individual ROI was multiplied with the t-score map corresponding to the individual contrast image to delineate the most activated vertices within the individually defined ROI. We purposely did not perform spatial normalization to a standard stereotaxic space  or smoothing of the images to allow for subsequent cortical thickness analyses within the individually mapped ROI in native space of the anatomic images, as needed for the use-dependent plasticity analyses. To determine variability of the location of the genital field and hand representation in S1 between women, coordinates of peak neural activation were transformed in MNI space. Barycentre and dispersion across individually mapped fields were computed by averaging individual coordinates in MNI space.After standard spatial preprocessing, fMRI data were analyzed using a general linear model (GLM). The two within-subject conditions of interest  were modeled using a boxcar function convolved with a canonical hemodynamic response function (HRF). Activation maps were calculated with 3. Furthermore, data were spatially smoothed with a 6-mm full-width at half-maximum isotropic Gaussian kernel. Whole-brain group-level analysis with t tests contrasting neural response to the two with-subject factors genital stimulation versus stimulation of the dorsum of the right hand was thresholded at p < 0.05 with FWE-correction for multiple comparisons. Coordinates of the group-based neural activation reflecting the genital field are given in standard MNI space.To additionally localize the genital representation field in S1 on the group level, a random effects GLM was estimated across subjects. For this, individual contrast maps were spatially normalized to a standard MNI template and resampled to an isotropic spatial resolution of 3 × 3 × 3 mmThese statistical analyses and figures were computed using MATLAB Automated image segmentation included (1) spatial registration (affine registration to tissue probability map); (2) initial SPM unified segmentation and skull stripping; (3) local intensity transformation to reduce tissue inhomogeneities  for SPM12 was used to perform cortical SBM of the anatomic scans. Image segmentation was conducted using an automated standard procedure. The individually defined ROIs for the clitoris and the dorsum of the right hand were separately mapped onto individual native space cortical surfaces of the left and right hemisphere. After cortical surface registration, mean thickness of the 10 functionally most active vertices within the individually mapped ROIs was separately calculated for each hemisphere in each woman. Cortical thickness at each vertex was calculated as part of central surface estimation , describcorr = 0.025). Using partial correlation analyses, we used age, years since onset of sexual contact, and whole-brain cortical thickness as covariates to control for effects of these variables on genital field cortical thickness. Furthermore, correlations and partial correlations between left-hemispheric cortical thickness of the representation field of the right hand and frequency of sexual intercourse for either time window were calculated to confirm for region-specificity of use-associated variation. These statistical analyses and figures were computed using R Project for Statistical Computing  and IBM SPSS Statistics .We assessed mean frequency of sexual intercourse per week using a standardized biographic questionnaire to quantify sexual intercourse within the past 12 months and in five-year ranges since the onset of the first sexual genital contact. As noted above, we excluded sexual anxiety, discontent or dysfunction as well as dissociation during intercourse using established questionnaires . To assoThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Custom MATLAB Code  for SPM12 and CAT 12 will be provided on request.Demographic and behavioral data are presented in Sensory-tactile stimulation of the clitoral region (relative to right hand) induced significant focal neural activations in S1. Sixteen women exhibited bilateral neural activations in S1. For four women, a significant activation was found in either the right or the left hemisphere only. We next mapped the individual representation of the dorsum of the right hand for use in subsequent cortical thickness analyses. Sensory-tactile stimulation of the dorsum of the right hand  induced significant contralateral focal neural activations in S1. x = −18, y = −34, z = 74; T = 7.72, pFWE-corr = 0.024; right hemisphere: x = 18, y = −40, z = 68; T = 10.26, pFWE-corr < 0.0001). Of note, no other significant neural activations were observed at the group level in response to the stimulation of the clitoral region, suggesting that the stimulation paradigm specifically targeted the genital field and was not overly arousing. When analyzed at the group level across all women, GLMs revealed significant symmetric dorsolateral neural activations in S1 in response to stimulation of the clitoris (relative to hand) in both hemispheres . Similarly, longer-term frequency of sexual intercourse estimated since the onset of sexual contact was significantly correlated with thickness of the individually-mapped left-hemispheric genital field in a partial correlation analysis . Partial correlation analyses between cortical thickness of the right-hemispheric genital field and frequency of sexual intercourse did not reveal any significant effects, suggesting lateralized use-associated structural variation. We mapped individual ROIs for the genital field  onto native cortical surfaces for each subject and estimated cortical thickness of the individual genital representation field  onto native cortical surfaces for each woman and estimated cortical thickness of the individual representation field of the hand (for individual data, see We present novel evidence on the precise location of the female genital representation field and its capacity for use-associated structural variation. Using functional mapping during sensory-tactile stimulation of the clitoral region, we show focal bilateral neural activations within the dorsolateral postcentral gyrus in S1. We show that the individual location of peak neural activations in response to clitoral stimulation varies considerably between women. We applied cortical surface analysis to the individually-mapped ROI to compute structural thickness of the genital field. Correlating the individually-mapped morphologic data with behavioral data on sexual contact, we provide first evidence that thickness of the genital field varies as a function of frequency of genital intercourse in the past 12 months and lifetime, in line with use-associated plasticity.Our results are noteworthy in several ways. To localize the female genital field, we measured neural response in a tactile-sensory stimulation paradigm that delivers a physiologically valid stimulus as opposed to a previous study using electrical stimulation of the clitoris . FurtherOur results suggest profound variability of the individual location of the genital field within the dorsolateral part of S1 with individual peak activations clearly deviating from the group mean. This means that any study looking at structural variation of the genital field as a function of certain conditions, such as sexual behavior, sexual abuse or sexual dysfunction, must necessarily implement individual mapping of the genital field and compute data, i.e., cortical thickness, on an individual level. Clearly, only by using individually-mapped ROIs, such studies yield precise reliable surface-based parameters for association with specific conditions.We computed data on structural thickness of the genital field in individually-mapped ROIs, based on the 10 most activated vertices per hemisphere for each woman. We show that individual thickness of the left genital field associates with frequency of sexual intercourse. The association was stronger for genital intercourse within the past 12 months. While less pronounced, the association was significant for lifetime genital contact. Frequency of genital intercourse was not associated with thickness of the representation field of the right hand nor with thickness of the entire cortical mantle, confirming a specific association between genital touch and genital field thickness. This is compatible with the idea that the female genital field has capacity for structural plasticity depending on its use, commensurate with the general “use-it-or-lose-it” principle of experience-dependent plasticity . While iSeveral mechanisms might contribute to dynamic use-associated structural plasticity of the genital field. Structural thickening of the mature cortex as a function of use most likely reflects formation of new synapses by axonal sprouting, dendritic arborization, and dendritic spine growth rather than induction of new neurons through neurogenesis . There iIt must be noted that use-associated variation of structural thickness of the female genital field in our study was limited to the left hemisphere. This lateralized effect is puzzling given that the neural representation of the clitoris is bilateral. Left-hemispheric dominance of neural plasticity has been reported for learning-dependent structural change after coordination and motor skill training . Such laWhile our localization of the female genital field was experimental in nature, our investigation of the capacity of the genital field for structural variation as a function of genital contact was cross-sectional and relied on retrospective self-report of genital intercourse. Our results align with the general principle of an association between frequency of genital intercourse and structural variation, albeit the direction of effect is a matter of discussion. It is conceivable that thickness of the genital field may drive frequency of sexual intercourse. Results from animal models provide causal that clitoral stimulation drives genital field thickness . Future In conclusion, we provide an unequivocal localization of the female genital field in S1 and support for use-associated plasticity of the human genital field. On a secondary level, our findings support the notion that studies investigating change of the human genital field must map the field individually. Our results pave the way for future research into the plasticity of the human genital field as a function of normal or adverse experience as well as genital field structure, function and plasticity in pathologic conditions, such sexual dysfunction, sexual deviation, or sexual risk-taking behavior.


Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal–fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID Here we report on animal studies elucidating mechanisms preventing the transfer of a double deletion mutant of a pestivirus to the fetus in pregnant heifers. This mutant lacks both known factors engaged in blocking the innate immune response to pestiviral infection. As shown also in earlier studies, this mutant was not detected in the fetuses at any of the tested time points in contrast to the wild-type (wt) virus. However, similar to the wt the mutant was detected in a large variety of different maternal tissues. The only exception was the placenta where only wt but not mutant virus was detected. Using gene array analyses we showed that infection of two cell lines derived either from the maternal or the fetal site of the maternal-fetal interface with the mutant virus induces a significant antiviral gene expression response. The reaction of cells from the maternal side was more complex and virus replication in these cells was reduced, almost completly blocking the mutant virus. These results support the hypothesis that replication of the mutant virus is blocked in the placenta due to a highly active innate immune response and the prevention of replication also blocks transfer of the virus to the fetus. Flaviviridae .Using a dilution series of an BVDV in-vitro transcribed RNA standard, the genome copies in the respective samples were determined to allow harmonization between PCR runs. A standard for BVDV was used to calculate number of genome copies. The RNA concentration was determined by spectrophotometry using a Nanodrop 2000c (ThermoFisher Scientific), and the corresponding number of genome copies was calculated based on the BVDV segment length using an online tool [in vitro-transcribed RNA were stored at working dilutions of 2×105 copies/μl at −20°C in RNA Safe buffer .The stock solutions of the To detect BVDV RNA, RNA in situ hybridization (ISH) was performed on tissues using the RNAScope 2–5 HD Reagent Kit-Red  according to the manufacturer’s instructions. The probe design was conducted by ACD for wt BVDV. A positive control probe peptidylprolyl isomerase B  and a negative control probe dihydrodipicolinate reductase (DapB) were used to verify specificity of the wt BVDV probe.As a first step we tested the new probes using paraffin embedded cell pellets established with tissue culture cells (MDBK-B2). The probe reacted well with the RNA of the wt virus and double deletion mutant present in cells that had been formalin fixed and embedded in paraffin whereas no staining occurred with mock infected control cells (not shown). Briefly, tissue samples taken from the animals were embedded in paraffin wax and cut at 5μm sections. Mounted sections were baked on a heating plate . After deparaffinization, FFPE tissue sections were pretreated with hydrogen peroxide (ACD) , washed in distilled water and incubated with Target retrieval solution (ACD). Slides were subsequently washed in distilled water and 100% ethanol. After creating a hydrophobe barrier around the tissue section, slides were treated with Protease Plus solution (ACD), incubated in a HybEZ oven  and finally washed in distilled water. Next, different hybridization steps were performed using the HybEZ oven according to the protocol provided by ACD. Positive signals were detected after treatment with RED solution (ACD). Slides were counterstained with hematoxylin.https://david.ncifcrf.gov/ and for the analyses of protein-protein interactions the “Search Tool for Retrieval of Interacting Genes/Proteins” STRING v11 https://string-db.org/.RNA from BCEC-1 and F3 cells infected with wt BVDV and ddBVDV was extracted and purified with a method based on the work of Chrigwin and colleagues  as descr


In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed. The engineering of viral genomes is important in many aspects of virus research, including studies of the structure and function of viral protein-coding genes and non-coding RNAs, virus–host interactions, gene therapy applications, and the development of recombinant vaccines. In addition to the classical recombination and selection of recombinant viruses in virus-infected eukaryotic cells used previously , three mThe CRISPR/Cas system is an important part of adaptive immune mechanisms of bacteria and archaea, which is used to resist foreign genetic plasmids and phage invasion. In 1987, the structure of CRISPR was first reported , and theThe CRISPR/Cas9 system can target any kind of gene or genomic region, allowing site-directed gene editing such as mutation, knockout, insertion, and deletion (indel). The functional complex consists of the Cas9 nuclease and a sgRNA, which is composed of a crRNA and a tracrRNA. The chimeric sgRNA directs the Cas9 nuclease to bind to the target DNA fragment followed by 5′-NGG-3′ (PAM) , and theIn the last decade, the number of publications describing CRISPR and gene-editing-related research in both life sciences and virology has dramatically increased . In partStaphylococcus aureus Cas9 (saCas9)/gRNAs composed of multiple gRNAs targeting the conserved region in LTR and viral domain of HIV-1 effectively removed the latent HIV-1 virus, inhibited virus reactivation, and significantly improved the efficiency of destroying the HIV-1 genome [As the CRISPR/Cas9 system can achieve efficient gene editing in biological organisms with cellular structure, its use for editing viral genes integrated into the host genome is attractive as a research tool. It is well known that genomes of HIV and other retroviruses can permanently integrate into the host genome, from where they can be activated to produce an infectious virus that can pose new threats to the host. In 2013, a specific gRNA and CRISPR/Cas9 nuclease complex was designed and transfected into Jurkat cells to target the integrated HIV-1 long terminal repeat (LTR) . The com1 genome . More re1 genome ,38,39,40For human beings, about one-fifth of tumors are caused by a virus infection, of which the oncogenic viral genes directly promote the occurrence and development of tumors . The hepHigh-risk human papillomavirus (HR-HPV) is recognized as the main cause of cervical cancer. HPV encodes the oncogenic genes E6 and E7, which play important roles in maintaining the malignant phenotype of cervical cancer cells. In 2014, specific CRISPR/Cas9-gRNA complexes targeting the HPV-16 E7 gene were first reported in HPV positive SiHa and Caski cells, of which the inhibition of E7 expression resulted in the up-regulation of tumor suppressor protein pRb, eventually inducing tumor cell apoptosis and inhibiting tumor cell growth . In addiAs a human mouse mammary tumor virus like-2 (HML-2) subgroup of human endogenous retroviruses (HERVs), the HERV-K is activated in several tumors and has been suggested to be related to prostate cancer progression and motor neuron diseases. In a recent study , the HERHerpesviruses are double-stranded DNA viruses with large genomes. In the infected host, herpesviruses have the distinct ability to escape the surveillance of the host immune system by establishing a life-long latent infection and causing recurrent diseases. Herpesviruses include a variety of important human pathogens, such as herpes simplex virus (HSV), Epstein–Barr virus (EBV), human cytomegalovirus (HCMV), and livestock and poultry pathogens such as pseudorabies virus (PRV), duck enteritis virus (DEV), infectious laryngotracheitis virus (ILTV), and Marek’s disease virus (MDV).The CRISPR/Cas9 system can achieve direct DSB in the genome of DNA viruses, using NHEJ and/or HDR pathways to introduce site-specific indels or insertion of heterologous genes with high frequency ,63. In 2α-4 gene transcription [The CRISPR/Cas9 system has recently been reported as an antiviral strategy to interfere with human herpesvirus infection in vitro . The gE cription . In addicription . These rSome researchers have explored the role of the CRISPR/Cas9 system in EBV gene editing. Seven gRNAs were designed and transfected into Raji cells with latent EBV infection. It has been demonstrated that once the corresponding functional sites of the EBV viral gene were destroyed, the cell proliferation and virus load were both significantly decreased, whereas the apoptosis pathway in the cells was restored . DeletioMany scholars have applied the CRISPR/Cas9 system to the gene editing of other human herpesviruses too. Three specific gRNAs targeting the UL122/123 gene of HCMV, a key regulator responsible for lytic replication and reactivation from latency, were transfected into primary fibroblasts and U-251 MG cells . A concoIn addition to human disease-related herpesvirus, the CRISPR/Cas9 system is also attractive for scientists focusing on animal herpesvirus. In 2015, some researchers co-transfected the purified PRV genomes with the constructed specific gRNA CRISPR/Cas9 complex into PK15 cells and obtained up to 100% viral gene editing efficiency . SimultaRecently, the CRISPR/Cas9-based gene-editing system has also been successfully applied in other DNA viruses, such as the vaccinia virus (VACV), JC polyomavirus (JCPyV), fowl adenovirus 4 (FAdV-4), and African swine fever virus (ASFV). The gRNA-guided CRISPR/Cas9 system has greatly increased the efficiency of generating mutant VACVs without evident off-target effects, providing a marker-free system that can be used for the efficient construction of VACV vectors armed with any therapeutic genes in the TK or N1L regions ,87,88,89Francisella novicida (FnCas9) has demonstrated the ability to target endogenous bacterial RNA, and the reconstructed gRNA targeting human +ssRNA virus (such as HCV) in eukaryotic cells inhibits the expression of viral proteins, providing a possibility for the treatment of diseases caused by RNA virus infection [At present, the CRISPR/Cas9 system is mainly used for DNA virus gene editing, and there are still huge technical obstacles in its application in RNA virus mutagenesis. A new Cas9 nuclease from the Gram-negative bacterium nfection . The latnfection . Infectinfection . These dRecently, genome-wide CRISPR/Cas9 screening has been used to identify host factors that are potentially involved in the replications of some viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , HIV 9797, arthrIn addition to the studies on gene function, gene therapy, and virus–host interaction, CRISPR/Cas9-based gene-editing technology has also been widely applied in vaccine research for its high efficiency, specificity, versatility, flexibility, simplicity, and low cost compared to the other viral genome editing techniques, which has demonstrated an efficient and powerful strategy for future development of genetically engineered vaccines. The latest progress in the application of CRISPR/Cas9 in virus genome editing and advancing orthopoxvirus (OPXV)-based recombinant vaccines and vectors has been well summarized by Okoli and colleagues . A CRISPRecently, the number of studies on animal anti-viral vaccine development utilizing the CRISPR/Cas9 system has been increasing . The CRIIn 2016, CRISPR/Cas9-mediated genome editing in the live attenuated vaccine vector herpesvirus of turkeys (HVT) was reported as the first application of this technology in avian herpesvirus mutagenesis . Based oSimultaneously, some scholars have applied this technology to perform similar research on other poultry herpesviruses. The CRISPR/Cas9 and HDR systems were used for the insertions of the HA gene of highly pathogenic AIV H5N1, the pre-membrane (PrM), and gE genes of duck Tembusu virus (DTMUV) into the UL27/UL26 and US7/US8 regions of the DEV C-KCE strain genome, to develop a potential trivalent vaccine to prevent the infections of H5N1-AIV, DTMUV, and DEV . SubsequCRISPR/Cas9-based genome editing has also been applied in generating a vaccine against canine infectious disease. For both wild and domestic carnivores, canine distemper (CD) is one of the most important infectious diseases caused by the canine distemper virus (CDV). Using the CRISPR/Cas9-based gene editing approach, a highly efficient recombinant virus, containing CDV virus-like particles (VLPs) and concurrently expressing the matrix (M), H, and F genes, has been recently constructed, which can assemble CDV-VLPs and provide faster seroconversion and higher rates of antibody positivity than the parental virus strain among foxes and minks . This isRecent advances in the development of CRISPR/Cas9-based gene-editing technology has brought immense opportunities for molecular interrogation of viral genetic determinants of virus–host interactions, pathogenicity, gene-deleted, and/or recombinant vaccines, through its special advantages of simple design, high efficiency, low cost, and wide application; however, some shortcomings, especially the possible occurrence of “off-target” effects, are causing some concerns. According to the principle of complementary base pairing, gRNA guides Cas9 nuclease to carry out strict site-specific cleavage of target genes; however, within the whole genome, the binding of gRNA to non-specific target sites may lead to an unexpected genomic modification, which is called “off-target”. Therefore, the possibility of “off-target” occurrences should be first considered to be avoided or reduced as much as possible when gRNAs are analyzed and designed. To resolve this problem, scientists have established various strategies to increase the gRNA specificity to eliminate the occurrence of “off-target” modification, such as using recombinant Cas9 nuclease and/or Cas endonuclease from different bacterial sources, shortening the length of the gRNA sequence, or making some changes in the secondary structure. All these measures have been able to reduce “off-target” modifications to a certain extent. We can also analyze, monitor, and prevent the occurrence of “off-target” modifications with the help of computational software prediction, intra- and extracellular detection, and whole-genome sequencing.Until now, most of the reports on the CRISPR/Cas9-based gene editing of viruses are still in the stage of laboratory research and are focusing on three main aspects: (1) using this technology to achieve the deletion of viral pathogenic genes as potential tools for gene therapy applications against diseases, although this still requires long-term research, observation, and practice to realize the clinical application; (2) editing some of the functional viral genes, as a simple and rapid genetic engineering technology to be applied for studying the virus pathogenesis and/or virally induced tumorigenesis; (3) modifying the viral genome by gene deletion, substitution, and/or insertion to construct vaccine candidate strains with various gene recombination, providing a new efficient approach for the research and development of recombinant vaccines. However, a long period is required to observe the precise editing site and the passage stability of reconstituted viruses. The emergence and development of any innovative technique is a key step in progressing both science and technology. Undoubtedly, CRISPR/Cas9-based gene editing technology provides endless possibilities for future scientific research and vaccine development to meet the significant outstanding challenges in biology.


Since their discovery in 2014, reptile nidoviruses  have emerged as significant pathogens worldwide. They are known for causing severe and often fatal respiratory disease in various captive snake species, especially pythons. Related viruses have been detected in other reptiles with and without respiratory disease, including captive and wild populations of lizards, and wild populations of freshwater turtles. There are many opportunities to better understand the viral diversity, species susceptibility, and clinical presentation in different species in this relatively new field of research. In captive snake collections, reptile nidoviruses can spread quickly and be associated with high morbidity and mortality, yet the potential disease risk to wild reptile populations remains largely unknown, despite reptile species declining on a global scale. Experimental studies or investigations of disease outbreaks in wild reptile populations are scarce, leaving the available literature limited mostly to exploring findings of naturally infected animals in captivity. Further studies into the pathogenesis of different reptile nidoviruses in a variety of reptile species is required to explore the complexity of disease and routes of transmission. This review focuses on the biology of these viruses, hosts and geographic distribution, clinical signs and pathology, laboratory diagnosis and management of reptile nidovirus infections to better understand nidovirus infections in reptiles. Nidovirales is a large group of diverse enveloped positive-strand RNA viruses  and Middle East respiratory syndrome (MERS-CoV) (The order  viruses . NidovirERS-CoV) , 3. ThisERS-CoV) .Following the emergence of these viruses in humans from animal sources, there is a renewed interest in animal nidoviruses including understanding the risk of cross species transmission from wildlife reservoirs. Novel viruses originating in wildlife reservoirs, especially bats, have also caused significant mortality and morbidity in animal populations, including swine acute diarrhoea syndrome coronavirus (SADS-CoV). This virus was implicated in the death of nearly 25,000 piglets –8. AlthoNidovirales continues to expand rapidly with the introduction of next generation sequencing (NGS) and metagenomics studies (Python regius) is not a new phenomenon, with some groups reporting a syndrome of unknown cause being observed by veterinarians since the late 1990's  since the 1990's. In 2016, the first nidovirus in a lizard was reported in this species, both with and without respiratory disease  (Myuchelys georgesi). In contrast to the infections in snakes and lizards, respiratory disease was not the predominant syndrome observed, but rather the most significant pathological changes were in the kidneys  . In fres kidneys .Batrachochytrium dendrobatidis)  –34, yet Serpentovirinae” OR “serpentovirus.” Searches were conducted without limits on publication dates or geographical location. A total of 455 articles were identified and 391 remained once duplicates were removed. Additional articles were excluded if the study was not available in English, a reptile was not referred to throughout the article (only an author surname or Python software© or programming language), unable to access or non-relevant articles leaving 213 articles to be reviewed. Additional articles were found manually from the citations of relevant articles. A summary of the search terms and results can be found in A literature search was conducted between January 2018 and June 2021 using the following databases: Web of Science , PubMed, Google Scholar to find peer-reviewed articles as well as Trove for thesis manuscripts. Search terms included “reptile AND nidovirus,” “python AND nidovirus,” “turtle AND nidovirus,” “lizard AND nidovirus,” and “Serpentovirinae spp. sequences, viruses identified by the International Committee on Taxonomy of Viruses (ICTV) (n = 8) were included (n = 41). The following search terms were used to identify these sequences; “Tobaniviridae,” “Serpentovirinae,” “unclassified Nidovirales,” “unclassified Torovirinae,” and “unclassified Serpentovirinae” (n = 49) and a remotovirus (bovine nidovirus) from the family Tobaniviridae can be found in To identify key published included  as well virinae” . A phyloNidovirales was composed of four families: Arteriviridae, Coronaviridae, Mesoniviridae, and Roniviridae . Th. ThNidovNidovirales exhibit significant diversity in their morphology. The recently revised family Tobaniviridae includes both of the previously known genera Torovirus and Bafinivirus. When examined by electron microscopy, toroviruses are known to be pleomorphic with bacilliform (rod-shaped), kidney shaped, and spherical virions often observed, while bafiniviruses have a rod-like or bacilliform appearance. A distinctive feature of both the previously known toroviruses and bafiniviruses are the surface projections that correspond to the club or petal shaped projections found on the surface of coronaviruses  , 21, 22.Nidovirales. Named from the Latin word “nidus” for nest, the order is characterised by a nested set of viral subgenomic messenger RNA's that are produced during infection  in Switzerland. This sequence was <85% identical to BPNV  that clustered with sequences found in boas, colubrids and mud snakes  tend to cluster . To datehon spp.  suggestihon spp. , 19, 20.d snakes  (20).In contrast, our knowledge of the reptile nidovirus diversity in lizards and turtles is limited to key publications with viruses detected from several reptile families including Scincidae (skinks), Chamaeleonidae (chameleons), and Chelidae (Austro-South American side-neck turtles) –23. DespReptile nidoviruses have been detected worldwide. This includes detections on four continents, including North America, Europe, Asia, and Australia. They have also been found in multiple hosts from the orders Squamata (lizards and snakes) and Testudines .M. viridis)  when compared with the ball python (P. regius)   based surveys of captive snake species , 41–43. 1, 5.1%) , 41, 42.In contrast to Pythonidae, the prevalence in snakes from the Boidae and Colubridae families appears to be significantly less with 2.4, 10.1, and 0.8% reported in captive Boidae species , 20, 41 Tiliqua rugosa) admitted to a wildlife rehabilitation centre. The virus was present in lizards with and without respiratory disease. Ongoing surveillance at two wildlife rehabilitation centres found 58.1% of T. rugosa admitted to this facility (for a variety of reasons) gave positive results in an SBNV PCR  experiencing respiratory disease associated mortalities (Pogona vitticeps), common leopard geckos (Eublepharis macularius), and ocelot geckos (Paroedura pictus). These animals were clinically healthy throughout this period and gave negative results for VCSV by PCR , in a single river system, and was implicated in the mortality of over 400 turtles (Emydura macquarii). Since 2015, there have been no confirmed clinical cases of BRV in wild M. georgesi, yet BRV was detected on conjunctival swabs from a small number of clinically normal M. georgesi (9 of 31 sampled) and E. macquarii (2 of 49 sampled) in a follow-up survey 6 months after the cessation of the outbreak (n = 360) from many reptiles, amphibians, arthropods, and fish failed to detect other species infected with BRV  juveniles (~6 weeks old) were exposed orally and intratracheally with a cell culture grown BPNV. Clinical signs were observed 4 weeks after exposure and included mucosal hyperaemia and profuse mucus secretion, followed by a progression to the appearance of petechial mucosal haemorrhages, open mouth breathing, and anorexia by 10–12 weeks post exposure. The presence of infectious virus was confirmed using virus isolation from oroesophageal swabs taken on the day of euthanasia; 5, 10, and 12 weeks post exposure  infected with SBNV can also present with respiratory disease. Similarly to P. regius, a respiratory syndrome has been observed since the 1990's that is characterised by excess mucous in the oral cavity, sneezing, serous to mucopurulent discharge from the eyes and nose, lethargy, inappetence, pale mucous membranes, depression, and emaciation  infected with VCSV, clinical signs included wheezing, vertical head tilting with gasping, increased mucus in the oral cavity, anorexia, and reduced water intake  infected with BRV were largely found as dead or moribund animals with bilateral ocular inflammation, poor body condition, and some had tan foci on the skin of the ventral thighs or hind limb paresis   , 18. Expneumonia . The proneumonia , 57.M. viridis) high viral loads have also been confirmed in the lung, but in contrast to the findings of Stenglein et al.  Dervas et al.  with SBNV. However, a single veiled chameleon  coinfected with VCSV-A and VCSV-B had both respiratory symptoms and histological lung lesions like those identified in snakes. This included an interstitial proliferative and catarrhal pneumonia, rhinitis, and tracheitis (n = 3) or mild non-specific lesions (n = 3) including focal xanthomatous mural enteritis with coelomic foreign body, severe heterophilic enteritis with mural granulomas, splenic lymphoid hyperplasia, mild lymphocytic portal hepatitis, rare mineralization of the tunica intima of large cardiac vessels, and hepatocellular vacuolization   . Subsequ regius) . This fi regius) . For exaMorelia spp.) that were nidovirus PCR positive for over 2 years with serial testing at ~4 month intervals remained asymptomatic for the duration  of infected pythons, 12.5% (1 of 8) of infected boas, and were absent in a single infected colubrid . Anotherduration .T. rugosa) infected with SBNV. In animals presenting to a wildlife care facility, SBNV was detected by qRT-PCR in 12% of apparently healthy individuals (4 of 33)  that did not develop respiratory disease over a 3 month period of monitoring prior to euthanasia (M. georgesi) an intensive field survey was undertaken 6 months after the cessation of the initial BRV outbreak and BRV was detected by qRT-PCR in 29% (9 of 31) of apparently healthy individuals  . Similarthanasia . In fresividuals . It is lividuals  that addA co-infection, defined as more than one pathogen infecting an individual, is not uncommon in reptiles . To dateC. calyptratus). These viruses only shared 53% nucleotide identity of ORF 1b, which is considered the most conserved region of the genome  of transmission of nidoviruses in reptiles remains unclear. The successful infection of several exposure . Subsequexposure , 22. In n = 26) and hatchlings (n = 18) of nidovirus positive python mating pairs. Eggs were “cleaned” by exposure to UV irradiation or a quaternary ammonium disinfectant and artificially incubated. Despite virus being detected in/on most eggs following hatching only a single offspring became infected. This infection was detected when the offspring was 8–12 months of age and had been sampled at 4-monthly intervals. The viral sequence was more similar to the infected male (>98%), than the female (84%) of the breeding pair raising the question of whether there had been true vertical transmission , equine arteritis virus (EAV), and gill-associated virus (GAV) –71. In asmission . To datein situ hybridisation (ISH). There are currently no assays available for the detection of nidovirus specific antibodies. Given the recent discovery of nidoviruses in reptiles there is limited validation, or standardisation of diagnostic methods, offering opportunities for future research.The diagnostic options currently available for the detection of reptile nidovirus infections largely reflect the methodology used for their discovery. To date, most methods of detection are directed at the detection of virus or its components with next generation sequencing (NGS) laying the foundation for the development of both real time and conventional reverse transcription polymerase chain reaction (RT-PCR) assays. Such molecular assays are the predominant diagnostic tool available. Other diagnostic tools include transmission electron microscopy (TEM), virus isolation in cell culture, immunohistochemistry (IHC), and NGS and viral metagenomics have been used to detect novel reptile nidoviruses from a range of samples including fresh tissues  and swabs in viral transport media . With advances in technology, partly as a result of the difficulties when undertaking virus isolation, most sequencing occurs on nucleic acid extracted directly from a tissue sample or swab , 15–17, in silico to optimise performance before being applied to routine testing. In addition to qualitative (positive/negative) results, quantitative PCR assays also provide insights into the amount of virus present which can be used to develop associations between the virus detected and a possible role as the cause of a disease process  assays have been used to detect reptile nidoviruses. To detect virus, PCR offers several advantages when compared to TEM, virus isolation in cell culture and ISH. This includes fast results and a high analytical sensitivity and, usually, specificity. One of the advantages of NGS and molecular assays such as PCR is that new assays can be designed and first evaluated  process , 18, 22. process , confirmMost PCR assays target the most conserved region of the virus, ORF 1a or ORF 1b, or the more variable region encoding for the spike protein , 55, 59.Several ante-mortem sample types including swabs, tracheal washes, faeces, and blood samples have been successfully used for PCR , 55. ForThe long duration of infection and viral shedding that is apparent in reptiles infected with nidoviruses facilitates the application of PCR based virus detection and surveillance. A longitudinal survey of pythons in a single collection over 28 months revealed that infection with a nidovirus can be chronic and definitive evidence of viral clearance was not observed . HoweverThis technology has been responsible for the detection of many viruses either in suspension or in sections of tissue. Although it has been in use for many decades, TEM finds merit in situations where currently available assays do not allow visualisation of virus morphology, tissue tropism, intracellular events associated with virus replication, assembly and release from cells. Consequently, TEM is a valuable tool for pathogenesis studies , 75. TraAlthough considered a “gold standard” for the laboratory diagnosis of viral diseases, when compared to options available for the testing of mammalian species, the choice of established continuous reptile cell lines is limited. Furthermore, consideration must be given to the cultural requirements, especially temperature, for both cell lines and virus replication due to the poikilothermic nature of reptiles . HoweverMorelia spilota spilota) cell culture. Cultures were inoculated with viral transport media from an oral swab collected from a P. regius and maintained at 30°C (M. viridis) liver and brain cells. Cultures were inoculated with homogenates of lung tissue from diseased snakes and maintained at 30°C  anti-serum at 3 days post inoculation, with all cell types except brain cells shown to be permissive for MVNV (M. viridis) using suspensions of liver, lung, kidney and intestine inoculated onto viper heart cells (VH-2) at 29°C  heart cell cultures, and two cell lines: iguana heart (IgH2) and viper heart (VH-2). Cultures were inoculated with fresh-frozen tissue homogenate  from a single chameleon  and maintained at 30°C  of infected animals died over a 28 month period  and thercroflora , 65, 94,croflora . Antimiccroflora , 41, 56.croflora . FurtherManagement practises that limit the introduction and transmission of a reptile nidovirus in captive collections are consistent with general recommendations for hygiene and biosecurity in all facilities that house reptiles. Quarantining new animals from collection for a designated period to enable appropriate health cheques and screening of pathogens is strongly recommended . The durA review of nidoviruses in reptiles reveals a relatively new field of research. The literature is dominated by the detection of novel nucleic acid sequences that broaden our understanding of viral diversity in reptiles. Opportunities exist to further our understanding of this diversity, especially in the lesser studied species by opportunistic screening of samples from disease investigations, or PCR based surveys of wild reptile species. There also remains exciting knowledge gaps to fill, especially in the linking of novel sequences to clinical disease and pathology through transmission trials and fulfilment of Koch's postulates in various species. This will also explore the complexities of the likely routes of transmission. As more sequences are detected further research into determining if there are clear host-specific lineages, and the apparent resistance of different reptile species to infection will provide additional insights into host susceptibility. Opportunities to explore the impact of reptile nidovirus genotypes and co-infections on pathogenesis or virulence are also numerous.Improvements in sequencing technology and analysis underpin the rapid development of targeted PCR assays. However, given the diversity of viruses detected in different species there may be significant advantages in moving toward the initial use of assays that are more broadly reactive to ensure different reptile nidoviruses or strains with minor genetic variation are not missed. The apparent long duration of nidoviral infection and shedding in snake species facilitates the use of PCR based monitoring. Conversely, asymptomatic infections, intermittent shedding, and varying viral loads, highlight the need for a serological assay to provide alternative diagnostic and surveillance approaches, especially for live animals. However, the development of such assays may prove challenging.The study of nidoviruses in reptile populations has been focused on research in captive populations. The nature of captive reptile collections (the acquisition of wild and exchange of captive reptiles) can provide an opportunity for the introduction of both known and unknown pathogens. High holding densities and high rates of transfer between collections can potentially increase pathogen exposure and lower barriers to transmission . FurtherUnfortunately, the prevalence and distribution of nidoviruses in wild reptile populations is still largely unknown. In the midst of a global SARS-CoV-2 pandemic, the importance of understanding wildlife disease not only for the species involved but also for the potential public health implications, is readily apparent. By their nature, detecting emerging or novel pathogens is difficult, yet with advancing detection methods it is imperative that research into the extent and distribution of reptile nidoviruses continues so that we preserve the priceless biodiversity and critical reptile populations worldwide.KP and LS conceived and designed this review. KP wrote the manuscript, analysed the data, and prepared figures and tables. EA, PK, and LS contributed to the concept and reviewed drafts of the manuscript. All authors contributed to the article and approved the submitted version.This review was supported by the College of Public Health, Medical, and Veterinary Sciences Higher Degree Research Enhancement Scheme, James Cook University, Australia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors, EA.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Boechera stricta (Brassicaceae). Over 6 years, we simulated climate change along an elevational gradient in the Rocky Mountains through snow removal, which accelerates snowmelt and reduces soil water availability. Costs of reproduction were greatest in arid, lower elevations, where high initial reproductive effort depressed future fitness. At mid-elevations, initial reproduction augmented subsequent fitness in benign conditions, but pronounced costs emerged under snow removal. At high elevation, snow removal dampened costs of reproduction by prolonging the growing season. In most scenarios, failed reproduction in response to resource limitation depressed lifetime fecundity. Indeed, fruit abortion only benefited high-fitness individuals under benign conditions. We propose that climate change could shift life-history trade-offs in an environment-dependent fashion, possibly favouring early reproduction and short lifespans in stressful conditions.Investment in current reproduction can reduce future fitness by depleting resources needed for maintenance, particularly under environmental stress. These trade-offs influence life-history evolution. We tested whether climate change alters the future-fitness costs of current reproduction in a large-scale field experiment of  For exaEmpirical studies have investigated costs of reproduction across diverse taxa –11, but Contemporary climate change exposes natural communities to increased temperatures, altered precipitation and more frequent and severe extreme events such as heatwaves, droughts and floods . These cEmpirical studies typically examine the costs of reproduction by quantifying how current reproductive effort affects future fitness components, such as survival, growth and fecundity . Yet, stBoechera stricta (Brassicaceae), a perennial forb native to the Rocky Mountains. This alpine habitat is dominated by iteroparous perennials with slow life cycles [Here, we test the hypothesis that novel, stressful climates could exacerbate costs of reproduction for populations of e cycles –40. We pe cycles . We examWe tested whether: (i) costs of reproduction are greater in arid low elevation sites compared with mesic high elevation locales, and (ii) snow removal augments the costs of reproduction within each garden. As climate change progresses, high elevation populations will experience warmer and drier conditions similar to current conditions at lower elevations ,42 and w. 2(a)Boechera stricta (Graham) Al-Shehbaz (Brassicaceae) is a short-lived perennial forb native to the Rocky Mountains, where it ranges in elevation from 700 to 3900 metres above sea level (m.a.s.l.) and latitudes from Utah to Alaska [o Alaska . We condo Alaska ,45. Thiso Alaska , making (b)B. stricta [n = 7094, from 104 maternal families and 43 source populations, of which n = 5824 survived the first winter and were included in analyses; n = 14–16 blocks, except in the highest garden, which had seven blocks). To minimize microenvironmental effects on fitness, we randomized one full sibling per family into each block and alternated snow removal and control blocks in each garden. Snowmelt timing, soil temperature and soil moisture varied minimally across blocks of the same treatment within each elevation and our multiyear experiment captured inter-annual climate variation representative of historical climates .We established five common gardens near the Rocky Mountain Biological Laboratory  at 2553, 2710, 2890, 3133 and 3340 m.a.s.l., reflecting the local elevational distribution of  stricta . In this stricta ,48. Priog. S1 in ). Transpg. S1 in . From ple winter  and a goIn the Rocky Mountains, climate change decreases winter snowpack and hastens spring snowmelt . Reduced(c)We monitored survival, flowering success and fecundity from April 2014 to October 2019 by visiting all individuals 2–3 times per week. For reproductive individuals, we measured fecundity by summing the length of all mature siliques (fruits) for each individual (a reliable proxy for seed set ). We als(d)To test whether simulated climate change conditions exacerbate costs of reproduction, we used generalized linear models that analysed how reproductive effort in the first year (successful and failed reproduction) affected three fitness components: longevity (plant lifespan), future probability of reproduction and future fecundity. We calculated first-year fitness as the total mature silique length produced by each individual in 2014, with values of zero for individuals that produced no mature fruits. We quantified first-year failed reproduction as the total number of failed siliques of each individual in 2014. We examined our three response variables as a function of first-year fitness, failed reproduction, garden elevation  and snow removal treatment, and all two- and three-way interactions of first-year fitness or failed reproductive effort with garden and treatment. Negative effects of first-year fitness or failed reproduction on future fitness components indicate a cost of initial successful or failed reproduction, whereas positive effects signify a benefit of reproduction or suggest that fruit abortion is adaptive. Significant interaction terms reflect shifts in costs of reproduction across abiotic environments.We standardized first-year fitness and failed reproduction to a mean of 0 and standard deviation of 1. All models included initial size as a covariate and random effects for maternal family and block to account for genotypic differences and microenvironmental variation. Previous analyses from this and complementary experiments documented local adaptation to elevation and geographical location of source populations ,49. To a(i)A mixed-effects Cox proportional hazards model  evaluat(ii)car package v. 3.0-7 in R [c). Therefore, we excluded the lowest elevation garden from fecundity analyses.We investigated fecundity costs in hurdle models that account for zero-inflation. First, we analysed how first-year fitness and failed reproduction affected the cumulative probability of reproducing in all subsequent years using a mixed model logistic regression with the aforementioned fixed and random effects . We ass0-7 in R  and of r(iii)To examine the lifetime consequences of allocating resources to failed fruits, we analysed lifetime fecundity as a function of the proportion of fruits that failed in the first year , garden elevation, treatment and all interactions, with the aforementioned covariates and random effects in a generalized linear mixed model with a gamma distribution and log link . We calTo evaluate the combined effect of first-year fitness and failed reproduction, we analysed lifetime fecundity as a function of initial silique length, initial failed silique number, garden elevation, treatment and all 2-, 3- and 4-way interactions with a covariate for initial size and random effects for block and genotype in a generalized linear mixed model with a gamma distribution and log link (glmer function of lme4 R package) . We visu(iv)R package MCMCglmm v. 2.26 [6 Markov chain Monte Carlo (MCMC) iterations with the first 30% of iterations (6 × 105) discarded for burn-in and 2000 iterations sampled for estimating the mean and 95% credible intervals of genetic correlations within each garden elevation individually, for control and removal treatments [gV) and block effects using us covariance structure [We calculated genetic correlations between initial reproduction and subsequent fitness components using generalized linear mixed models in the  v. 2.26 . These g v. 2.26 . We modeeatments . These Beatments . We extrtructure . We test. 3(a)c). Thus, our 6-year long experiment captured the full lifespan of most plants. The significant interaction between garden elevation and first-year fitness indicated that the longevity cost of reproduction varied across elevations . We found no influence of first-year fitness on longevity at the highest and lowest elevation gardens, but a pronounced longevity cost of reproduction emerged at the second-lowest garden (2710 m) . By contrast, at mid-elevation gardens (2890 and 3133 m), greater first-year fitness lengthened lifespan .Excluding plants that died during the first winter and those destroyed by gopher tunnelling, experimental individuals lived an average of 3.23 years (1182.1 ± 189.8 days) and 7.5% of the transplants survived until October 2019 c. Thus, χ24 = 65.7, p < 0.0001; electronic supplementary material, table S1) occurred because failed reproduction severely reduced longevity at the lowest elevation gardens (2553 and 2710 m), increased longevity at mid-elevations (2890 and 3133 m) and did not influence longevity at the highest elevation .The significant interaction between garden elevation and first-year failed reproduction ((b)χ23 = 17.37, p = 0.00059; electronic supplementary material, table S3). We found a significant cost of first-year fitness on future odds of reproduction at high and low elevations  in both treatments . By contrast, the probability of subsequent reproduction increased with first-year fitness in the control treatment at the mid-elevation (2890 m), but snow removal entirely eliminated this benefit of initial reproduction .The probability of reproduction after the first season varied as a function of a significant three-way interaction among first-year fitness, garden elevation and treatment . At mid-elevation (2890 m), the future probability of reproducing increased with first-year failed reproduction under control conditions . By contrast, at low elevation (2710 m), high initial failed reproduction lowered future reproductive odds . First-year failed reproduction had no effect on the future probability of reproducing for all other garden elevation by treatment combinations .Garden elevation and experimental treatment influenced the costs of first-year failed reproduction (number of failed siliques in 2014 × garden elevation: (c)χ23 = 12.31, p = 0.0064; electronic supplementary material, tables S6 and S7), which was driven exclusively by a fecundity cost of reproduction under control conditions in the highest elevation . We detected no fecundity cost of failed reproduction .The fecundity costs of first-year fitness differed across garden elevation by treatment ((d)χ24 = 20.96, p = 0.0003; electronic supplementary material, table S8). Lifetime fecundity declined with the proportion of failed fruits across all environments, except in the control treatment at the second-highest elevation (3133 m); the negative effects of high proportional production of failed siliques were most pronounced in low elevations and under snow removal at high elevations .The lifetime costs of resource allocation to first-year failed fruits differed as a function of garden elevation by treatment . In four of the five elevations, lifetime fecundity peaked under high allocation to both successful and failed initial reproduction, but few individuals occupied that part of the fitness landscape (figure 3f–j). For individuals with lower first-year fitness, allocation to failed initial reproduction was detrimental in the lowest elevation gardens . By contrast, the lifetime costs of initial failed reproduction dampened at higher elevation gardens (2890 and 3133 m). We found no significant interaction in the highest elevation.The synergistic effect of successful and failed reproduction on lifetime fecundity varied as a function of garden elevation, (successful first-year fitness × first-year failed reproduction × garden elevation: (e)All patterns that emerged at the phenotypic level were also present in genetic correlations, validating that costs of reproduction detected in phenotypic analyses result from genotypic variation in resource allocation and acquisition. At low and high elevations (2710 and 3340 m), we detected trade-offs between first-year fitness and failed reproduction for the probability of future reproduction under both control and snow removal . We foun. 4Our large-scale field experiment demonstrated that environmental conditions can fundamentally alter the cost of reproduction, a key life-history trade-off that underlies the evolution of perenniality. Concordant with predictions, costs of reproduction were more apparent in arid, lower elevation sites. For some fitness components, costs of reproduction also emerged under control conditions at our highest elevation. By contrast, in the mid-elevations, high investment in first-year reproductive effort lengthened lifespan (both treatments) and increased future fecundity (control treatment only). Snow removal at these mid-elevation sites eliminated this positive association, generating pronounced fecundity costs of reproduction for one site in genetic correlation analyses. These results suggest that climate change could alter life-history evolution across the elevational range of the species.B. stricta in a greenhouse experiment [B. stricta populations are already in decline at low elevations [The initial investment in reproduction decreased future fitness at the lower elevations  even under ambient conditions. Climate change has increased aridity in this system through reductions in snowpack and accelerations of snowmelt ,50,58, wperiment . We suggevations , and heiBy contrast with the costs of reproduction at low elevations, at mid-elevations (2890 and 3133 m), longevity increased with initial investment in reproduction and individuals with high fitness in the first growing season were more likely to reproduce again in control treatments. These mid-elevations experience the mild climates, with greater snowpack, later snowmelt, lower temperatures and reduced evapotranspiration than low elevation site, without the shorter growing seasons of higher elevations (see table S1 in ). We proDactylorhiza lapponica. Snow removal extended the growing season by an average of 9.8 days at this elevation [At the highest elevation (3340 m), the probability of future reproduction declined with initial reproduction in both treatments. Phenotypic and genetic models also revealed fecundity costs of reproduction under control conditions but not snow removal. As this site has the highest water availability along this elevation gradient , the coslevation . If the levation .(a)Plants regularly produce flowers and fruits that fail. Selective fruit abortion could enable plants to resorb and reallocate resources to alternative functions, or resources allocated to failed fruits could be lost, intensifying costs of reproduction . We founGiven the costs of reproductive failure, why would plants produce surplus reproductive structures? Proposed hypotheses include: uncertainties in pollination success; predation of flowers, fruits and seeds; the unpredictable nature of future resource availability; and the potential to improve seed quality by the selective abscission of low-quality fruits . Althoug(b)Erysimun capitatum (Brassicaeae) populations had higher mortality at all life stages compared to those from higher elevations, and low elevation plants reproduced more quickly and were more frequently semelparous than alpine plants [Climate change influences vital rates that shape life-history strategies –62. It fe plants . As adule plants , long-lie plants . Yet, ouAs climate envelopes move upslope in mountainous regions and toward the poles latitudinally, local populations may no longer perform optimally in new environmental conditions at their home sites ,64,65. I. 5In our long-term experimental manipulation, we demonstrate that climate change can shift costs of reproduction in a perennial subalpine forb. Costs of reproduction were prevalent in arid, lower elevations and under the short growing seasons at the highest elevation. Additionally, pronounced costs of reproduction emerged under climate change simulations at mid-elevations. We hypothesize that shifts in costs of reproduction could alter life-history evolution, potentially reducing longevity in all but the highest elevation locales. We encourage future studies to examine how shifts in costs of reproduction under climate change will affect plant population dynamics.Click here for additional data file.


Moreover, thermodynamic analysis demonstrated the spontaneous and endothermic nature of the dye uptake. Importantly, even when subjected to five regeneration cycles, the gel beads retained 87% of their original adsorption value, suggesting their suitability as an efficient and reusable material for dye wastewater treatments.Porous sustainable cellulose/gelatin/sepiolite gel beads were fabricated via an efficient ‘hydrophilic assembly–floating droplet’ two-step method to remove Congo red (CR) from wastewater. The beads comprised microcrystalline cellulose and gelatin, forming a dual network framework, and sepiolite, which acted as a functional component to reinforce the network. The as-prepared gel beads were characterized using FTIR, SEM, XRD, and TGA, with the results indicating a highly porous structure that was also thermally stable. A batch adsorption experiment for CR was performed and evaluated as a function of pH, sepiolite addition, contact time, temperature, and initial concentration. The kinetics and isotherm data obtained were in agreement with the pseudo-second-order kinetic model and the Langmuir isotherm, with a maximum monolayer capacity of 279.3 mg·g Nowadays, wastewaters from dye-using industries, such as clothing, leather, synthesis, and electroplating, pose a major challenge to global society . Of the Considerable effort has been put into developing techniques to remove CR from effluents, including photolysis , coagulaβ-(1→4)-linked d-glucose units. Since the successful preparation of cellulose aerogels by Tan et al. in 2001, the field has progressed rapidly with third-generation cellulose aerogel materials today being widely used in adsorption, energy storage, thermal resistance, electromagnetic interference shielding, and biomedicine . Th. ThS°) i follows :(8)ln were used as the adsorption conditions, as shown in Reusability is a vital factor for potential practical applications and can provide further insights into the adsorption mechanism. The adsorption–desorption process was performed five times, and the results are shown in −1 for CR at 303 K. Thermodynamic analyses illustrated that the CR adsorption onto MGS beads was spontaneous and endothermic. After five adsorption/desorption cycles, the MGS beads were found to retain 87% of their initial adsorbability, demonstrating themselves as an efficient and renewable candidate for dye wastewater treatment.This work reports the fabrication of cellulose/gelatin/sepiolite (MGS) gel beads via a simple and efficient two-step ‘hydrophilic assembly-floating droplet’ method. During the MGS gel bead preparation, microcrystalline cellulose (MCC) and gelatin (GEL) worked as dual network frameworks, and sepiolite (SEP) acted like a ‘crosslinker’ to connect and reinforce the dual network. A series of characterizations demonstrated that the MGS beads were lightweight and highly porous, and the incorporation of SEP, not only increased the adsorption capacity, but also made the beads more thermally stable. The adsorption behavior followed the pseudo-second-order model and Langmuir isotherm, with a maximum monolayer capacity of 279.3 mg·g


Bemisia tabaci- transmitted cucumber vein yellowing virus  is endemic in the Mediterranean Basin, where it causes significant losses in cucurbit crops. In this study, the role of the coat protein (CP) of CVYV for B. tabaci transmission and plant infection was investigated using a cloned and infectious CVYV cDNA and a collection of point and deletion mutants derived from this clone. Whitefly transmission of CVYV was abolished in a deletion mutant lacking amino acids in position 93–105 of the CP. This deletion mutant caused more severe disease symptoms compared to the cDNA clone representing the wild-type (wt) virus and movement efficiency was likewise affected. Two virus mutants carrying a partially restored CP were transmissible and showed symptoms comparable to the wt virus. Collectively, our data demonstrate that the N-terminus of the CVYV CP is a determinant for transmission by the whitefly vector and is involved in plant infection and symptom expression.Most plant viruses rely on vector transmission for their spread and specific interactions between vector and virus have evolved to regulate this relationship. The whitefly  Cucumber vein yellowing virus (CVYV) causes significant damage to cucumber and other cucurbit crops, such as zucchini and melon. The virus was first described in 1960 in Israel ,8,9,10. Ipomovirus (family Potyviridae) [Ipomoviruses are distinct from other viruses in the family Potyviridae and virus species in this genus have unique genome arrangements [CVYV belongs to the genus viridae) . The filviridae) ,14,15 (Fngements . The CVYngements .Bemisia tabaci whiteflies [Potyvirus and specific (DAG) motifs were identified as determinants for virus transmission [CVYV is transmitted in a semi-persistent manner by iteflies ,18. For iteflies ,20,21,22smission ,24,25. Ismission . Similarsmission . For thesmission .B. tabaci vector transmission of members of the genus Ipomovirus, for which transmission efficiencies vary considerably among the species. CVYV and squash vein yellowing virus (SqVYV) are transmitted efficiently [Nothing is known about virus genes involved in iciently ,18,29,30iciently ,32 and ticiently ,34,35,36Ipomoviruses, we noticed striking similarities between the CP of CVYV and that of the U/CBSV. Particularly, the core and carboxy-terminal regions of the CP were very similar  and presumably accounting for the observed serological cross reactions between U/CBSV and CVYV [Ipomovirus CP in the transmission process, we questioned whether the highly divergent N-termini (~38% aa identity) of the CP may explain the differences in transmission efficiencies between the viruses.When comparing the otherwise diverse polyproteins of the and CVYV . ConsideTo study the role of the CP in WF transmission, we generated a collection of CP substitution and deletion mutants in an infectious cDNA clone of CVYV and analyzed their effect on vector transmission and pathogenesis. Our results demonstrate that the N-terminal region of the CVYV CP harbors amino acids involved in WF transmission, virus movement and symptom development in cucumber. Specifically, a deletion in amino acid position 93–105 (clone CVYV_CP_Del3) abolished vector transmission and resulted in delayed but more severe symptoms. Collectively, our data advance the knowledge about CVYV transmission and identify a CP region that plays a key role in the infection process.Ipomoviruses revealed striking diversity in the N-terminal regions of the proteins ; a region of 33 aa in position 73–105 showed the highest variability  were stable, while others reverted to the original sequence . The mutAgroinfiltration of the deletion mutant clones CVYV_CP_Del1 and CVYV_CP_Del2, encoding a CP with a deletion of 10 aa at position 73–82 and 83–92, respectively, were not infectious, while the deletion mutant clone CVYV_CP_Del3 was infectious, and the introduced deletion was stable in successive mechanical transmission series. However, there was a delay of 7–10 days compared with the wt cDNA clone for symptoms to develop, and those became apparent ~21 d.a.i. In early infection stages, the symptoms resembled those of the wt, however during infection, vein yellowing symptoms became more severe  and exteIn whitefly transmission experiments, the three stable alanine-substitution mutants were transmissible, causing typical symptoms similar to the wt approximately 9–12 d.a.i. In contrast, CVYV_CP_Del3 was not transmissible and even high numbers of whiteflies and a prolonged acquisition- and inoculation access period (AAP/IAP) did not result in infections . AbsenceC. sativus 14 d.a.i. for CVYV_CP_Del3+8, whereas CVYV_CP_ Del3+5 had a further delay of about five days. This was also evident when CVYV from agro-infected plants was mechanically transmitted to cucumber resulting in symptom phenotypes comparable to the wt (not shown). Furthermore, whitefly transmissibility was restored, and verification of the CP by direct sequencing of RT-PCR products confirmed the expected sequence.In order to fine map the CP region involved in whitefly transmission, the deletion region was shortened by inserting either eight or five amino acids into CVYV_CP_Del3  to obtaiIpomovirus differ from other genera within the family Potyviridae. While SPMMV and ToMMV still represent typical potyviruses, all other viruses lack HC-Pro, a critical gene with suppression of silencing function and involved in vector (aphid) transmission [The genomes of viruses in the genus smission ,39,40. Ismission , to carrIpomoviruses by B. tabaci is in a semi-persistent manner, with transmission efficiencies varying largely with each virus species. The SPMMV was originally described as a whitefly-borne virus from Kenya, Uganda, and Tanzania [B. tabaci was confirmed as vector of ToMMV. Whitefly transmission studies with CVYV isolates from Israel and Jordan showed plant infections already after an AAP as short as 30 min [Transmission of Tanzania , but latTanzania . For ToMTanzania ,32,43,44Tanzania ,32, B. ts 30 min ,18. A trs 30 min . Our exps 30 min . Our whis 30 min . HoweverB. tabaci being the insect vector for Ipomoviruses, a mechanistic explanation for its virus transmission however is still pending, and neither receptors in the vector nor motifs in the virus it transmits are known.With The surface-exposure of N- and C-termini of potyvirus CPs has been known for a long time , emphasiThe restoration of transmissibility observed for virus constructs CVYV_CP_Del3+8 and CVYV_CP_Del3+5, supports that transmission competence was not assigned to a unique minimal motif in the Del3 CP region, as both reversions of eight and five amino acids in length resulted in transmissible viruses. Our results rather suggest that the CP region in position 93–105 is part of a structural domain that is necessary for interaction with the vector. Alternatively, disruption of the Del3 region could affect the folding in the N-terminus and prevent the correct assembly of CP structures required for transmission, as demonstrated in other virus/vector interactions ,55. CompA negative effect of CVYV_CP_Del3 infection on the behavior of the WF when feeding on infected plants was not evident by visual examination during the transmission experiments. In this context, a possible effect of virus-induced symptoms or alterations of the plant physiology on host attractiveness and vector feeding, as reported in other systems ,59,60, dThe disruption of a putative vector-binding domain on the surface of the CVYV virion did not alter infectivity and movement functions of the capsid protein, since the construct CVYV_CP_Del3 was able to infect cucumber systemically. Mutants carrying a deletion in position 73–82 and 83–92 were not able to infect the plant and this correlates with the role of the N-terminal region of the CP of potyvirids in short- and long-distance movement ,62,63,64The effect of amino acid substitutions in the viral CP on symptom expression has been documented for several viruses ,66,67,68Taken together, here we present evidence of the role of the N terminus of the CVYV CP for vector transmission and plant infection, and demonstrate that a region of 13 aa is required for whitefly transmission but is dispensable for systemic movement in the host plant. Further studies precisely revealing structural alterations of the CP and the localization of the virion in the vector can contribute to clarify the CVYV transmission process and provide insights into the puzzling transmission of other members of the genus.Cucumis sativus var. Vorgebirgstraube were grown at 18 °C and kept at 22/23 °C for agroinfiltration, to be transferred after one week for maintenance at 25/26 °C.An infectious cDNA clone of the CVYV isolate DSMZ PV-0776 , driven Ipomovirus was performed in Geneious Prime® v. 2019.0.4  using the alignment program Clustal Omega, on selected species: Cassava brown streak virus , Ugandan cassava brown streak virus , Coccinia mottle virus , Squash vein yellowing virus , Tomato mild mottle virus , Sweet potato mild mottle virus . The prediction of the secondary structure and solvent accessibility of the CVYV CP was performed using the protein modeling function of the Swiss-Model software  and superimposing on the Potato virus Y CP .Escherichia coli strain DH5α, and plasmid DNAs were prepared from 3 mL overnight cultures using a Nucleospin Plasmid kit (Macherey-Nagel). Plasmid pCB_CVYV_TK was used as template to generate the desired CP mutant clones, using the primers reported in ® Master Mix, New England Biolabs GmbH, Germany) with two additional fragments obtained by PCR amplification of pCB_CVYV_VK using primer combinations CVYV_Open_3UTR_F/pDIVA_TrfA_R and CVYV_Open_NIb_R/pDiVA_TrfA_F. Mutant clones carrying a deletion in the N-terminus of the CP  were generated by Gibson Assembly using the full-length wt clone as template and primers reported in ® 10-beta chemical competent E. coli cells according to the manufacturer′s instructions and bacterial colonies were grown for two days at 30 °C. Plasmids were purified from liquid cultures using the monarch plasmid miniprep kit (NEB). All generated constructs were verified by Sanger sequencing to proof read the desired mutation in the CP sequence.For alanine-scanning mutagenesis of the selected residues, a plasmid carrying the 3′ region of the CVYV genome, including the CP ORF, was constructed. For this purpose, the full-length cDNA clone pCB_CVYV_VK representing the wild-type was used as template in a PCR reaction  using primers CVYV_CP_F/CVYV_CP_Teilklon_R . The PCRR. radiobacter C58C1 cells, and infiltrated at OD600 nm = 0.8 in agroinfiltration buffer  into the first true leaf of three week-old cucumbers. To favor infections, the needle of a syringe was used to prick the stem and inject the agrobacterium solution, while cotyledons were punctured and subsequently infiltrated using a 1 mL syringe. Mechanical inoculations were performed using sap obtained from infected leaf tissues ground in 0.05 M phosphate buffer, supplemented with carborundum and silica powders. The plants were grown up to four weeks post-inoculation to monitor symptoms development and for molecular tests.Plasmids were transformed in ® software .Total RNA from cucumber leaves was extracted using the RNeasy Plant Mini Kit , according to manufacturer′s instructions. First-strand cDNA was synthetized using the SuperScript™ IV Reverse Transcriptase , with the specific reverse primer CVYV_RT_R (5′-TTTTATAACTTTACGCATAAAGG-3′). Two microliters of first-strand cDNA reaction were used for PCRs with primers CVYV_CP_F/R  and the B. tabaci (MEAM 63) colonies were maintained on tomato and cucumber plants in insect-proof cages at 26 °C, with a 16/8 h light/dark period. For the WF transmission assays, at least ~50/60 WF adults were transferred onto a symptomatic leaf of an infected cucumber for an AAP of at least 4 h. Two healthy cucumbers at one true-leaf stage were used as recipient plants for an IAP of at least 4 h. WF were removed and the plants were monitored for symptom development for 30 days. The stable alanine substitution- and deletion mutants  were tested for transmission, and the wild-type (CVYV_CP_wt) was used as control. To exclude a low transmission efficiency of CVYV_CP_Del3, a further transmission assay was conducted using ~150 WF with AAP and IAP on two recipient plants of 24 h each. Symptom development provided proof for whitefly transmission and virus infection, which was further verified by RT-PCR using primers CVYV_CP_F/R (Non-viruliferous V_CP_F/R .