The poultry industry is the largest source of meat and eggs for human consumption worldwide. However, viral outbreaks in farmed stock are a common occurrence and a major source of concern for the industry. Mortality and morbidity resulting from an outbreak can cause significant economic losses with subsequent detrimental impacts on the global food supply chain. Mass vaccination is one of the main strategies for controlling and preventing viral infection in poultry. The development of broadly protective vaccines against avian viral diseases will alleviate selection pressure on field virus strains and simplify vaccination regimens for commercial farms with overall savings in husbandry costs. With the increasing number of emerging and re-emerging viral infectious diseases in the poultry industry, there is an urgent need to understand the strategies for broadening the protective efficacy of the vaccines against distinct viral strains. The current review provides an overview of viral vaccines and vaccination regimens available for common avian viral infections, and strategies for developing safer and more efficacious viral vaccines for poultry. Mycoplasma gallisepticum. Control of virus outbreaks through vaccination on poultry farms will curb the risk of zoonotic infection, limit the exchange and introduction of novel pathogens into wild stock and the establishment of a wildlife reservoir.The poultry industry is the largest and one of the most important animal protein producers for human consumption. In 2020 alone, the global consumption of poultry meat was estimated to be more than 130 million metric tons, with egg production exceeding 86.67 million metric tons. The development of high-density poultry farms due to the industry’s rapid expansion also increases the risk of disease outbreaks. Numerous etiological agents have been isolated from farmed stock, ranging from bacteria such as acute coliform bacillary and chronic tuberculosis, ecto-and endo-parasites, and fungal pathogens to at least eleven virus species that can be transmitted either horizontally, vertically, or both. Coinfection of farmed birds with multiple pathogens is common in poultry husbandry. Prior infections with avian respiratory viruses have been documented to predispose birds to secondary bacterial infection. Indeed, a study by Sid et al. [Viral outbreaks are one of the leading causes of economic losses for poultry industries worldwide . Viral eThe rapid emergence of new virus variants highlights the urgent need for improved disease control and management and, in turn, drives the search for new or improved vaccine strategies. The turn of the millennium has seen the advent of a few innovative approaches to developing universal viral vaccines for humans against influenza A subtypes ,5. A uniAmong the different vaccine platforms detailed in this section, experimentally proven vaccine technologies such as live and inactivated vaccines have a long history of use on commercial farms as a prophylactic against economically important poultry viral diseases with global significance.The culture of native seed virus in embryonated chicken eggs (ECE) or in cell cultures is one of the oldest methods of vaccine preparation. Cultured viral particles are typically inactivated via physical  or chemical  means to destroy infectivity while preserving immunogenicity. Inactivated vaccines are considered a safer alternative to live vaccines due to their inactive status. However, inactivated viral vaccines are low in immunogenicity and require booster doses and formulation with adjuvants such as oil, saponins and aluminum hydroxide for longer lasting immunity. Immune responses triggered by inactivated vaccines typically consist of humoral immunity with a slow onset period and are unsuitable for a DIVA strategy . In addiLive attenuated (LA) vaccines have a long history of use against several poultry diseases. Conventional attenuation is typically achieved through serial passage of the wildtype (WT) virus in either irrelevant cell culture or host. However, the attenuation process requires a long turnaround time. Production of LA vaccine is cost-effective owing to minimal scale-up and downstream processing costs. Generation of LA vaccines by reverse genetics (RG) can bypass laborious conventional methods for developing attenuated strains. Attenuated viruses are generated from an infectious clone and engineered in vitro through RG, which is a tractable and rapid approach ,9,10. OtThe whole pathogen is not essential to confer complete protection against the disease, and fractionating virus proteins in chemically defined formulations has been one of the regulatory norms for minimizing side effects from vaccine components other than the immunogen. Recombinant subunit protein vaccines are among the most well-established, stable and DIVA compatible . RegardlVLPs are structural proteins with morphological features that resemble virus structures. Due to the similarity in structure, VLPs have successfully been utilized as novel vaccines against several viral pathogens. VLPs can be assembled using either prokaryotic or eukaryotic expression systems. Experimental studies have shown VLPs to confer high levels of protection against viral infections such as infectious bronchitis virus and avian influenza in chickens. The utilization of VLPs as a vaccine is considered a safer alternative to inactivated and attenuated vaccines due to the lack of genetic material. The safety profile, immunogenicity, protective efficacy and host immune response have also been comprehensively described . In addiAnother approach to vaccination is through the use of DNA vaccines. DNA vaccines can be engineered to encode genes for the expression of a specific or multivalent antigen in the transfected host. DNA vaccines elicit both cellular and humoral immunity and can be designed to trigger specific cell mediated immune (CMI) responses to facilitate lymphoproliferation and cytokine secretion. Nevertheless, DNA vaccines come with their own set of challenges. The first is the difficulty in integration of the DNA vaccine  into the® and Innovax®) and IBDV and ILTV (Farmune®-HVT-IBD-LT) into rHVT for the production of trivalent vaccines [Viruses such as fowl pox virus (FPV), turkey herpesvirus (HVT), adenovirus, ILT and MDV are established recombinant viral vectors used in the production of poultry viral vaccines ,17. Amonvaccines ,24.Recent advances in genome editing using CRISPR-Cas9  have paved the way for developing multiplex rHVT vector poultry vaccines. CRISPR-Cas9 can be employed for targeted mutagenesis of viral vectors such as HVT ,26,27, ISeveral viral vectors have been developed to express heterologous antigens in vitro as well as displaying them on the viral surface. Among these, baculovirus (BV), an insect pathogen, has been widely used as a tool for protein production in insect cells and is an excellent tool for displaying foreign proteins on the viral envelope . AdvantaAvian influenza virus is one of the most contagious viruses in farmed poultry and a significant concern due to its zoonotic potential. The poultry pathogen influenza A (Type A) is a segmented, negative-sense RNA virus. Influenza A viruses are categorized into different subtypes depending on their surface antigens known as hemagglutinin (HA) and neuraminidase (NA). Eighteen different subtypes of HA have been documented along with a total of eleven subtypes recorded for neuraminidase (NA) antigens. Combining the two antigens could theoretically produce 198 subtypes, most of which have already been identified in nature . PhylogeProtection against avian influenza AIV is primarily mediated by the humoral immune response, which produces neutralizing antibodies against the HA and NA antigens. However, immune responses triggered by both antigens are not considered to be broadly protective. Additionally, the cell mediated immune response involving cytotoxic T-lymphocytes is known to play a vital role in clearing infection and providing protection between antigenic distinct AIV ,45.Modern poultry AIV vaccines are primarily developed with platforms such as inactivated WT or RG; subunit or virus-like particles; recombinant virus vectors; DNA vaccines; and defective replicating alphaviruses, i.e., defective Venezuelan equine encephalitis (VEE) expressing HA protein or a DNA vaccine with a H5 AI virus gene insert. Currently, there is no universal vaccine against AIVs for poultry . A comprThe LPAI was later replaced by HPAI virus seeds (H7N3 or H5N1 Gs/GD lineage), but the highly demanding biocontainment and stringent quality control that comes with the use of an HPAI are major hurdles for their use . NonetheLive attenuated AI vaccines (LAIV) developed from ‘WT’ strains are not recommended for poultry by the OIE/FAO due to the potential risk of reversal of the attenuated strain into an HPAI by reassortment or mutation. A stable LAIV is an attractive target since it can be formulated for needle-free mass vaccination through nasal spray. Efforts along this line resulted in the development of cold-adapted/temperature-sensitive (ca/ts) variants to serve as LAIV vaccines . InteresViral vectors such as FPV, HVT, adenovirus, ILT and MDV have been evaluated as recombinant vectors for AIV vaccines . CurrentThe BV system is another alternative for the development of avian-based influenza vaccines, adapted from the established model for human influenza vaccine production ,67,68. T® or cationic derivatives trimethylpolyprenylammonium iodides (PTAI) and the Esat-6 gene from Mycobacterium tuberculosis as an adjuvant and utilized for immunization against H5 influenza virus in chickens [The US Department of Agriculture (USDA) conditionally approved the first DNA avian flu vaccine against H5N1 for chickens in 2017 ,80. Plaschickens ,82. ProtNDV is one of the most important contagious diseases affecting the respiratory, nervous, and digestive systems of poultry worldwide, resulting in severe losses to the poultry industry. The virulence, pathotypes, disease manifestations and its panzootic potential in poultry are well described in the literature . The majLive attenuated vaccines developed from lentogenic NDV strains, especially LaSota or inactivated oil emulsion vaccines , are comThere are at least 13 different recombinant viral vector vaccines licensed commercially to protect poultry against NDV. The first generation consisted of two FPV vectors, followed by six second generation and five third generation vaccines incorporating an HVT vector with the Fusion (F) protein or the hemagglutinin-neuraminidase (HN) protein of NDV. Most of these vaccines are administered via subcutaneous routes and in ovo ,24. HoweThe etiological agent of Marek’s disease (MD) is an alphaherpesvirus belonging to the genus Mardivirus, in the family Herpesviridae. MDVs are classified into three different species based on their serotypes: Gallid herpesvirus 2 , Gallid herpesvirus 3  and Meleagrid herpesvirus 1  . MDV-1 cThe MDV-2 serotype vaccine includes the SB-1 strain  or 301B/The MDV-3 serotype vaccine consists of another apathogenic strain, MDV-3 (HVT) FC126 cloned into an HVT vector . The HVTILT is an avian respiratory disease caused by a Gallid herpesvirus-1 (GaHV-1) belonging to the genus Iltovirus, and the subfamily Alphaherpesvirinae within Herpesviridae family. Infection with ILT is associated with decreased egg production, weight loss and mortality, and the transmission of disease typically occurs through aerosols and fomites . The spe® FP LT + AE, CEVA Biomune), gI and gD  or gB and UL-32  [ILT recombinant vaccines currently available for commercial use utilize either FPV or HVT vectors with expression of an array of ILT genes, such as gB and UL32 (VectormuneBiomune) . In addiBiomune) , modifieBiomune)  and recoBiomune) . AdditioBiomune) .The advantages associated with the use of recombinant vaccines includes the inability of the live strain to transmit between chickens, recombine and revert to the wildtype virulent form ,128. A bNumerous immunization routes have been evaluated for ILT vaccines, with the most practical route being in ovo vaccination of broilers with HVT, MDV, or the GaHV-1 ‘meq’ gene-deleted vector. Layers can be vaccinated at day 1 of age with HVT, MDV, FPV, NDV vector vaccines or GaHV-1 ‘meq’ gene-deleted strains followed by vaccination with live attenuated ILT strains . AdminisBirnaviridae. Infection with IBDV results in clinical symptoms of depression, diarrhea, dehydration and neoplasia of the bursa of Fabricius. IBD outbreaks have resulted in high morbidity with significant economic losses worldwide. VP2 is a structural polypeptide of IBDV and the major IBD antigen targeted for vaccine development [IBD, or Gumboro disease, is a highly contagious immunosuppressive viral disease of young chickens. The etiological agent of IBD is a non-enveloped, double-stranded bi-segmented, A and B, RNA-bearing avibirnavirus belonging to the family elopment . IBDV haelopment . There aelopment .Protection against IBD is achieved through a combination of passive MDA inherited from immunized parent stock and active immunization of chicks with modified live vaccines (MLVs). Most IBD MLVs originated from attenuated strains of IBDV serotype 1. In the late 1990s, immune complex vaccines containing live intermediate plus strains were mixed with hyperimmune sera against IBDV to produce virus-antibody complex vaccines that were administrated on day 1 or in ovo. The presence of high levels of anti-IBDV MDA does not interfere with protection against vIBDV and vvIBDV . MLVs arBesides live vaccines, several commercial recombinant vector IBDV vaccines have also been developed. However, the first generation, employing an FPV vector, was found to provide low protection. Currently, two second generation rHVT-IBD vaccines with host protective VP2 adapted from Faragher 52/70 (Vaxxitek HVT + IBD) and variant Delaware E (Vectormune IBD) are available. Third generation rHVT vaccines, Innovax IBD-ND and Ultifend IBD ND, were similarly formulated to include the VP2 immunogen from IBDV and the F gene from NDV. Another combination is a trivalent vaccine (Ultifend ND IBD) against IBDV, NDV and MD. Two more vaccines were produced and licensed in 2020 using a rHVT vector (Vaxxitek HVT + IBD + ND and Vaxxitek HVT + IBD + ILT) containing VP2 from IBDV, F from NDV genotype VII 1.1 and gD from ILTV USDA. The vaccines have been optimized for administration in ovo or via the subcutaneous route to provide long-lasting immunity. Another experimental study successfully displayed VP2 (BV-VP2) from IBDV on the BV viral surface. Vaccination of chickens with the BV-VP2 vaccine elicited high levels of VP2-neutralizing antibodies and stimulated antigen-specific lymphocyte proliferation. A challenge experiment of BV-VP2 vaccinated chickens with vvIBDV HZ strain was found to reduce clinical signs of disease and mortality . The useCoronaviridae family [IBV is a highly infectious, positive sense, single-stranded, enveloped RNA gammacoronavirus from the e family . Outbreae family ,136. Gene family . SurveilThe current approach to commercial vaccination typically takes the form of immunization with two antigenically diverse IBV variants selectively complemented with an inactivated vaccine for layers . CommercVaccine candidates typically target the four major virion assembly structural proteins known as spike (S), membrane (M), small envelope (E) and nucleocapsid (N) . Among tInactivated IBV vaccines used affinity-purified IBV antigens or formaldehyde or β-propiolactone-inactivated viruses, and in particular, the Mass-type (GI-1) genotype for immunization. The use of multivalent inactivated vaccines containing Mass antigens in association with live vaccines was shown to boost antibody titers and enhance protection against two homologous and three antigenically distinct heterologous IBV strains . A studyLive IBV vaccines are notably superior to inactivated vaccines . Cross-pIBV recombinant vaccines generally adopt immunogens such as the S1 and S2 subunit of the spike protein to create subunit vaccines. Immunogens are commonly purified from cell lines or expressed by baculovirus vectors . SeveralAnother potential path of control for IBV is with VLPs. An advantage of using VLPs over live IBV vaccines lies with its inactive status. VLPs consisting of S and M protein were found to produce functional antibody levels comparable with animals vaccinated with inactivated H120 . Most imChimeric recombinant and novel bivalent viral vector vaccines created through ‘RG’ are ideal for creating vaccines with dual protection against IBV and an alternative viral pathogen. RG vaccines require a shorter production process and have improved safety profiles to circumvent recombination events when used in the field. Ellis et al.  found thOrthoreovirus, under the family Reoviridae. Chickens infected with aRV usually display signs of viral arthritis (tenosynovitis), malabsorption and enteric symptoms with up to 10% mortality [The viruses discussed above are the primary pathogens of poultry; however, other viruses, such as adenovirus and reovirus, are generally considered to be secondary invaders of the upper respiratory tract of chickens . Avian rortality ,166. Conortality .The protective coverage and efficacy of a vaccine can be expanded through three major steps, as specified by Prabakaran et al.  and showThe strategy of modifying existing neutralizing epitopes to expand protection is illustrated with an example of H5N3 (A/duck/Singapore/3/1997) modification to confer cross-protection to an antigenically diverse range of human H5N1 viruses circulating from 1997 to 2004. The original H5N3 HA epitopes located at the 140th loop and 190th α-helix were modified to encompass the H5N1 clade 2 variants. The resulting reassortant virus strains showed reactivity and cross-neutralizing efficacy similar to the reference serum against H5N1 clade 2 viruses. In addition, mice immunized with the H5N3 mutant produced cross-neutralizing antibodies and cross-protected against distinct H5N1 viral infections. Thus, this strategy, which reduces the need for biosafety level 3 (BSL3) containment facilities, was proven to favor the development of broadly protective vaccines ,168. EarFor a broadly protective vaccine to be created, efforts are needed to understand the molecular aspects of the protective antigen with the effects to magnify the humoral response to the target antigen, reduce ‘off-target’ antibody responses and, more importantly, augment the response to the target epitopes . In the Several studies have also focused on inducing broadly reactive antibodies against both HA head and stalk regions. The conserved amino acids of an immunogenic antigen were first deduced with a phylogenetic analysis of all related virus variants. The conserved amino acids were then designed into a consensus immunogen to produce broadly neutralizing antibodies that neutralize viruses displaying the conserved epitopes. The consensus of the vaccine can range from micro-consensus, whereby the vaccine is designed to protect only against a branch of the phylogenetic tree, to a centralized consensus vaccine, where the antigen is expected to be effective against genetically diverse variants across the phylogenetic tree. However, ancestral and consensus-based conventional antigen designs are intrinsically influenced by the input sequences used to generate the synthetic molecule, and as such, are subject to sampling bias. To overcome these limitations, computationally optimized broadly reactive antigen (COBRA) is used to generate unique immunogens with exclusive spatiotemporal accuracy B. PrevioAntigenic cartography (AC) is a new, powerful mathematical and computational method to quantify and visualize fine-grain phenotypic differences among strains of viruses . The tecAdjuvants can be used in vaccine delivery agents to induce and promote a greater protective immune response against pathogens. They are traditionally divided into two categories. The first comprises immunostimulatory molecules, such as cytokines, Toll-like receptors (TLRs) and bacterial products that enhance and protect the immunogen from degradation in vivo. The second comprises of biodegradable particles such as polymeric nanoparticles, chitosan, liposomes and biomacromolecular compounds like sodium alginate, and gums derived from natural sources. The advantages of biomolecular compounds are that they are non-toxic, biodegradable and possess antiviral properties ,189,190.The mucosal site is a popular entry point for multiple viruses, and as such have to be taken into special consideration when it comes to the production of an efficacious and broadly protective viral vaccine against poultry diseases. Mucosal vaccination is the optimal formulation for mass vaccination and currently, there are only a handful of vaccines available for both human and animal applications. Numerous papers have been written on the effects of adjuvants and their unique properties for enhancing mucosal immune responses. Adjuvant for formulation with a broadly protective viral vaccine should ideally stimulate both cellular and humoral responses to induce antigen-specific IgA and systemic IgG ,189.Vaccines adjuvanted with TLR agonists or synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs can bind to TLRs present on immune cells to initiate an immune cascade for the production of proinflammatory and Th1 cytokines. The Th1 immune response is an important mediator for protection against poultry viruses . AnotherAmidst the increased demand for poultry meat and products worldwide, commercial and backyard farms are tasked with the continual challenge of producing the all-important supply of animal protein for human consumption. Vaccination is our only line of defense to ensure the demands for poultry products are met worldwide. With the advent of new technology, vaccine design can now be refined through the use of consensus-based algorithms, target conserved epitopes and neutralizing epitopes-based approaches to produce better vaccines against viral pathogens with genetically diverse lineages. Vaccine optimization with techniques such as antigenic cartography also allows for real-time immunological analysis of field and vaccine strains for quicker and more straightforward selection or update of immunogens. Additionally, newer generation of vaccines are being increasingly produced with protective immunogens from multiple species to provide simultaneous protection against co-circulating viral species. Advancement in adjuvant formulation and delivery strategies have likewise seen a shift to mucosal adjuvants for better stimulation of both systemic and mucosal immunity. While many vaccine design strategies for the control of AIV are in place, attention is needed to tackle evolving strains of poultry viruses such as the MDV and NDV. The development of a broadly protective vaccine against MDV would greatly benefit from a combination of improved formulation through the use of a mucosal adjuvant and optimization of vaccine targets based on the growing evidence from experimental studies. Considering the success of new vaccine strategies, more techniques must be channeled into the practical creation of efficacious, safer, and more cost-effective broadly protective viral vaccines.


Associations of chromatin with the nuclear lamina, at the nuclear periphery, help shape the genome in 3 dimensions. The genomic landscape of lamina-associated domains (LADs) is well characterized, but much remains unknown on the physical and mechanistic properties of chromatin conformation at the nuclear lamina. Computational models of chromatin folding at, and interactions with, a surface representing the nuclear lamina are emerging in attempts to characterize these properties and predict chromatin behavior at the lamina in health and disease. Here, we highlight the heterogeneous nature of the nuclear lamina and LADs, outline the main 3-dimensional chromatin structural modeling methods, review applications of modeling chromatin-lamina interactions and discuss biological insights inferred from these models in normal and disease states. Lastly, we address perspectives on future developments in modeling chromatin interactions with the nuclear lamina. Genome organization relies on chromosomal interactions  and at ts (LADs) . Some LAs (LADs) , underscComputational modeling of chromatin structure  creates Here, we highlight the heterogeneous nature of the NL and LADs, outline the main 3D chromatin structural modeling methods currently used, review computational models of chromatin-NL interactions, and discuss biological insights deducted from these models in normal and pathological conditions. Lastly, we address perspectives on applications of modeling interactions of chromatin with the NL with the aim of better appreciating the multiple facets of functional genome organization.via LADs, and thereby radially organize the genome  and B-type lamins   . The NL 2 genes) . Studies2 genes) . Additio2 genes) . Interes2 genes) . Whether2 genes)  remains 2 genes)  and provIn mammalian cells, hundreds of LADs have been mapped throughout the genome  using vaLADs are a general feature of genome organization, but not all LADs have similar properties . Some arLMNA gene  polymer physics models chromatin as a semi-flexible polymer chain that can adopt many configurations within physical constraints applied to the chain; 2) restraint-based modeling represents chromatin by beads in a Euclidian space with restrained interactions between them commonly determined from chromosome interaction data. We next provide an account of these modeling approaches in light of their relevance for modeling chromatin configuration at the NL. For details on 3D genome modeling methods, we refer to an excellent exhaustive review .P of the polymer—that is, the length under which the polymer behaves as a rigid rod and above which it behaves as a flexible chain. The repeating units of chromatin, modeled as monomers in the polymer chain, further limit the number of conformations the polymer can adopt during simulations; this limitation is typically achieved by introducing a self-avoidance effect to prevent clashes between monomers  of similar epigenomic signatures, or “colors”  Figure . Self-avhromatin . Despitehromatin .a priori knowledge of the modeled chromatin region, such as chromatin states or transcription factor binding profiles. Block copolymer simulations can also model the behavior of a chromatin chain that can assume various thicknesses and compositions, providing a more realistic view of chromatin  that mediate these interactions  Figure . Again, hromatin . Polymerhromatin .Other chromatin 3D modeling approaches use restraint-based methods to infer spatial information directly from experimental data and reconstruct structures without assumptions on folding mechanisms. Contact matrices derived from Hi-C other 3C-sequencing data are used to identify pairs of interacting domains as primary constraints. One restraint-based approach is to reconstruct a single consensus structure representing the average of 3D conformations in the cell population under study . ConsensTo enable this, other methods simulate many structures. Resampling methods carry out a large number of independent optimizations from the same input data. Optimizations start from, most commonly, a random chromosome configuration and use the same scoring function aiming to reach a state where no constraint violations remain, producing a quasi-stable structure  Figure . Of noteWe have also reported ensembles of chromatin structures  relying Chromatin folding at, and interactions with, the NL have been modeled in attempts to identify physical processes driving these events, infer mechanisms of chromatin association with, and dissociation from, the NL, and provide more accurate spatial genome structures at the nucleus level.C 360 nm representing a ∼50 kb region to enable modeling interactions of small vLADs or euchromatic sub-LAD regions  or heterochromatin (higher persistence length) at the NL can be approximated  surface representing a NL, fitted with an attraction potential towards the polymer . Chromat regions . The poled chain . Furtheroximated .Data from simulations indicate that in the absence of attraction potential from the NL surface, the polymer must be pinned by at least one bead to enable any interactions with the surface. Second, a flexible chain explores a greater area than a semi-flexible or rigid chain, so one may infer that a euchromatic region can explore a greater space near the NL than a more rigid heterochromatic domain . Third, Without attraction potential, polymer interactions with the surface are only transient and barely detectable (except at the anchors). This is consistent with other models where turning off an attraction potential between the NL surface and a chromatin polymer promotes polymer detachment . In contThe absence of attraction potential towards a NL may still enable expected polymer positioning relative to a physical constraint. Indeed, when a self-avoiding polymer is confined to a nucleus sphere, non-specific entropic forces alone can remarkably shape and position chromatin polymers in the sphere and approximate high-order localization of loose (thin) and compact (thick) segments in the sphere center or periphery, respectively .These findings have been extended by modifying the polymer-surface interaction parameter as a fraction of the chromatin polymer bound to the NL, and introducing a chromatin volume fraction (modeling a hydration effect) and an intra-chromatin attraction potential . Tuning in vivo chromatin imaging observations and, with only a small number of parameters, account for changes in phase separation (chromatin vs. aqueous) that may drive mesoscale chromatin reconfiguration in developing Drosophila larvae  and degradation of the NL, which releases heterochromatin from the nuclear envelope and elicits SAHFs . To disein silico by introducing LBR as a “binder” mediating these interactions in block copolymer models , which are only detectable in later passage . NonetheData from simulations are not uncommonly interpreted as “explaining” a biological observation, as exemplified in some recent studies . WhereasIntegration of genomic datasets from wet-lab experiments into restraint-based models of 3D genome structure have provided new spatial insights into genomic consequences of pathological states, which may open to new therapeutic avenues. For instance, statistical analyses of Chrom3D models of human fibroblast genomes indicate that UV-induced DNA lesions are predominantly detected in LADs, suggesting greater UV-susceptibility of chromosomes at the nuclear periphery . Even moCorroborating these findings, 3D genome models generated from Hi-C and radial positional information of loci reveal a decrease in the frequency of single nucleotide polymorphisms from the nuclear periphery towards the nucleus center, especially of those associated with melanomas or lung cancer . This isThese studies provide seminal examples of how restraint-based genome models may generate predictions on consequences of pathological insults or mutations on genome integrity with a 3D perspective. Currently, these models cannot explain a pathology, but hypotheses generated from statistical analyses of modeled structures open the door to better designed studies aiming to target specific regions in a 3D nucleus space which would not be predicted from one-dimensional data.A powerful outcome of computational models of chromatin behavior is the opportunity to generate testable hypotheses on the physical properties of chromatin. A recent elegant example is the use of block copolymer models of chromatin to make predictions on the nature and dynamics of homotypic chromatin interactions occurring in microphase-separated compartment throughout the nucleus . TestingDespite their increasing complexity and power, chromatin models still ignore some information inherent to chromosome structure important for nuclear function, such as additional physical properties of chromatin. For example, chromatin domains can intermingle or partly invade the NL. So chromatin cannot simply be considered as a hard entity (non-penetrable beads in models). Instead, beads may be fitted with a soft permeable outer zone and an adjustable penetrability factor to enhance flexibility of the chromatin chain and better approximate chromosome compaction . SimilarRecent analyses of NL structure and organization  suggest Polymer models of chromatin folding at the NL in progeroid cells  predict Further developments of 3D genome modeling to yield mechanistic insights into changes in nuclear architecture linked to disease would also be beneficial. Evidence implicates lamin mutations in alterations in LADs and chromatin conformation, and nucleus size and shape are also affected in cancer cells . IntroduDNA repeats constitute more than half of the human genome and play a role in re-wiring epigenomes and gene expression programs in a variety of developmental and pathological conditions . Repeat in situ hybridization techniques allow their visualization in nuclei  due to their repeat nature, repeat sequence reads are often discarded from analyses, 2) they are not relevant for the questions asked, or 3) they can simply be modeled as monomers (beads) alike any other chromatin domain. The latter is illustrated by the hidden inclusion of L1 repeats as LAD-associated topological domains (beads) in restraint-based whole-genome 3D models . Today hn nuclei . Altogetn nuclei .Recent evidence indicates that the NL constrains enhancers at the nuclear periphery, within LADs  and betwin situ hybridization (Combinations of more sophisticated computational approaches with rapidly evolving wet-lab technologies such as high-throughput genome editing , live chdization  and biopdization , will ex


Immune checkpoint inhibitor (ICI) is one of the most important tumor treatment methods. Although the therapeutic efficiency of immune checkpoint inhibitor mono-therapy is limited, the combination of chemotherapy plus immune checkpoint inhibitors has shown great advantages in cancer treatment. This is mainly due to the fact that tumor reactive T cells could fully provide their anti-tumor function as chemotherapy could not only cause immunogenic cell death to increase antigen presentation, but also improve the immunosuppressive tumor micro-environment to synergize with immune checkpoint inhibitors. However, traditional chemotherapy still has shortcomings such as insufficient drug concentration in tumor region, short drug duration, drug resistance, major adverse events, etc, which might lead to the failure of the therapy. Nano chemotherapeutic drugs, which refer to chemotherapeutic drugs loaded in nano-based drug delivery system, could overcome the above shortcomings of traditional chemotherapeutic drugs to further improve the therapeutic effect of immune checkpoint inhibitors on tumors. Therefore, the scheme of nano chemotherapeutic drugs combined with immune checkpoint inhibitors might lead to improved outcome of cancer patients compared with the scheme of traditional chemotherapy combined with immune checkpoint inhibitors. The development of immune checkpoint inhibitors (ICI) has brought new opportunities for tumor patients . Mainly The rationale of immune checkpoint inhibitor therapy is based on the anti-tumor function of tumor reactive T cells (CD8+ cytotoxic T lymphocytes)  . ApoptosSecond, tumor reactive T cells that could recognize tumor antigens need to contact with tumor cells to provide their anti-tumor effect. However, tumors develop multiple mechanisms to escape the “hunt” from tumor reactive T cells. One way is to exhaust tumor reactive T cells to impair their anti-tumor function, like expressing immune checkpoint molecules (such as PD-L1) to induce tumor reactive T cells apoptosis or exhaustion by activating immunosuppressive pathways , or recrAnother way is to reduce the number of tumor reactive T cells at the tumor site. The failure to attract tumour-reactive T cells to the tumour could be caused by the lack of appropriate chemokine secretion from the tumor  . The actTherefore, if tumor antigen presentation and tumor reactive T cells infiltration could be ensured, the therapeutic efficiency of immune checkpoint inhibitors on tumors could be improved as tumor reactive T cells could fully act its anti-tumor effect.Immune checkpoint inhibitor combined with chemotherapy has been proved to increase the efficiency of tumor treatment in a variety of clinical studies. For example, nivolumab (PD-1 antibody) plus first-line chemotherapy resulted in significantly longer overall survival than chemotherapy alone in patients with advanced esophageal squamous-cell carcinomaincluding esophageal cancer (13.2 months vs 10.7 month) ; the comOn the one hand, traditional chemotherapy could lead to immunogenic cell death (ICD) of tumor cell , which cOn the other hand, traditional chemotherapy could reverse the immunosuppressive tumor micro-environment, including the depletion of immunosuppressive cells, like CD4+CD25+FOXP3+ regulatory T (Treg) cells, myelogenous suppressor cells (MDSCs) and M2 like tumor associated macrophages (TAMs) , 25, andBased on the above analysis, the combination of chemotherapy and immune checkpoint inhibitors could be called a “Golden Combination” as tumor reactive T cells to fully exert their anti-tumor effects. This is because chemotherapy could promote tumor antigen presentation and tumor reactive T cells infiltration while the immune checkpoint inhibitor could block the immunosuppressive pathway.However, the disadvantages of traditional chemotherapy might affect this “Golden Combination”. First of all, traditional chemotherapy is usually administrated by a single dose at a regular interval . Drug concentration at tumor region would rapid decrease after chemotherapy (within 3 days after chemotherapy), which might reduce the tumor’s response to chemotherapy . Such a Second, traditional chemotherapy often causes serious side effects in cancer patients . TraditiNano chemotherapeutic drugs, which refer to chemotherapeutic drugs loaded in nano-based drug delivery system, are developed to overcome the shortcomings of traditional chemotherapy, including low bio-availability, low local concentration, short duration, major systemic side effects, etc. There are mainly two kinds of nano chemotherapeutic drugs in clinic currently, including Nab-paclitaxel and liposomal chemotherapeutic drugs Table 1.Nab-paclitaxel is a 130 nm particle formulation comprising albumin nanoparticles and paclitaxel with non-covalent bonds, which could largely reduce the adverse effect of solvent-based paclitaxel, including bone marrow suppression, allergic reactions, neurotoxicity and systemic toxicity . Food anLiposomal chemotherapeutic drugs are chemotherapeutic drugs loaded in liposomes (a revolutionizing nano carrier). Their success is attributed to stable drug loading, extended pharmacokinetics, reduced off-target side effects, and enhanced delivery efficiency to disease targets . There aIn the combination therapy of chemotherapy plus immune checkpoint inhibitor, only local therapeutic effect of chemotherapy in the tumor region is needed. Jie Mei et al. has confirmed this concept in patients with hecepocellular carcinoma (HCC), which is normally considered chemotherapy insensitive . ResearcFirstly, concentration of nano chemotherapeutic drugs in tumor region would be higher than traditional chemotherapeutic drugs, improving the therapeutic effect. Compared with the traditional doxorubicin, Doxil  showed 4 to 16 times the doxorubicin concentration in the tumor regions of patients using the drug . Other sSecondly, nano chemotherapy drugs could reduce systemic toxicity. Almost all the clinical trials of nano chemotherapeutic drugs have proved this point. In a study comparing traditional paclitaxel with nab-paclitaxel, although there was no significant difference in the incidence and degree of side effects between the two drugs, almost every patient in the traditional paclitaxel group received steroid treatment; moreover, patients in the traditional paclitaxel group were more likely to have granulocytopenia above grade 4 . A meta-In 2019, nab-paclitaxel combined with PD-L1 inhibitor was written into the treatment guidelines for metastatic triple negative breast cancer due to its good therapeutic effect with an ORR rate of 56%, much higher than historical ORR of nab-paclitaxel or PD-L1 inhibitor alone . In a phin vivo experiments. Kuai et al. constructed liposomes loaded with doxorubicin to stimulate the immune system and enhance the efficiency of immunotherapy  effect might be the mechanism for the local aggregation . There iAt present, there are few choices of nano chemotherapeutic drugs, which might be due to the limited therapeutic effect of mono-chemotherapy in many advanced tumors, resulting in the slow development of new nano chemotherapeutic drugs. The current treatment scheme of chemotherapy combined with immune checkpoint inhibitors has given new value to nano chemotherapeutic drugs. It does not necessarily lie in the direct anti-tumor effect, but in improving the tumor local immune micro-environment to enhance cancer treatment. Many nano chemotherapeutic drugs use nano vehicles that have already been proved by the FDA, such as liposomes . If the Nano chemotherapy drugs combined with immune checkpoint inhibitors might be a better combination to improve the efficiency of the current scheme of traditional chemotherapy plus immune checkpoint inhibitors in the treatment of tumors. This combination could not only increase the local therapeutic effect of chemotherapy, including increasing antigen presentation and improving the immune micro-environment, thus increasing the therapeutic effect of immune checkpoint inhibitors, but also reduce T cells depletion and systemic toxicity of chemotherapy drugs so that patients would better tolerate this treatment regimen, which would benefit cancer patients ultimately. Current evidence from clinical trials are limited; further validation for its safety and efficiency is needed. Also, novel kinds of nano-chemotherapeutic drugs with better tumor targetability could be expected to improve the therapeutic effect of nano-chemotherapy plus immune checkpoint inhibitors.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.XQ, WH and JY conceptualized, wrote, and reviewed the manuscript. All authors approved the submitted version.Start up Fund for Talent Researchers of Tsinghua University (No. 10001020507).We thank Dr. Qian Lu for many fruitful discussions.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.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.


Epstein–Barr virus (EBV) is an oncogenic human herpesvirus infecting approximately 90% of the world’s population. The oral cavity serves a central role in the life cycle, transmission, and pathogenesis of EBV. Transmitted to a new host via saliva, EBV circulates between cellular compartments within oral lymphoid tissues. Epithelial cells primarily support productive viral replication, while B lymphocytes support viral latency and reactivation. EBV infections are typically asymptomatic and benign; however, the latent virus is associated with multiple lymphomas and carcinomas arising in the oral cavity. EBV association with cancer is complex as histologically similar cancers often test negative for the virus. However, the presence of EBV is associated with distinct features in certain cancers. The intrinsic ability of EBV to immortalize B-lymphocytes, via manipulation of survival and growth signaling, further implicates the virus as an oncogenic cofactor. A distinct mutational profile and burden have been observed in EBV-positive compared to EBV-negative tumors, suggesting that viral infection can drive alternative pathways that converge on oncogenesis. Taken together, EBV is also an important prognostic biomarker that can direct alternative therapeutic approaches. Here, we discuss the prevalence of EBV in oral malignancies and the EBV-dependent mechanisms associated with tumorigenesis. Epstein–Barr virus (EBV) is a ubiquitous human tumor virus that resides as a lifelong infection in the oral epithelium and B lymphocytes that involves latency and productive viral replication . B lymphEpithelial cells also support the productive phase of the viral lifecycle following a coordinated viral gene expression cascade resulting in amplification of the viral genome and production of progeny virions . LatencyInfection with EBV is typically acquired asymptomatically in childhood. However, delayed infection until adolescence often causes infectious mononucleosis, a self-limiting lymphoproliferative response to primary EBV infection . AlthougEBV is primarily transmitted through saliva. Early studies showed that infectious EBV is secreted in saliva from patients with infectious mononucleosis and in healthy adults . In healEpithelial cells in the oral cavity are the major producers of EBV found in saliva . EBV infThe importance of epithelial cells in the lifecycle of EBV has proven difficult to study due to challenges associated with in vitro epithelial infection. Infection of primary epithelial cells in monolayer culture typically is not efficient and viral DNA is rapidly lost within days . In addiBamHI fragment A rightward long non-coding transcripts (BARTs) and BART microRNAs. EBV-infected germinal center B cells express the default/latency II program restricted to EBNA1, LMP1, LMP2, and non-coding RNAs. Infected memory B cells that are dividing express only EBNA1 and viral non-coding RNAs (latency 1), while non-viral gene products are detected in non-dividing memory B cells (latency 0). The infected memory B cells provide a long-lived cellular reservoir for EBV persistence and life-long infection. Terminal differentiation of the memory B cell to a plasma cell in response to antigenic stimulation reactivates the EBV productive lifecycle [EBV infection of circulating resting B lymphocytes in the oral cavity initiates a journey for life-long persistence in the memory B cell compartment. It is also important to note that EBV may directly infect germinal center or memory B cells . Entry iifecycle  Expressiifecycle .EBV infects over 90% of adults worldwide, and EBV is associated with 1% of all cancers . EBV immEBV latent infection is a shared feature among EBV-associated malignancies . The EBVHigh viral loads due to deregulated viral replication can also contribute to the tumorigenic process. Individuals with infectious mononucleosis typically exhibit higher viral loads in blood and saliva for months after diagnosis and are at an increased risk for developing Hodgkin’s lymphoma . FurtherEBV infection has been shown to increase genomic instability by various mechanisms . EBNA-1 EBV induces epigenetic alterations that regulate the expression of tumor suppressor expression and apoptotic responses. Nasopharyngeal carcinoma and EBV-associated gastric carcinoma display CpG island hypermethylator phenotypes that silence tumor suppressor gene expression. Common tumor suppressors silenced in NPC and EBV-associated gastric cancer include: RASSF1, CDKN2A/p16, CDH1, and PTEN . ImportaIn this review, we will describe various molecular mechanisms utilized by EBV to promote oncogenesis. Specifically, we will direct our attention to EBV-associated lymphomas and carcinomas affecting the tissues and organs of the oral compartment either directly or indirectly. While many of the precise mechanisms remain unclear our goal is to present a current summation of established links between EBV and cancer development and to identify the questions that remain. EBV driven malignancy is a complex process that differs between the various EBV-associated cancer types. It is important to understand both the similarities and differences in the role of the virus across different cancers to prevent and treat EBV-related neoplasms.Lymphoma is a disease of the lymphatic system characterized by malignant outgrowth of lymphoid cells or lymphoid precursors . Oral lyBurkitt Lymphoma (BL) is an aggressive non-Hodgkin B cell lymphoma currently classified into three variants based on both clinical features and cancer epidemiology: Endemic, sporadic and immunodeficiency-associated . Dennis Common to all BL variants is translocation of MYC into the immunoglobulin heavy chain or light chain loci. MYC is placed under the control of the immunoglobulin enhancer resulting in overexpression of the MYC oncogene. Translocation of MYC on chromosome 8 into the immunoglobulin heavy chain locus on chromosome 14 is the most frequent, occurring in 75% of cases . The traEBV is detected in the majority of endemic BL cases carrying a clonal EBV genome in tumor cells indicating that viral infection preceded cellular transformation . How EBVPlasmodium parasites have been epidemiologically linked to endemic BL. The mechanistic interplay between malaria, EBV, and BL is complex and still unresolved. Repeated malarial infections have been suggested to impair cytotoxic T cell responses potentially affecting immune control of EBV [Plasmodium falciparum infected mice exhibited abnormal expression of AID in B cells apart from the reactions of the germinal center [P. falciparum infected red blood cells were also shown to enhance AID transcription and protein levels in tonsillar B cells [Plasmodium chabaudi induced mature B cell lymphomas with AID-dependent translocations that included MYC/IgH [The malarial l of EBV . Chronicl of EBV . Loss ofl of EBV . Plasmodl center . Extract B cells . Importa MYC/IgH . Thus, EWhether EBV is essential for the development of BL has been questioned by the prevalence of EBV-negative sporadic BL and immune-associated BL cases. However, two recent studies identified EBER-negative cases that where EBV microRNAs were detected, raising the possibility that EBV-negative BL may be derived from EBV-infected B cells . An alteDiffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma, accounting for 30% of all lymphoma cases . DLBCL cEBV-positive DLBCL (NOS) are rare malignancies that typically present in immunocompetent individuals over 50 years of age, but this cancer can also affect children and younger adults. EBV-positive DLBCL (NOS) accounts from 3–15% of DLBCL cases, being more prevalent in Asia and Latin America . EBV-posEBV-positive DLBCL (NOS) display unique molecular features compared to EBV-negative DLBCL. Overall, EBV-positive DLBCL (NOS) have a lower mutational burden suggesting that EBV can replace mutations otherwise required to drive the oncogenic phenotype . ConsistHodgkin lymphoma is a B cell malignancy characterized by the presence of few malignant cells surrounded by many non-neoplastic inflammatory cells. Based on histological and immunophenotype presentation, Hodgkin’s lymphoma (HL) is classified into two major types: known as classical HL and nodular lymphocyte predominant HL. In classical HL, the malignant cell is the Hodgkin and Reed/Sternberg cell (HRS). In nodular lymphocyte predominant HL, the malignant cell is the lymphocyte predominant cell (LP) . As noduClassical HL account for 90% of all HL. The HRS malignant cell is derived from a germinal center B cell that has undergone somatic hypermutation of immunoglobulin genes and clonal expansion . The HRSEBV is associated with approximately 20 to 50% of classical HL cases. The presence of the virus in HRS cells is typically identified via in situ hybridization (ISH) to EBV DNA or EBV EBER RNA. Among the classical HL subgroups, EBV is common in the nodular sclerosis and mix cellularity classical HL subtypes, with rare detection in lymphocyte rich or depleted types . An EBV Although the HRS cells originate from germinal center B cells carrying clonal immunoglobulin rearrangements , these HEBV latency factors has been suggested to compensate for the loss of BCR and support tumor growth and survival. High levels of LMP1 are detected in classical HL. LMP1 mimics CD40 signaling and activates JAK/STAT, PI3K, and NF-kB signaling pathways . Forced The tumor microenvironment in classical HL is composed of inflammatory cell infiltrates suggested to create an immunosuppressive environment for HRS cells to escape immune surveillance and/or support the growth and survival of HRS cells . T cellsAlthough EBV has been implicated in supporting HRS and development of HL, the presence of EBV-negative HL has suggested multiple avenues that can lead to lymphoma. EBV may substitute for mutations and promote oncogenic phenotypes that support growth, survival, and immune evasion. EBV-positive classical HL display distinct genetic alterations from EBV-negative classical HL, similar to what is observed in EBV-positive DLBCL . WhetherOver 90% of oral cancers are squamous cell carcinomas . Oral sqOral squamous cell carcinomas (OSCC) comprise a heterogenous group of carcinomas that arise at various anatomical sites in the oral cavity. Oral cavity squamous cell carcinoma (OCSCC) arises from lips, anterior tongue, roof and floor of mouth, gingiva, and buccal mucosa. Oropharyngeal squamous cell carcinoma (OPSCC) involves the tonsils, posterior third of tongue, and soft palate. OPSCC due to infection with human papillomavirus has rapidly emerged as a distinct entity and will be discussed separately.OSCC accounts for an estimated 2.5% of all cancers worldwide . Based oEBV has been detected at higher levels in OSCC compared to healthy tissues with many studies detecting EBV DNA, RNA and protein in tumor cells ,128. HowThe viral gene expression program in EBV-positive OSCC is not well established. Various studies have detected EBER expression in OSCC tumor cells ,131. AltPorphyromonas gingivalis and Porphyromona endodalis have been shown to produce butyric acid, an HDAC inhibitor that can reactivate latent EBV [In the oral cavity, the microbiome can influence the prevalence of herpesviruses  as well as the degree of inflammation . In chrotent EBV . Such miAlthough EBV infection is not sufficient to immortalize epithelial cells in culture, viral infection can confer oncogenic features to already immortalized/transformed cells lines similar to what is observed in EBV-associated NPC and gastric carcinoma. EBV infection of hTERT-immortalized gingival epithelial cells induced a DNA hypermethylator phenotype , which iReductions in smoking and alcohol consumption has decreased OSCC incidence worldwide. Nonetheless, the past thirty years has seen the emergence of OPSCC associated with human papillomavirus (HPV) infection in developed countries ,145,146.The majority of HPV-positive OPSCC occur at the tonsils or base of tongue, lymphoid rich regions where EBV also resides. Moreover, HPV-positive OPSCC has distinct clinical features not evident in other HPV associated cancers . HPV-posAs a potential cofactor, EBV is detected in 5–20% of HPV-positive OPSCC ,156,157.Although the contribution of EBV to HPV-positive OPSCC carcinogenesis still needs to be defined, similar mechanisms suggested for EBV driven NPC carcinogenesis may be involved. EBV is a well-established etiological agent in nasopharyngeal carcinoma (NPC), a HNSCC occurring in the nasopharynx. The proximity to the oropharynx and presence of a lymphoid infiltrate in NPC tumors provide EBV access to nasopharyngeal epithelial cells . Early gHPV-immortalization of epithelial cells may create an environment that favors latent EBV infection rather than productive replication. Epithelial interactions between EBV and HPV have been studied in epithelial organotypic raft culture, a physiologically relevant culture producing stratified and differentiated epidermal layers that support HPV and EBV productive lifecycle. HPV-immortalized keratinocytes grown in organotypic rafts inhibited EBV replication following de novo infection . An aborPIKCA, ZNF750, CASZ1, PTEN < CYLD, and DDX3X, while mutations in FAT1, CDKN2A, NOTCH1, CASP8, and HRAS are associated with HPV-negative OSCC [At the molecular level, HPV-positive OPSCC display distinct genetic changes compared to HPV-negative OSCC . HPV16-pive OSCC . The disOral hairy leukoplakia (OHL) are benign oral lesions that presents as elevated white patches on the dorsum and lateral borders of the tongue, and in rare cases the soft palate, pharynx, or esophagus are involved . Other fOHL is a result of productive EBV replication within oral epithelial cells. EBV infects oral squamous epithelial cells and replicates in the absence of cell-mediated immunity. Manifestation of OHL is one of the earliest signs of AIDS, and OHL is strongly correlated with decreased CD4+ counts . A deficAlthough OHL is associated with lytic EBV replication, several latently associated factors (except the EBERs) are expressed simultaneously with lytic genes . EBNA2 iAlthough EBV growth promotion is frequently associated with latent gene products, OHL epithelial hyperplasia implicates replicative gene products in promoting carcinogenesis. This is supported by B cell immortalization being less efficient in the absence of BZFL1. Presence of BZLF1 also enhanced lymphoma development in humanized mice models . SeveralLymphoepitheliomas are undifferentiated carcinomas associated with a dense lymphoid infiltrate that occur at various anatomical sites being associated with EBV. These epitheliomas resemble undifferentiated NPC also having a lymphoid infiltrate. Salivary gland lymphoepitheliomas involving the parotid gland are rare oral tumors that are associated with EBV. A high incidence is observed among native people in Greenland, Alaska, and some Asian populations . In thesEBV EBERs are detected strictly in malignant epithelial cells and not significantly detected in the infiltrating lymphocytes or surrounding benign tissues . DetectiIn the more than 55 years since its discovery, EBV has been shown to play a complex role in the pathogenesis of several human malignancies. Though EBV associated oral cancers are rare, the oral cavity is central to the life cycle of the virus within its human host. The oral anatomy provides proximity between the B lymphocytes and epithelial cells in which the virus completes its life cycle. The persistence of EBV within the tissues of the oral cavity is a risk factor for cancers in the oral compartment and at other anatomical sites. The current understanding of EBV mediated oncogenesis is summarized in


Several smartphone applications have been developed for the purpose of low-cost and convenient assessments of vegetation canopy structure and understorey illumination. Like standard hemispherical photography, most of these applications require user decisions about image processing, posing challenges for repeatability of measurements. Here I report a test of CanopyCapture, an application that instantaneously estimates percentage canopy gap fraction without any input from the user, and has the added advantage of an intuitive levelling mechanism.Nothofagus) stand and an old-growth podocarp/broadleaf forest. These comparisons were repeated using a wide-angle adapter to enhance the smartphone camera’s field of view from 45 to 65°. I also asked if CanopyCapture results depended on sky condition (sunny vs. overcast) and on the type of smartphone used.Gap fraction estimates by CanopyCapture were compared with gap fraction values computed by the LAI-2200C Canopy Analyzer, in two contrasting evergreen temperate forests in New Zealand: an even-aged southern beech (2 = 0.39), and use of the wide-angle adapter lifted this value to 0.56. However, CanopyCapture output was not significantly correlated with LAI-2200C output in the even-aged Nothofagus stand, where there was less spatial variation in canopy structure. Despite being much less sensitive to variation in gap fraction than the LAI-2200C, CanopyCapture was nevertheless able to detect differences in average gap fraction between the two forests studied. CanopyCapture results beneath intact canopies were not significantly affected by sky condition, but reflection of direct light off tree trunks in sunny weather caused slight overestimation of gap fraction beneath broken canopies and gaps. Uneven or patchy cloud cover can also cause erroneous readings beneath large canopy openings. Three different models of smartphone gave different results.CanopyCapture output was significantly correlated with gap fraction computed by the LAI-2200C (RCanopyCapture offers a rapid and repeatable proxy for comparisons of average canopy gap fraction in multiple stands/forests, provided large sample sizes are used. Measurement under even overcast skies is recommended, and studies involving multiple operators will need to standardize smartphones to ensure comparability of results. Although wide-angle adapters can improve performance, CanopyCapture’s low sensitivity prevents high-resolution comparisons of the light environments of individual understorey plants within a stand. Ecologists and foresters often have need of high-quality data on forest canopy structure and/or understorey light availability . Uses ofThe LAI-2200C Canopy Analyzer is a powerful state-of-the-art tool for quantification of canopy structure and understorey light availability . HoweverHemispherical photography obviates the need for access to a clearing and is a more affordable method, especially since the development of digital cameras. The potential for distinguishing between the direct and diffuse components of irradiance offers the additional advantage of very detailed information about understorey light environments . HemisphDensiometers are an affordable low-tech option that can detect broad patterns of spatial and temporal variation in canopy openness . HoweverSeveral free smartphone applications have been developed recently with a view to providing affordable, rapid, and convenient assessments of canopy structure and understorey light availability. These include PocketLAI , Canopeoclosure i.e. the percentage of the sky obscured by vegetation when viewed from a singular point, like a camera lens. Moreover, standard smartphone cameras produce rectilinear images, which like any other projection that is not perfectly equisolid, will distort the relative areas of gaps in the canopy as a function of zenith angle  with fewer Knightia excelsa (Proteaceae), Laurelia novae-zelandiae (Atherospermataceae) and Litsea calicaris (Lauraceae). The treeferns Dicksonia squarrosa (Dicksoniaceae) and Cyathea smithii (Cyatheaceae) are common in the understorey, and sometimes form dense thickets in treefall gaps (Ripogonum scandens (Ripogonaceae) forms dense tangles in places. Although some stands were selectively logged during the mid-20th century, most forest in the reserve approximates an old-growth structure, including treefall gaps in varied stages of regeneration. Nomenclature follows An old-growth podocarp/broadleaved forest was sampled in the Lake Okataina Scenic Reserve . The podocarps all gaps . The monNothofagus) stand was sampled on a poorly-drained site near Mamaku on the Patetere Plateau . Nothofagus truncata up to 22 m tall formed most of the canopy, accompanied by fewer and slightly smaller N. menziesii. An approximately normal distribution of N. truncata diameters between 15 and 70 cm, and the presence of occasional larger cut stumps in an advanced state of decay, were consistent with establishment of an even-aged Nothofagus stand after logging of the previous forest. Weinmannia racemosa (Cunoniaceae) and Ixerba brexioides (Strasburgeriaceae) were common in the subcanopy and understorey. There were only a few small canopy gaps, resulting from standing deaths of N. truncata trees. Access to this forest was provided by Hancock Forest Management (OTPP_91459) and Department of Conservation (97727-RES).A southern beech (G) is based on averaging the logarithms of diffuse light transmittance in the five angular bands, weighted by their relative areas:The LAI-2200C quantifies the attenuation of diffuse sky radiation in five angular bands within a quasi-hemispherical field of view, using five sensors arranged in concentric rings. One of two wands was mounted on a tripod and used to take measurements 1.5 m above each sampling point. The other wand was placed in a large clearing that offered an unobstructed view of the sky over the 148° field of view of the instrument, and programmed to take readings at 30 s intervals. Combining data from the two wands enables calculation of diffuse non-intercepted irradiance beneath a forest canopy as a proportion of simultaneous irradiance outside the forest. Computation of gap fraction , Bi is diffuse irradiance reaching the understorey wand from each of the five zenith angle arcs, Ai is diffuse irradiance reaching the clearing wand from each of the same arcs, and Wi is the weighting of each of the five zenith angular bands according to their relative areas.where the subscript CanopyCapture installed on a Samsung Galaxy A21s smartphone was used to estimate gap fraction at the same sampling points. The 13 MP front camera used by CanopyCapture has a 45° field of view, meaning it samples a much smaller area of the forest canopy than the LAI-2200C. After each measurement with the LAI-2200C, the phone was placed on the same tripod and CanopyCapture measurements taken with and without a clip-on 37 mm wide-angle HD lens  fitted to the phone camera. This lens increased the camera’s field of view to 65°. Measurements were made under overcast conditions, between 11:00 and 15:00 h on summer days .Measurements were taken at 30 points in each forest, randomly spaced 11 to 15 m apart on transects run through the forest. This spacing meant that successive sampling points were unlikely to fall beneath the crown of the same canopy tree, as most crown diameters in New Zealand forests are ≤10 m . All traDacrycarpus dacrydioides that mostly established after 19th-century logging; and Beilschmiedia tawa, some of which are undoubtedly older (D. dacrydioides (some exceeding 1 m diameter) that predate logging. The other common trees are Laurelia novae-zelandiae, Alectryon excelsus (Sapindaceae), and Melicytus ramiflorus (Violaceae). Recent tree deaths have left a few small canopy gaps. Research in Jubilee Bush was authorized by Hamilton City Council.CanopyCapture installed on a Samsung Galaxy A21s smartphone was used to compare canopy closure values obtained under blue and overcast skies. This aspect of the work was carried out in Jubilee Bush , a 5-ha urban remnant of evergreen temperate forest in Hamilton, New Zealand. The present forest canopy in Jubilee Bush is dominated by ly older . There aSampling points were randomly spaced between 11 and 15 m apart along two transects run parallel to the longest axis of the reserve. At each of 27 sampling points, three flat-topped 25 cm plastic stakes were driven into the soil, to provide a level sampling platform 15–20 cm above the forest floor. CanopyCapture measurements were taken from these sampling platforms under overcast and blue skies between 09:30 and 15:00 h in August (winter) 2021.CanopyCapture installed on three different smartphones was used to estimate canopy closure in a wooded area of the University of Waikato Hamilton campus . These consisted of a Samsung Galaxy A21s (13 MP front camera with 45° field of view), a Samsung Galaxy S20 , and a Sony H4113 . Fifteen sampling points were spaced at random intervals along a 100-m long transect run along the longest axis of the wooded area. Measurements were made under an overcast sky. The eight different tree species present in the canopy included two deciduous species that were leafless at the time of sampling (August 2021), resulting in wide spatial variation in canopy gap fraction.smatr R package , using the  package . SMA , and use of the wide-angle adapter lifted this figure to 0.56 , and post-hoc pairwise comparisons showed the mean value recorded on the Samsung A21s was significantly higher than those obtained with the other two phones  = 0.17, P = 0.85), despite the narrower range of values obtained with the Sony H4113 phone , with the caveat that large numbers of points should be sampled to compensate for the narrow field of view and low sensitivity.CanopyCapture was able to distinguish between two forests that differed significantly in average gap fraction . Leaf arcf. nearly two orders in the old-growth forest at Okataina  are one likely cause of discrepancies between different phones; however, this factor is not a sufficient explanation, as mean estimated canopy closure differed most clearly between two Samsung phones with cameras that shared the same field of view but differed in resolution (Significant discrepancies between CanopyCapture results obtained with different phones  show thasolution . Differesolution  have alssolution .CanopyCapture offers a rapid and repeatable proxy for comparisons of average canopy cover and/or understorey illumination in multiple forest stands, provided large sample sizes are obtained from each stand. The repeatability resulting from the lack of user input suits CanopyCapture well to studies integrating data obtained by multiple users working in different locations, provided all participants use the same model of smartphone. The intuitive levelling mechanism is also convenient, obviating the need for the operator to carry a tripod. Although enhancement of smartphone cameras’ field of view with wide-angle or fisheye adapters has the potential to improve performance, CanopyCaptures’s low sensitivity prevents high-resolution comparisons of the local light environments of individual plants within a stand.10.7717/peerj.13450/supp-1Supplemental Information 1Click here for additional data file.


The severe acute respiratory virus covariate-2 (SARS CoV-2) that causes Corona Virus Disease 2019 (COVID-19) has affected more than 194 million people worldwide and has attributed to or caused more than 4 million deaths. Acute kidney injury (AKI) is a common complication of COVID-19. Point of care ultrasonography (POCUS) can be a useful tool for the nephrologist. POCUS can be used to elucidate the cause of kidney disease and then also help to manage volume status. Here, we review pearls and pitfalls of using POCUS to manage COVID-19 associated AKI with special attention to kidney, lung, and cardiac ultrasound.  A trained nephrologist with a portable ultrasound can make important contributions without exposing additional staff like transporters or technicians. A recent review of the literature has found that most publications on the role of POCUS in COVID 19 have been limited to case series and opinions Mr. R is a 46-year-old man with no medical history who presents to the emergency room with difficulty breathing following 10 days of fever, diarrhea, and cough. He is found to have COVID-19 and a serum creatinine of 2.0 mg/dL (176.84 µmol / L). The first task for the nephrologist is to differentiate AKI from chronic kidney disease (CKD). Hirsch et al. reported that among patients with COVID-19 and AKI, baseline serum creatinine was known in only 15%. Ultrasound at the point of care can help to differentiate between AKI and CKD. The combined thickness of the cortex and medulla on ultrasound is known as the parenchymal thickness and in normal individuals is up to 1.5 cm The next question for the nephrologist is whether Mr. R’s AKI is attributable to a reversible cause, like urinary obstruction. Classic epidemiological studies of AKI in hospitalized patients estimate that urinary obstruction accounts for 1% of cases of AKI in the hospital and rates among COVID-19 associated AKI are similar Acute tubular necrosis (ATN) is the most common finding among patients with COVID-19 associated AKI In summary, urinary obstruction in COVID-19 associated AKI is rare and kidney ultrasonography is of limited utility for diagnosing ATN—the most common form of COVID-19 associated AKI. The most important information clinicians are likely to get from kidney POCUS is differentiating AKI from CKD in an otherwise undifferentiated patient with no known baseline kidney function. Acute kidney injury on CKD in hospitalized patients with COVID-19 is associated with a higher likelihood of requiring dialysis upon discharge Despite best medical practices, including volume resuscitation, Mr. R gets intubated. He quickly becomes oliguric and his oxygen saturation is dropping. Is Mr. R’s oxygen saturation dropping because he is developing pulmonary edema from renal failure? Or is Mr. R’s oxygen saturation dropping because his disease is worsening, and his oliguria is due to days of insensible loss from fever, poor oral intake, and diarrhea? POCUS can be a valuable tool in these scenarios and nephrologists should understand how lung and cardiac ultrasound can be used to augment the traditional physical examination. When an ultrasound wave meets a water-thickened interlobular septum, a linear reverberation artifact called a B-line juts away from the pleural surface Lung ultrasonography might be an attractive tool for evaluating extravascular lung water and thereby volume status in patients with COVID-19 associated AKI. Yet, there are potential pitfalls. It is critically important for the POCUS practitioner to be able to distinguish between lung ultrasound findings consistent with pulmonary edema and those findings consistent with COVID pneumonia. Figure 1 demonstrates typical lung ultrasound findings in COVID-19.In a case series by Yasukawa in Washington D.C., 10 patients presenting to the emergency department with COVID-19 all had a diffusely irregular pleura, all the patients had areas of confluent B-lines and half also had subpleural consolidations A case series of 3 physicians with COVID-19 on home isolation showed a typical pattern in mild disease from an irregular pleura with scattered B-lines and subpleural consolidations that progressed to confluent B-lines on day 5 to 7 of illness followed by resolution back to A-line pattern Given the high mortality associated with volume overload identifying volume status in patients with COVID-19 associated AKI is critical. Lung ultrasound findings might be unreliable or misleading. This is where using your standard four cardiac views are helpful. The parasternal long axis can be used to look at mitral valve excursion and overall left ventricular function. The parasternal short axis can be used to look for left ventricular contractility and wall motion abnormalities. The apical four chamber view can be used to look for left ventricular and right ventricular size and function comparison, as well as pericardial effusion. The subxiphoid can be used to look at the inferior vena cava (IVC). An early case series from Wuhan, China described three cases where focused cardiac echo was used in patients with COVID-19 to identify new onset global left ventricular dysfunction and acute myocardial infarction Imaging of the inferior vena cava is may be useful in managing critically ill patients with AKI requiring dialysis and this remains true in COVD-19 Despite best efforts, Mr. R's saturation drops below 80% on 100% FiO2 and therefore he requires extracorporeal membrane oxygenation (ECMO).Patients with severe COVID-19 pneumonia and severe acute respiratory distress syndrome (ARDS) may remain hypoxemic despite best ventilator practices. These patients may require ECMO for life support if they remain hypoxemic. Depending on the type of failure the patient has, they may require veno-venous (VV) ECMO for primary respiratory failure, or veno-arterial (VA) ECMO for primary cardiac and respiratory failure. According to the worldwide Extracorporeal Life Support Organization registry, of the 2,132 cases of ECMO for COVID-19 reported thus far, 95% (2032) have been for VV-ECMO, reflecting the severe respiratory failure COVID-19 patients experience To understand the role POCUS and transesophageal echocardiography plays in managing patients on ECMO, we must explain the different configurations of ECMO. In VV-ECMO, a large outflow/drainage cannula (21-25 Fr) is placed at the IVC/RA junction, typically via the femoral vein, along with an inflow cannula (17-21 Fr) into the SVC/RA junction. Sometimes femoral vein to femoral vein configurations are used and so it is important to know which configuration your patient has POCUS is helpful in managing ECMO patients purely from a circuitry standpoint. For both VV- and VA-ECMO patients, POCUS helps identify atrial and ventricular size and function, valvular regurgitation, thrombus, the presence of pericardial effusion, and, most importantly, cannula position Nephrologists who use POCUS need to be aware of the limitations of interpreting IVC size and variability in patients on ECMO. The drainage cannula often sits in the IVC. This catheter can be anywhere from 21-25 Fr, meaning as much as 8.33 mm in diameter. Drainage cannulas are typically multi-stage cannulas, meaning they are perforated on the edges to allow for maximal drainage of the IVC. Therefore, the IVC must remain larger than the cannula diameter in order to ensure continuous, uninterrupted flow for the ECMO circuit Sometimes, the cannulas may not be visualized using transthoracic echocardiography. These patients may need transesophageal echocardiography (TEE) to see if the cannulas are positioned appropriately. Transesophageal echocardiography can also be used to elucidate the cause of hypoxemia. Both trans-thoracic and trans-esophageal echocardiograms can be used to identify a right to left intra-cardiac shunt, such as due to a patent foramen ovale, that might explain a patient's hypoxemia. Transesophageal lung ultrasound (TELUS) is increasingly recognized as a tool to diagnose hypoxemia in patients who are too critically ill to move to a CT scanner and in whom other traditional imaging modalities are not practical or safe While on ECMO, Mr. R required intermittent boluses of colloid due to fluctuating ECMO flows and an IVC collapsed around his drainage cannula, even though he clinically appeared euvolemic. POCUS showed improved aeration of his lung bases and anterior lung fields. He was able to be weaned off VV-ECMO. His IVC appeared dilated with no respiratory variation post-VV-ECMO, and fluid was restricted. His acute kidney injury resolved, and urine output improved. Point of care ultrasonography has a variety of uses for the nephrologist evaluating COVID-19 associated AKI (Table 1). A nephrologist well trained in ultrasound can minimize exposure from additional staff such as technicians and patient transporters that are required for formal radiology studies. Kidney ultrasound can help identify obstruction and CKD. Many patients during the pandemic arrive with no known baseline kidney function. Chronic kidney disease is associated with a higher mortality and higher likelihood of requiring dialysis upon discharge. Therefore, POCUS can give the nephrologist important prognostic information. Ultrasound-based volume assessment is important in managing hypervolemia. Lung ultrasonography needs to be interpreted with caution because B-lines often arise from an irregular pleura due to the underlying pulmonary process, and can also be seen with subpleural consolidations. When patients require ECMO, TEE and TELUS can be useful for evaluating volume status. As COVID-19 continues to spread across the globe, nephrologists should become more informed about how POCUS can improve the care of their patients. The authors have no conflicts of interest to disclose.