Gorham-Stout disease (GSD) is a rare disease that causes massive osteolysis and proliferation of abnormal lymphangiomatous tissues. Patients with GSD often experience pain associated with bone fractures and chylothorax. However, bleeding caused by abnormal lymphangiomatous tissue or hematological dysfunction rarely occurs.A 22-year-old female patient with GSD presented with severe left hip and lower limb pain. The GSD had disappeared her right pelvic bone and femur, but no abnormalities were found in the bones at the site of the pain.The patient presented with a chylothorax and cerebrospinal fluid leakage. She was treated with sirolimus and an epidural blood patch, and her symptoms resolved. Computed tomography and magnetic resonance imaging revealed an epidural hematoma extending from L3 to the caudal region, and blood results revealed a consumption coagulopathy.We presumed that the hematoma caused pain and prescribed pregabalin and morphine. The pain gradually subsided.An unexpected liver subcapsular hemorrhage occurred 4 months later, and the patient went into hemorrhagic shock. Transcatheter arterial embolization was promptly performed, and the patient recovered.GSD infrequently causes bleeding related to abnormal lymphangiomatous tissues and coagulopathy, yet it can lead to serious events if it occurs. The condition of massive idiopathic osteolysis was first described by Jackson in 1838, GSD most commonly affects children and young adults, with an average age at diagnosis of 25 years.,5 The etiology of GSD is unclear. Some studies have suggested that interleukin-6, macrophage colony-stimulating factor, vascular endothelial growth factor (VEGF), and receptor activator of NF-kappaB ligand play a crucial role in osteoclast function and promoting lymphangiogenesis.,7 GSD may involve any bone of the whole body, but it is commonly found in the upper part of the body, vertebrae, ribs, and pelvic bones.It has been reported that GSD is not associated with race or sex.,9 Patients with GSD commonly experience osteolysis and a decline in activities of daily living, which leads to pathological fractures. However, the proliferation of lymphangiomatous tissue around the affected lesion can cause various complications, including cerebrospinal fluid (CSF) leakage and chylothorax, leading to symptoms such as headache and dyspnea.The most common symptom of GSD is localized pain in osteolytic lesions.In this report, we describe a case in which proliferating lymphangiomatous tissue in the spinal canal caused severe headache and lower limb pain in a patient with GSD. Consumption coagulopathy may lead to bleeding.A 22-year-old female patient with GSD presented with severe pain in the left hip, lower limb, and head. She was diagnosed with GSD based on an osteolytic lesion biopsied at 12 years of age. She experienced right lower limb osteolysis at 2 years of age, which gradually progressed to the right femur, pelvic bone, and lumbar vertebrae. Despite treatment with interferon-alpha, bisphosphonate, and vitamin D, the disease progressed, and the patient’s condition worsened. At 14 years of age, the patient presented with a pleural effusion and chylothorax. Pleurodesis was performed, and sirolimus was administered, following which fluid accumulation in the pleura decreased. However, the osteolytic lesions gradually extended into the sacrum. At 18 years of age, the patient complained of severe orthostatic headaches. Examination revealed a recurrent bilateral acute subdural hematoma. Radioisotope inspection showed CSF leakage and accumulation in the lymphangiomatous tissue at the dura mater of L3; thus, she was diagnosed with cerebrospinal fluid hypovolemia due to a CSF-lymph fistula. After multiple burr hole irrigation and drainage, an epidural blood patch was performed. The dyspnea and headache gradually disappeared.9/L , fibrinogen 144 mg/dL , d-dimer 71.9 µg/mL .At 22 years of age, she complained of headaches and severe pain in the hip and lower limb and was admitted to the hospital. The pain continued to worsen, and the patient often lost consciousness. The pain was confined to the lumbosacral region and the left side of the lower limb and head, but no osteolytic lesions were observed in the left limb Fig. A. The paWe presumed that the pain was caused by intracranial hypotension due to minor CSF leakage and nerve root compression due to a lumbar epidural hematoma. After pregabalin was prescribed, rapid improvement was observed in the lower limb pain and headache. The patient experienced tolerable mild dizziness and somnolence. The lower limb pain worsened 2 months after treatment. However, this time, morphine was prescribed, which relieved the pain without any severe adverse events. Finally, oral administration of 120 mg morphine and 300 mg pregabalin daily helped her perform daily activities. However, the coagulopathy persisted, and the anemia and thrombocytopenia gradually progressed. She sometimes required transfusions of red blood cell products.Four months later, the patient suddenly presented with severe abdominal pain and went into shock with severe anemia. Computed tomography revealed a massive subcapsular liver hemorrhage Fig. A, but noThis report describes a young female patient with GSD who developed intractable headaches and pain in her hip and lower limb, probably caused by a lumbar epidural hematoma related to abnormal lymphangiomatous tissues. She also experienced sudden hepatic hemorrhage due to consumption coagulopathy caused by the progression of GSD.,7,11 Furthermore, the augmented number of macrophage-like cells in GSD lesions produce VEGF-A, VEGF-C, and VEGF-D that can stimulate differentiation into osteoclasts and lymphangiogenesis. Conversely, some studies have suggested that bone resorption may be secondary to lymphangiogenesis. Growth factors secreted by the lymphatic endothelial cells may affect osteoclasts and osteoblasts. In GSD patients, it has been shown that lymphatic vascular endothelial hyaluronan markers are present on endothelial cells, and the levels of circulating platelet-derived growth factor-BB, which promotes lymphangiogenesis, are elevated. The endothelial cells express VEGF receptors, especially VEGF receptors-3, which is activated by VEGF-C, VEGF-A, and VEGF-D promoting angiogenesis and lymphangiogenesis in the bone marrow of GSD patients.,16 As a result, bone resorption might be caused by mechanical compression of abnormal lymphangiomatous tissue.The etiology and pathogenesis of GSD remain unknown. The main features of GSD are bone absorption and lymphangiogenesis. Osteoclasts play a crucial role in bone resorption. Humoral factors, such as interleukin-6, macrophage colony-stimulating factor, and receptor activator of NF-kappaB ligand, seem to promote osteoclastogenesis and the survival of osteoclasts. The pain can arise spontaneously or be caused by pathological fractures. Although all bones may be affected, the ribs, spine, and skull are frequently affected by GSD. The type of bone affected by the disease can negatively affect various daily activities. However, the present report showed that the painful lesion was not an osteolytic lesion; thus, the pain was probably not caused by osteolysis.Complaints in patients with GSDs are associated with osteolysis. The most common clinical presentation in a case series comprising 64 Chinese and 3 English patients included localized pain . Abnormal lymphangiomatous tissues proliferate and invade the surrounding tissues in the affected petrous bones, temporal bones, and vertebrae. Consequently, CSF leakage is caused by fistulae formation in the dura mater. CSF leakage can induce intracranial hypotension and lead to chronic subdural hematomas, as observed in this report. Furthermore, bleeding and hematoma have been reported in the affected regions following the injury of fragile proliferated lymphangiomatous tissues.In patients with GSD, both lymphatic and vascular proliferations are observed in the osteolytic lesions. The abnormal lymphatic and blood vessel tissues easily collapse. Intrathoracic osteolytic lesions, such as those in the clavicle, rib, or thoracic vertebrae, can result in pleural effusions, including chylothoraces. Regarding GSD, 2 cases with serous effusion presented with LIC in a previous report. LIC might cause refractory serous effusions such as pleural effusion and bleeding. The presented report showed the features of LIC, an epidural hematoma, and subcapsular liver bleeding. From the above, the bleeding events in this report might have been caused by the combined factors of the vulnerability of abnormal lymphangiomatous tissues and LIC.Furthermore, consumption coagulopathy may occur in patients with GSD. It has been commonly reported that patients with lymphatic and venous malformations, such as complex lymphatic anomalies, have localized intravascular coagulopathy (LIC), which manifests as anemia, thrombocytopenia, the elevation of the D-dimer level, and coagulation dysfunction.As shown in the current case, GSD often causes events related to bone vulnerability, including pain. Although infrequent in patients with GSD, bleeding associated with abnormal vascular disruption and coagulopathy can be life-threatening. Thus, we focused on symptoms other than those related to the affected bones in GSD.www.editage.com) for English language editing.We would like to thank Editage (Conceptualization: Yasutomo Kumakura, Norio Hasuda, Kazuki Akita.Data curation: Yasutomo Kumakura.Supervision: Tetsuya Iijima, Takashi Matsukawa.Writing – original draft: Yasutomo Kumakura.Writing – review & editing: Yasutomo Kumakura, Norio Hasuda, Kazuki Akita, Tetsuya Iijima, Takashi Matsukawa.


Geminiviridae and the genus Begomovirus, are DNA viruses that are transmitted by whitefly Bemisia tabaci (Gennadius) in a circulative persistent manner. They can easily adapt to new hosts and environments due to their wide host range and global distribution. However, the factors responsible for their adaptability and coevolutionary forces are yet to be explored. Among BGVs, TYLCV exhibits the broadest range of hosts. In this study, we have identified variable and coevolving amino acid sites in the proteins of Tomato yellow leaf curl virus (TYLCV) isolates from Old World  and New World . We focused on mutations in the coat protein (CP), as it is highly variable and interacts with both vectors and host plants. Our observations indicate that some mutations were accumulating in Old World TYLCV isolates due to positive selection, with the S149N mutation being of particular interest. This mutation is associated with TYLCV isolates that have spread in Europe and Asia and is dominant in 78% of TYLCV isolates. On the other hand, the S149T mutation is restricted to isolates from Saudi Arabia. We further explored the implications of these amino acid changes through structural modeling. The results presented in this study suggest that certain hypervariable regions in the genome of TYLCV are conserved and may be important for adapting to different host environments. These regions could contribute to the mutational robustness of the virus, allowing it to persist in different host populations.Begomoviruses, belonging to the family  Aleyrodidae) being the most prevalent. Among the whitefly-transmitted viruses, BGVs (Geminiviridae), criniviruses (Closteroviridae), and torradoviruses (Secoviridae) are considered the most destructive. Begomoviruses (BGVs) are DNA viruses that are categorized as monopartite or bipartite, having circular single-stranded DNA genomes , and being encapsulated in twinned icosahedral capsids [Bemisia tabaci, or other whiteflies [B. tabaci whitefly complex comprises over 35 cryptic species that cannot be distinguished morphologically or by traditional classification methods, and this complex is capable of transmitting over 200 species of BGVs [B. tabaci species, and hundreds of crop species and varieties [The greatest proportion of emerging diseases that pose a threat to agriculture on a global scale are transmitted by insects, with those transmitted by whiteflies  between the OW and NW begomoviral species . This raThe distinction between OW and NW BGVs is based on their geographic distribution and reflects the evolutionary history of these viruses. It is believed that these two groups arose because of geographic isolation, with OW BGVs being mainly found in the regions of Africa, Asia, and Europe, while NW BGVs are predominantly found in the Americas. The separation of these groups likely occurred a long time ago, and it is thought that their divergence was driven by various factors, including geographical barriers and differences in the host plant populations. While OW and NW BGVs share some genetic similarities, they have evolved independently over time, leading to differences in their genomes. Despite these differences, OW and NW BGVs are still considered to be the same species due to their ability to recombine and exchange genetic material. Furthermore, studies have shown that some OW and NW BGVs can infect the same host species, indicating that they may have similar biological interaction properties.Several approaches have been used to control BGVs and other viruses in crops, including the use of resistant cultivars, the introduction of resistance (R) genes, RNA interference (RNAi), recessive genomic mutational methods, and pesticides to control the insects’ vectors ,34. The Comparing the OW and NW BGVs can provide insights into the mechanisms driving virus evolution and adaptation to different environments. For example, comparing the genetic diversity and recombination patterns between these two groups can reveal how different environmental factors have shaped their evolutionary trajectories. Additionally, studying the host ranges and interactions of these viruses with their insect vectors can provide insights into the co-evolutionary processes driving virus–host interactions.Tomato yellow leaf curl virus (TYLCV) into categories based on their occurrence in OW and NW geographical regions by parsing the GenBank files, as the virus is found in both regions. SNPs were identified from both groups, and their distribution was analyzed throughout the genome of TYLCV. The findings demonstrated that hypervariable regions are present in different parts of the genome in TYLCV from both OW and NW regions. Comparative analysis between TYLCV isolates from the OW and NW revealed that the highest variation was observed in the replication-associated protein (Rep), C3, C4, and coat protein (CP), with sites under positive selection. Notably, the degree of variation was significantly greater in TYLCV isolates from the OW.To evaluate the model, we classified Given Begomoviral CP vital importance, both in terms of their infectivity and antibody-based resistance ,44,45,46Additionally, in this study, we conducted a variation analysis and identified several sites that are positively selected. One of these sites, S149N (78%), was found to have originated in China but rapidly spread to Europe and underwent structural changes. Moreover, we detected a densely clustered group of amino acids in the CP of OW viruses, which may represent a new evolutionary paradigm. The increased structural flexibility of the CP may provide mutational robustness and allow for the maintenance of biological functions. Consequently, mutations in the viral coat protein sequences, particularly at specific sites, could lead to alterations in vector-dependent transmission, which could increase the likelihood of the emergence of a resistant viral strain ,48. The https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/ (accessed on 30 August 2021). To allow for comparison, only the DNA-A component was considered. Out of the total 269 OW species and 153 NW species identified as full-length DNA-A sequences, only these were included for further analyses. The average length of DNA-A sequences from both groups ranged from 1950 to 2067 nucleotides in length. Chargaff’s purine–pyrimidine equilibrium was detected in both groups with a ratio of 1.0, and the average purine content (G + A) was 49.82% for OW and 49.47% for NW, while the pyrimidine content (U + C) was 50.17% and 50.53%, respectively, with no significant difference. The GC percent was identified as 43.17% for OW and 43.89% for NW.A total of 12,332 and 3943 genomic DNA sequences for OW and NW bipartite genomes, respectively, were obtained from NCBI virus database Tomato leaf curl New Delhi Virus (TLCNDV), African cassava mosaic virus (ACMV), East African cassava mosaic virus (ACMV), and Mung bean yellow mosaic India virus, with Pi values of 0.58, 0.55, 0.55, and 0.54, respectively, which means that >50% of the nucleotide positions in their genome are polymorphic. However, 33 NW counterpart species showed significant genomic variation in their genome with highest variation (Pi = 0.29) identified in Pepper golden mosaic virus (PGMV) (Euphorbia mosaic yellow mosaic virus (EYMV), Bean golden mosaic virus (BGMV), TYLCV, and Potato yellow mosaic virus, with Pi’s of 0.29, 0.29, 0.26, and 0.25. Overall, the Pi analyses for the DNA-A revealed that mean nucleotide diversity of BGVs from the OW is more than twice that observed for BGVs from the NW  is shown by the Kolmogorov-Smirnov test in (Ageratum yellow vein virus (AYVV), Clerodendrum golden mosaic China virus (CGMCV), Corchorus yellow vein virus (CYVV), Cotton leaf curl Gezira virus (CLCGV), Sida yellow mosaic virus (SYMV), Squash leaf curl virus (SLCV), Sweet potato golden vein associated virus (SPGVV), Sweet potato leaf curl Georgia virus (SPLCGV), Sweet potato leaf curl Sao Paulo virus (SPLCSPV), Sweet potato leaf curl Spain virus (SPLCSV), Sweet potato leaf curl virus (SPLCV), Sweet potato mosaic virus (SPMV), and Watermelon chlorotic stunt virus (WCSV). In parallel to comparison of similar components, i.e., DNA-A across OW and NW species, DNA-B was compared with the DNA-A from both groups. Results obtained from our analysis suggest that DNA-B component is more variable than the DNA-A component of NW species (The nucleotide diversity (pi) method  was founs (PGMV) B. The otm the NW A,B. A si test in C. Compar species . As TYLCp-value ≤ 0.001) .Genetic variability in viral proteins is often influenced by the surrounding environment and the range of hosts available . TYLCV h≤ 0.001) B. This i≤ 0.001) C. The pap-value ≤ 0.05) displayed the presence of higher number of codons sites under positive selection and especially prominent in the V1, C1, and C1/C4 cistrons   . This siWe examined the amino acid content at each protein and profiled amino acid substitutions to examine the effect of genomic variation on protein variation. The relative amino acid abundance was calculated for TYLCV isolates from OW and NW. Interestingly, the amino acid abundance differed in each protein A. The mohttp://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (assessed on 20 August 2021) [To evaluate the effect of mutation in CP structure of TYLCV from both groups, the mutation was analyzed using Phyre2  server (st 2021) . The CP st 2021) ,68 (Suppst 2021) ,69. Selest 2021) . Stabilist 2021) . FurtherWhile we embarked on CP analysis, our inspiration was to distinguish mutations that might be of potential concern. In a situation of very low genetic diversity, conventional methods of identification of functional mutations have imperfect statistical power. However, a rich NCBI data set offers a possibility to look more deeply into the evolutionary relationships among the TYLCV sequences in the context host and geography . We founThis methodology of backtracking the mutation over phylogeny revealed that TYLCV isolates bearing the coat protein mutation S149N or S149T are replacing the original Spain form of the virus rapidly and repeatedly across the globe A. We do To know if there is a structural diversity in CP of OW isolates, we divided the sequence into three clades with respect to reference strain from Spain, i.e., S-clade, N-clade, and T-clade B. AccordThe inherent variability observed in the coat protein (CP) of TYLCV implies its significant involvement in the process of evolution and diversification. To investigate this further, we constructed separate phylogenetic trees using the CP sequences of OW and NW TYLCV, which allowed us to compare the evolutionary patterns of the two groups. By analyzing the CP protein sequences of both groups, we gained a better understanding of the evolutionary history of TYLCV and how it has evolved over time. To create the phylogenetic trees, we added information about the host plant species and the country of origin of the TYLCV isolates. The resulting phylogenies had different structures, with the OW phylogeny showing more clades and branches compared to the NW phylogeny. This suggests that the CP of OW TYLCV has accumulated more mutations over time than the CP of NW TYLCV. Additionally, the OW isolates showed higher nucleotide diversity and a greater range of host types, suggesting that maintaining flexibility in host adaptation is an important factor in TYLCV evolution. Interestingly, the host geography appeared to be a more important factor in determining the clustering of TYLCV isolates than the host plant species. In contrast, the NW isolates showed less genetic divergence, indicating a more recent common ancestor compared to the OW isolates. This could be due to a smaller population size, lower mutation rate, or more recent emergence of the virus in the region. These findings support the idea that mutations in the CP are responsible for the emergence of new TYLCV strains and are a major contributor to its evolution. However, further research is needed to fully understand the evolutionary patterns of TYLCV in different regions.  and identified hypervariable regions of the genome that exhibit a preference for nucleotide substitutions and positive selection  [Results obtained with our in-silico analysis demonstrated a different evolutionary pattern, with significant differences in variation that DNA-A segment of OW and NW BGVs, DNA-A segment, and DNA-B segments of NW BGVs, as well as across different cistrons level . To inve (TYLCV) ,77, for Tomato leaf curl viruses [−3 subs/site/year [The two-virion sense open reading frames (ORFs); CP/V1 and V2 and four complementary sense ORFs, i.e., C1, C2, C3, and C4, are interspaced by an intergenic region (IRs), which comprises a sequence capable of forming a stable hairpin loop structure, including the motif 5′-TAATATTAC-3′ common to all geminiviruses. The intergenic region is furthermore recognized as a site of the origin of DNA replication (ori). This region is particularly prone to variation and specifies a sensitive guide to differences between isolates. Previous analysis has shown that IRs in  viruses  and otheite/year . Our anaNicotiana benthamiana plants [The V2 from BGVs encodes a multifunctional protein, which is required for full infection and to suppress gene silencing at the transcriptional (TGS) and post-transcriptional (PTGS) level ,81. Furta plants . The V2 a plants . In preva plants ,86. For a plants . Our varBGVs are mainly transmitted by their whitefly vector, and the CP is the only protein that is exposed to whitefly tissues with specific motif, enabling interaction with insect receptors or other proteins that assist or enable virus translocation into or through insect tissues . The CP The C3 protein is known to enhance the replication of geminivirus DNA accumulation. Despite its known ability to enhance geminivirus DNA replication, studies have indicated that even with mutant C3 replicons, replication can still occur, although to a lesser degree, in both single cells and plants . InteresThe AC2 protein of BGVs is highly conserved and plays a critical role in viral infectivity . InactivTomato yellow leaf curl virus isolates has the potential to impact its replication in diverse host environments. This, in turn, can play a significant role in the virus’s adaptation and evolution.The replication-associated protein (Rep) encoded by the AC1 ORF  in bipartite geminiviruses and by C1  in monopartite geminiviruses (except mastreviruses) is known to be conserved in sequence, position, and function and is expressed under the control of a bidirectional core promoter in the IR . The RepC4, also identified as symptom determinants, has drawn much attention in recent years. Being the tiniest and one of the least-conserved proteins encoded by geminiviruses , C4 protIn summary, the results of this study suggest that the Rep, C1, C3, and C4 proteins of OW TYLCV isolates may play critical roles in the virus’s adaptation and evolution in different host environments. The novel discovery of co-evolving amino acids in the coat protein region of TYLCV isolates from OW underscores the significance of investigating the dissemination of OW TYLCV isolates. These differences may enhance their adaptability to host plants and vectors, contributing to their extensive host and vector range. The identified variation profiles in this study could also have important practical applications, including the development of diagnostic tools for BGVs, breeding BGV-resistant crops, and identifying genes susceptible to BGVs. Further research is necessary to fully understand the functional implications of these variations and their potential impact on virus-host interactions.https://biohpc.cornell.edu/lab/lab.aspx) (assessed on 20 August 2021) at Cornell University, Ithaca, NY, USA. Scripts created specifically for this study are available upon request.All the computation work was performed on high-performance computing nodes of BioHPC (http://www.ncbi.nlm.nih.gov/) (assessed on 20 August 2021), using custom-made scripts available under Entrez Programming Utilities . The GenBank files were parsed based on the geographical location, and species were categorized as OW and NW groups. For each species, an accession defining the complete genome, and coordinates for each cistron, were used as a reference genome  of BGVs species represented in GenBank  (assessed on 20 August 2021). These alignment files in nexus format for all genomic sequences were used to establish pairwise nucleotide diversity (Pi) in a 50-nt sliding window using the Tajima’s D test in DnaSP 5.10.1 [https://github.com/sanger-pathogens/snp-sites) (assessed on 20 August 2021). The type i.e., Transition (Ts) or Transversion (Tv), and position of each nucleotide substitution, were mined in a variant call format (VCF). By exploiting a method, --SNPdenisty, from the package VCFtools [The genomic sequence alignment  file were separately obtained for OW and NW isolates with multiple sequence alignment (MSA) using MAFFT  between the species of OW and NW, we quantified the difference using the D-statistic of the two-sample Kolmogorov-Smirnov (KS) test . D-statihttp://www.lcqb.upmc.fr/BIS2Analyzer/submit.php) (assessed on 20 August 2021) [http://caps.tcd.ie/) (assessed on 20 August 2021) [p-value ≤ 0.05) of identified coevolving sites and false discovery rate were evaluated [Full length nucleotide and protein sequences of each begomoviral species corresponding to NCBI GenBank accessions  were usest 2021)  and CAPSst 2021) . Amino ast 2021) , polaritst 2021) , charge st 2021)  and dipest 2021) . The stavaluated . Each covaluated .http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (assessed on 20 August 2021) under intensive mode [http://services.mbi.ucla.edu/SAVES) (assessed on 20 August 2021) for evaluation and quality checking. Models (in.pdb format) for two viruses or isolates were superimposed using Chimera v1.13 (https://www.cgl.ucsf.edu/chimera/ (assessed on 20 August 2021) [A custom bash script was used to extract the CP amino acid sequence based on the coordinate information in GenBank records. The three-dimensional structure of the CP of the two TYLCV isolates viz. ALN12561.1 from OW and QCG74731.1 from NW with the most variation was generated using Phyre2 (ive mode . Predictst 2021) . The TM-st 2021) .


As rightly stated by the author Mira Grant in her novel Countdown, “There is nothing so patient, in this world or any other, as a virus searching for a host”. Be it smallpox, influenza, or the coronaviruses, as history dictates, viruses have emerged and re-emerged to prove that something as small as being invisible to the naked eye is capable of causing human suffering. Through the years, viruses have evolved so as to see eye-to-eye with human evolution to exploit and survive within human host cells. It is simply commendable how deeply and clearly a virus understands its host cell, such that it interacts with it and convinces it to assist the virus in its pathogenesis. Therefore, an understanding of such host–virus interactions gives insight into the planning and development of antiviral strategies. With regard to the success of our previous issue on “Host Cell-Virus interaction”, we continued to receive some interesting articles from researchers all around the world, giving us an idea of the rigorous attempts being made globally to maneuver the interactions between the host and the virus, to ultimately help the body fight against an infection. Amongst the various articles received, 12 articles  outcompeted the others to make a place in this special issue of ours. As obligate intracellular parasites, the exploitation of the target cell for its own survival is of utmost necessity, and one of the ways in which it does so is by fighting against the host defense system. Our first article in the issue unveils a mechanism by which the Nipah virus disrupts the autocrine Interferon (IFN) signaling in the infected cells, emphasizing even more the role of inclusion bodies in Nipah infections, a study reported by Becker and Maisner [Moving on to the findings reported by Kundharapu and Chowdary, which shed light on the structural discoveries pertaining to the non-structural proteins of the Dengue Virus (DENV), the advantages of bioinformatics have been adopted to uncover a plausible mechanism behind the enhancement of helicase activity of the DENV-NS3 protein .The next article accepted for publication only points out the fact that, though HIV research has been holding its ground for a long time, it is far from reaching its near end. Here, Jin et al. emphasized the interaction of the human immunodeficiency virus (HIV)-1 Gag protein with the Multi-Aminoacyl-tRNA Synthetase Complex through the bi-functional human glutamyl-prolyl-tRNA synthetase (EPRS) subunit, discussing the effects of such an interaction on the virus and the host . With Human Papilloma virus (HPV) research gaining ground, Rattay et al. proceed to state the results of their study on the HPV-8 proteins, E1 and E2, being responsible for the suppression of the innate receptor RIG-1-like MDA5, an event that might be crucial for HPV persistence in the host .The next set of articles includes original research, a comment, and a reply pertaining to the research article, where Prezioso and their group account for the findings of their study on JC Poliomavirus (JCPyV) replication and microRNA expression after infection using archetypal and rearranged NCCR viral strains in COS-7 and SVGp12 cell lines. Their results highlighted the presence of viral miRNA in the exosomes of the infected supernatants, which could be carried on to uninfected cells, suggesting that the miRNAs may regulate the events of viral reactivation . FollowiIn another review, Dass et al. commendably describe the role of viral miRNAs and cellular miRNAs in human herpesvirus infections. With an elaborative take on the significance of the miRNAs in Herpes simplex viruses (HSVs), the authors continue to describe the regulatory effects of these miRNAs at different stages of HSV infection, such as replication and latency, while giving an account of the other 7 classes of herpesviruses. Also, the possibility of these miRNAs as biomarkers in herpesvirus infections has been discussed, along with their limitations .With the advent of new technology such as CRISPR, genetic modifications that may be customized for genome-wide screens are being welcomed as a means to identify factors that participate in host–virus interactions. A review discussing the implementation of CRISPR for the study of genome-wide screens of Flaviviruses has been published by Kanojia et al., which marks the tenth article in this special issue .In light of the fact that SARS-CoV-2 continues to wreck global health havoc, Sun et al. give a detailed account of the cellular sensors discovered and reported, along with the countermeasures undertaken by the virus to bypass or overpower these censors, in the fourth review of this special issue .In the final article, which is also the last review article in the issue, Stephenson-Tsoris and Liang describe the proteins of both the host and the virus that contribute to the virus–host interaction events in the stages from entry until egress of the Hepatitis D Virus . This special issue covers a diverse set of articles in terms of the techniques used, the approach to solving the question at hand, as well as the category of viruses. Even so, all the articles contribute to the understanding of virus–host interactions, which has been the prime objective of this second volume of the special issue. As Guest Editors of this Special Issue, we thank all the contributors to “Host Cell-Virus Interaction 2.0: Viral Stratagems of Immune Evasion, Host Cellular Responses and Antiviral Counterattacks”. We will be back soon with the third volume of this Special Issue with further interesting studies, cutting-edge research, and novel understandings on virus–host interactions and anti-viral drug discoveries.


Phenotypic plasticity plays a key role in adaptation to changing environments. However, plasticity is neither perfect nor ubiquitous, implying that fitness costs may limit the evolution of phenotypic plasticity in nature. The measurement of such costs of plasticity has proved elusive; decades of experiments show that fitness costs of plasticity are often weak or nonexistent. Here, we show that this paradox could potentially be explained by condition dependence. We develop two models differing in their assumptions about how condition dependence arises; both models show that variation in condition can readily mask costs of plasticity even when such costs are substantial. This can be shown simply in a model where plasticity itself evolves condition dependence, which would be expected if costly. Yet similar effects emerge from an alternative model where trait expression itself is condition-dependent. In this more complex model, the average condition in each environment and genetic covariance in condition across environments both determine when costs of plasticity can be revealed. Analogous to the paradox of missing trade-offs between life history traits, our models show that variation in condition can mask costs of plasticity even when costs exist, and suggest this conclusion may be robust to the details of how condition affects trait expression. Our models suggest that condition dependence can also account for the often-observed pattern of elevated plasticity costs inferred in stressful environments, the maintenance of genetic variance in plasticity, and provides insight into experimental and biological scenarios ideal for revealing a cost of phenotypic plasticity. Phenotypic plasticity occurs when the same genotype produces different phenotypes in response to different local or developmental environments. Plasticity, when adaptive, allows organisms to track an environment-dependent optimum within a single generation, permitting the expression of adaptive phenotypes in a new environment and preventing maladaptation in temporally or spatially variable environments . For theHowever, no organism or trait is completely plastic, despite the clear benefits of altering development in response to the environment, and in some cases plasticity fails to evolve entirely. For example, in a synthesis of plant reciprocal transplant studies, A fitness cost of plasticity is expected to manifest as a reduction in fitness of a plastic genotype compared to a non-plastic genotype that otherwise expresses the same trait value in a given environment see . EmpiricThus, the inference of costs of phenotypic plasticity depends upon the interpretation of covariance between fitness in one environment and trait expression in another. Yet such fitness costs of plasticity are rarely observed ; often nThis often “missing” cost of phenotypic plasticity represents an important unsolved paradox in evolutionary ecology , and hasThe methods for detecting costs of plasticity may fail for a variety of reasons, all of which relate to unidentified sources of variation. For example, if unmeasured aspects of the genotype affect both plasticity and fitness in the focal environment, then costs may be underestimated or even overestimated. Likewise, if this shared effect varies between test conditions in the same population, then cost estimates will vary. Life history theory has addressed similar obstacles in detecting trade-offs among fitness components, beginning with the well-known work of We begin by describing a causal model for fitness, and how such a causal model of fitness is related to a statistical regression model based on trait data to infer costs of plasticity. We focus on the case of two environments. We assume individual fitness in a focal environment is a function of trait expression in that environment  as well as the cost of having a plastic genotype,C is the cost of such plasticity. We refer to C (a population parameter fixed across environments). Thus, we assume costs are fixed within a genotype and are paid across all environments, which is perhaps the simplest form of a cost of plasticity. We make no assumptions about selection on z in other environments. where b cannot be measured with trait data from only one environment, and so is instead typically inferred from trait data from the same genetic backgrounds expressed in two or more environments in a GxE design. In this approach, b is assumed to be proportional to phenotype plasticity; that is, trait expression in another environment, Importantly, genotype plasticity proposed  to inferwhere the estimate We first describe trait expression in two environments independent of condition,i, i.Where C is estimable in a multiple regression of the formWhen trait expression is plastic but condition-independent as described in equation 3, the cost parameter if all covarying traits affecting fitness are included in the multiple regression. Thus, in a GxE experiment where fitness data in at least one environment is combined with trait expression across two environments for a set of genotypes, fixed costs of plasticity for condition-independent traits can be inferred from such a multiple regression, as has been widely proposed and implemented , and represents only one way that condition may impact variance in trait expression across multiple environments.We have introduced this simple model, a special case of van Noordwijk and de Jong’s general life history model, to illustrate a likely explanation for why the costs of plasticity are rarely observed. However, it is noteworthy that this model deviates substantially from the causal fitness model envisioned by past workers measuring costs of plasticity , in thatIn our second model, we assume that fitness is caused solely by the trait and by plasticity as in equation 1, repeated here,with no independent causal path for condition. Thus, this causal model of fitness is the same as that assumed in typical analyses of the cost of plasticity. We now assume trait expression is the result of both plastic resource allocation and the total pool of resources an individual has available to allocate, or condition. Thus we have assumed the trait is costly, in terms of resources, to express. We can modify our description of trait expression accordingly,i in environment 1, with a corresponding term for environment 2. In this model, condition affects fitness only indirectly via its effects on the expression of z. Variations in condition may be genetic or non-genetic. Importantly, in this model, two separate components contribute to the phenotypic plasticity , which can be described as the ability to match allocation strategy to the environment, and (b) plasticity arising from variation in resource acquisition R, or condition (which we have assumed is cost-free), which is simply the number of resources an individual has available to allocate to traits. As we show below, if a substantial amount of variation in plasticity is determined by this second component, variation in condition, then costs will be difficult to infer even if they exist.where z.This general model of trait expression is shown in As before, we can expand a or b and R and whereC needed to generate negative b).For simplicity assuming no covariance between R, is available, or a measure of b controlling for condition, then an appropriate multiple regression could be fit to obtain an estimate of the cost parameter C. Alternatively, if a measure of condition is available then the genetic covariance in condition Importantly, Model II shows that when trait expression is condition dependent across two environments, the observed degree of phenotypic plasticity in the trait itself will be in-part condition-dependent, and variance in phenotypic plasticity will be in part determined by (co)variance in condition across the two environments. If a measure of individual condition, R will affect both the measure of plasticity   . These eWe have explored the case where costs of plasticity are a fixed property of a genotype, paid in all environmental contexts, although inference of these costs may depend on the environmental conditions (as shown above). Alternatively, costs of plasticity may only be incurred when a plastic trait is actually expressed. For example, a “production cost”  of a plaPhenotypic plasticity is expected to often be costly, as without costs or some other constraint organisms would often evolve to be completely plastic, which they do not. Costs of plasticity are also expected on the simple basis of investment into mechanisms required to shift developmental trajectories or behavior in response to the environment . These iOur models can potentially account for several features of empirical evidence for the costs of plasticity. First, our finding that variance in plasticity generated by variance in condition masks costs of plasticity is consistent with the empirical finding of low or nonexistent costs of phenotype plasticity . Indeed,Together, these results suggest that care should be taken in experimental design to restrict within-environment variance in condition, or resource acquisition (to the extent this is possible) and to contrast environments that differ in quality, if the aim of the experiment is to infer costs of plasticity. Although such a restriction may be difficult when variation in condition has a genetic basis, some breeding designs  may help to reduce associations between condition and plasticity even if the variance in condition cannot be eliminated. Alternatively, GxE designs can be combined with other approaches, such as experimental evolution, to understand the nature of the costs of plasticity. For example, Empirical evidence suggests that both forms of condition dependence that we have explored may be commonplace. In general, many of the traits that have been the focus of previous studies of plasticity are traits that are closely related to life history and components of fitness. This includes traits such as the timing of reproduction or maturation, growth rate or size at maturity, body size, and defense against natural enemies ; traits z. However, our development of simplistic fitness models was purposeful; our aim was to understand how condition dependence can influence the inference of plasticity costs even when the true causes of fitness are similar to (Model I) or identical to (Model II) that envisioned in past studies (e.g., As a caveat, our assumed models for fitness are simplistic, particularly in Model II where we assume directional selection in the focal environment , and makes e.g., .A further caveat is that we have assumed no feedback between plasticity and condition in our models, in that plasticity itself does not feedback to influence condition. This is a simplistic view, as many plastic traits may themselves influence resource acquisition and thus condition. For example, this could be via a morphological trait involved in resource procurement e.g.,  or resouOthers have suggested that resource acquisition and condition dependence, as well as missing traits that affect fitness, may all make it difficult to measure the costs of plasticity . Our anaWe have shown that costs of plasticity may be especially hard to infer when the cost is paid at some point along the path from genotype to phenotype, rather than at the final level of phenotype expression itself. We believe that most previous ideas surrounding the causes of potential costs of plasticity fit this assumption, that costs are paid on one of several components of the path from genotype to phenotype. For example, If plasticity is adaptive, an open question is how genetic variance in plasticity is maintained (which appears to often be the case; In many ways, the study of phenotypic plasticity has moved on from the uncertainty surrounding costs that reached a zenith over a decade ago . Yet the


We evaluated metagenomic nanopore sequencing (NS) in field-collected ticks and compared findings from amplification-based assays.Forty tick pools collected in Anatolia, Turkey and screened by broad-range or nested polymerase chain reaction (PCR) for Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Jingmen tick virus (JMTV) were subjected to NS using a standard, cDNA-based metagenome approach.Eleven viruses from seven genera/species were identified. Miviruses Bole tick virus 3 and Xinjiang mivirus 1 were detected in 82.5 and 2.5% of the pools, respectively. Tick phleboviruses were present in 60% of the pools, with four distinct viral variants. JMTV was identified in 60% of the pools, where only 22.5% were PCR-positive. CCHFV sequences characterized as Aigai virus were detected in 50%, where only 15% were detected by PCR. NS produced a statistically significant increase in detection of these viruses. No correlation of total virus, specific virus, or targeted segment read counts was observed between PCR-positive and PCR-negative samples. NS further enabled the initial description of Quaranjavirus sequences in ticks, where human and avian pathogenicity of particular isolates had been previously documented.NS was observed to surpass broad-range and nested amplification in detection and to generate sufficient genome-wide data for investigating virus diversity. It can be employed for monitoring pathogens in tick vectors or human/animal clinical samples in hot-spot regions for examining zoonotic spillover. Genomic identification of microbial pathogens has established a substantial role in the diagnosis and monitoring of infectious diseases within the One Health concept  are among the most widely used NGS platforms, due to their relatively low cost and portability , sheep (Ovis aries), and dogs (Canis familiaris). They were morphologically identified to species level using appropriate taxonomic keys  using Bullet Blender 24 Gold . The lysate was centrifuged, and the supernatant was extracted using the IndiMag Pathogen Kit  with KingFisher™ Flex Purification System,  according to the manufacturer's protocol. Purified nucleic acids were treated with ezDNase (TFS) and subjected to cDNA synthesis using NEBNext Ultra II RNA First-Strand and Non-Directional RNA Second Strand Synthesis modules, utilizing random primer mix  according to manufacturer recommendations. Double-stranded cDNA was cleaned up using Agencourt AMPure XP reagent  and quantified using the Qubit dsDNA HS Assay Kit (TFS).A Ligation Sequencing Kit  and NEBNext End repair and Quick Ligation Modules (NEB) were used as directed by the manufacturer's protocol. Libraries were quantitated by Qubit (TFS). Samples were barcoded with the Rapid Barcoding Kit 96 (ONT) for combined sequencing. An epMotion 5075  was used for automated liquid handling. Sequencing libraries, combined as 16 and 24 barcoded pools, were loaded on GridION Mk1C (ONT) sequencer and run for 72 h.Basecalling and demultiplexing were performed on the GridION with the MinKNOW operating software v21.11.7 (ONT) and Guppy v 5.1.13  Reference Sequence (RefSeq) database using DIAMOND v2.0.14 , and target segment reads (for each PCR) were individually recorded, and proportions relative to the total number of reads or specific virus reads were calculated. Furthermore, the samples were compared in a binomial manner, where the results were scored as either 0 (PCR/NS negative) or 1 (PCR/NS positive). The Z-statistics obtained were then compared to the critical values for 95 and 99% two-tailed tests .Rhipicephalus bursa (70%), R. sanguineus sensu latu (17.5%), R. turanicus (7.5%), and Haemaphysalis parva (2.5%) species  comprised the majority of the mapped reads .A total of 301 ticks were sequenced in 40 pools, which comprised  species . The seq species . A total species . SequencFlaviviridae, species Mogiana tick virus) sequences were identified in 24 pools (60%) (Jingmen tick virus (family ls (60%)  (Simmondls (60%) . JMTV isls (60%) , sometimls (60%) . JMTV anls (60%) . In the ls (60%) . Near-cols (60%) .R. bursa ticks were suggested as the primary vector. Based on reports from Turkey and Iran, AIGV is a human pathogen, with the capacity to produce mild or severe CCHF-like disease  of the pools . Mapped We further compared JMTV and CCHFV detection rates by PCR and NS . Six pooMivirus is a recently established genus within the Chuviridae family   of the tick pools . The seqOrthomyxovirus (Quaranjavirus), a Peribunyavirus (Ixodes ricinus bunyavirus-like 1 virus), and an unclassified Riboviria (Butler's Creek virus) in three tick pools , tick species with detectable CCHFV or AIGV may overlap significantly in endemic regions, and genetic exchange resulting in altered virulence is also possible. Hence, adequate tools to diagnose and monitor AIGV are required, and our findings further indicate NS as a robust approach for this purpose.Due to the public health threat potential emergency and the lack of efficacious therapeutic or preventive measures, CCHFV is included among the pathogens targeted in the World Health Organization research and development blueprint  are included in the in vivo experiments failed to propagate these viruses, they are currently considered non-virulent residents of the tick virome , four phleboviruses , and other RNA viruses . InteresSeveral shortcomings of the current study should be addressed. First, information on the number of infected individuals in each pool was lacking; therefore, assessments on individual ticks or mixed infections could not be performed. We did not use a CCHFV quantitative-PCR approach, as it may not ensure accurate detection of virus genotypes including AIGV in circulation and multiple genotypes. A more informative comparison would have been possible by parallel sequencing of spiked pools with a known copy number of viral genomes. During NS, we did not carry out a real-time data interpretation for virus detection, but an end-point analysis of the sequencing data instead. NS was reported to be capable of generating 12% of the Ross River virus reads in a single infected mosquito after 10 min and 87.3% in 10 h  can be found below: KE, ED, and Y-ML: conceptualization. KE and ED: data curation. KE, ED, SAJ, BPB, SPN, MOT, BO, IS, and OFG: formal analysis. NLA, JPG, and Y-ML: funding acquisition and resources. ED, SPN, MOT, BO, IS, OFG, and DDR-W: investigation. H-ML and LJ: methodology. Y-ML: project administration and supervision. KE: writing—original draft. KE and Y-ML: writing—review and editing. All authors contributed to the article and approved the submitted version.


Over the past 2 years, the world has faced the impactful Coronavirus Disease-2019 (COVID-19) pandemic, with a visible shift in economy, medicine, and beyond. As of recent times, the emergence of the monkeypox (mpox) virus infections and the growing number of infected cases have raised panic and fear among people, not only due to its resemblance to the now eradicated smallpox virus, but also because another potential pandemic could have catastrophic consequences, globally. However, studies of the smallpox virus performed in the past and wisdom gained from the COVID-19 pandemic are the two most helpful tools for humanity that can prevent major outbreaks of the mpox virus, thus warding off another pandemic. Because smallpox and mpox are part of the same virus genus, the Orthopoxvirus genus, the structure and pathogenesis, as well as the transmission of both these two viruses are highly similar. Because of these similarities, antivirals and vaccines approved and licensed in the past for the smallpox virus are effective and could successfully treat and prevent an mpox virus infection. This review discusses the main components that outline this current global health issue raised by the mpox virus, by presenting it as a whole, and integrating aspects such as its structure, pathogenesis, clinical aspects, prevention, and treatment options, and how this ongoing phenomenon is being globally approached. A serieFurther epidemiological investigations took place within that same period of time in the Democratic Republic of Congo, when an mpox-like virus was isolated for the first time in 1970 from a 9-month-old patient whose symptoms were suspected to be part of the smallpox clinical picture . This caIn this narrative review, we are summarizing the main clinical manifestations of the mpox disease, while also presenting the structure of the virus and including a classification of the main strains. Furthermore, we include a useful analysis on how this public health emergency is being handled globally, in terms of prevention, which is comprised the two vaccines recently approved by the US Food and Drugs Administration (FDA), ACAM2000 and JYNNEOS, and also in regard to the recommended treatment with antivirals, its main representant being tecovirimat. The latter is also known as ST-246 or TPOXX and it constitutes the first choice in treating mpox virus infections, due to the lack of major side effects, opposed to the other antivirals approved by the US FDA, called brincidofovir and cidofovir.2.Global research efforts were deployed once the cases of patients infected with an unknown member of the Poxviridae family were slowly increasing during the 1970s, a decade which was also marked by the cessation of smallpox vaccine production. Therefore, a vaccine which was once used to ensure cross-immunity to the mpox virus as well , was nowThe general structure of poxviruses  was alreThe intracellular proteins that help modulate the actions of the mpox virus are first represented by the virotransducer ones, which interact with the apoptotic pathways. Secondly, the virostealth proteins are involved in the process of downregulation of immune recognition molecules, such as major histocompatibility complex class I and CD4. Thus, they lower the chance of the virus being identified by the immune system. The extracellular viral modulators, also known as viromimic proteins, consist of viroreceptors, reducing the host’s cytokines and chemokines impact and virokines, which are in a quest to imitate the host’s cytokines and chemokines . Under tAlthough the mpox virus shares its linear double-stranded DNA genome with variola and vaccinia viruses , the forAnother particularity that differentiates the mpox virus structure from the rest of the orthopoxviruses is given by the rather incomplete inhibition of E3L and K3L orthologs, derived from genes of the vaccinia virus. These mutations are considered to be the source of attenuation of mpox virus in a wide range of hosts, thus improving its transmission rates .3.So far, two distinct clades of mpox virus have been declared, represented by the African regions with their highest prevalence . The CenFurther epidemiological studies revealed a parallel presence of antibodies among unvaccinated patients in both the Western and Central regions, although the Congo area held more than 90% of these cases, including the only fatal ones. The Central African mpox presents a greater rate of morbidity and human-to-human transmission, also confirmed by the 2003 US outbreak, which had no case-fatalities, since the initial strain was West African .The mechanisms behind this high virulence reside in a suppression of the inflammatory cytokine production in human cells. This phenomenon is caused by preventing T-cell activation  and by hThis peculiarity of the virus being able to suppress the host’s T-cell response , which f4.Although it was first discovered in monkeys, mpox virus does not originate in these mammals  and has Although the main host reservoir of mpox is still unknown, small animals, such as rope squirrels (Funisciurus sp.), sun squirrels (Heliosciurus sp.), and Gambian giant rats  proved to be almost 10 times higher than the ones with the Western African (clades IIa and IIb) mpox variation. Moreover, the more virulent clade also proved to spread into the respiratory, genito-urinary and digestive systems more harshly than the other one, which could be considered milder in terms of manifestation .What should be noted is that the clinical picture of the mpox virus consists of symptoms which are characteristic of smallpox , such asRegarding the incubation period, two intervals were outlined during the 1980s mpox virus observations. One of them, which was approximately 10–14 days, the asymptomatic time frame, was measured from the exposure moment until the fever onset. The other one was estimated to last until the rash onset, and it was considered to take about 12–16 days. However, the period that lasts from the fever episodes until the rash burst also proved to be longer in the case of non-vaccinated patients .The rash burst marks the moment in which the fever level lowers. The lesions begin by appearing slightly confluent, but can evolve into specific maculopapular and vesiculopustular phases, which could be a differentiating criterion from other diseases that present the same skin manifestation . VaricelSeparating the mpox virus diagnosis from the variola virus one can only be performed through laboratory extended analysis . Other lPerhaps, the sign with the highest incidence among mpox virus patients is represented by lymphadenopathies that appear in the cervical and submaxillary areas . They usOne difficulty arises in the process of diagnosing patients with mpox who already have the human immunodeficiency virus infection and syphilis, due to the fact that they can present with atypical clinical aspects. A thorough screening for mpox should be initiated among patients with skin affections, therefore decreasing the risk of spreading mpox in hospitals .Overall, mpox cases last approximately 2–4 weeks , from thTo sum up the classification mentioned before, mpox is considered to have the following major clades: clade I, or the Congo strain; clade IIa, which exists in West Africa and presents low mortality; and clade IIb, which is currently being spread worldwide through human-to-human transmission .In a long-term follow-up study that included clade IIb mpox patients, the residual morbidity after they were healed was studied. During their mpox infections, they presented with nonspecific prodromal symptoms and localized lesions of the skin and mucosae, surrounding the anal and genital regions. Two-thirds of them still had anorectal pain and genital issues at their follow-up, 3–20 weeks after healing, besides persistent fatigability, which was also seen in other infectious diseases such as COVID-19 . The con6.For the past 2 years, the world has faced the impactful COVID-19 pandemic, with tremendous impact upon so many aspects of our lives, such as health, economy, and tourism . As the Given this situation, considered to be a multi-country issue of high risk, the Director-General of WHO declared the ongoing mpox outbreak as a public health event of international concern .Since the beginning of the year 2022 and as of 22 August 2022, 41,664 mpox cases and 12 fatalities were confirmed in about 96 countries, in all the six WHO regions, in an epidemiological update from WHO regarding the multi-country ongoing outbreak of this disease . The repFurther epidemiological investigations confirmed the fact that most of the affected patients, in a proportion of 95.8% of the cases reported with sexual orientation to the WHO, are represented by men aged 20–50, who identify themselves as men having sex with men (MSM), without any recent journeys to mpox endemic countries from Africa . HoweverDue to the increasing number of infected cases in their country, Portuguese scientists managed to release the first draft of the genome sequence of the mpox virus strain that had been recently identified in the non-African outbreak. After gathering genetic data, it was proved that the 2022 mpox virus is part of the West African mpox virus clade .The clinical management suggested by the WHO represents an advisory for Member States to pursue their national immunization technical advisory groups and develop recommendations regarding immunization for mpox, for groups of high risk in their countries .Immunocompromised people, pregnant women, and young people present a more prevalent risk of exposure. The recently approved vaccines for prevention of mpox virus are JYNNEOS and ACAM2000, as mentioned by the US Centers for Disease Control and Prevention CDC; . The latRegarding potential therapeutics, on May 2019, 2022, United States, Canada, and Europe confirmed that tecovirimat (Tpoxx) can be used for means of treatment of both patients with smallpox and patients with mpox .Another issue of important matter in facing the mpox phenomenon resides in misinformation of people, causing a so-called “misinfodemic” , with maAlthough measures are being globally taken, most challenges occur in the areas which are perhaps most in need, with the highest numbers of cases. People from African endemic regions have to deal with issues such as the expenses that arise from the use of healthcare facilities and limit their intentions of accessing them, further leading to underreporting of cases, with an impact on worldwide surveillance. Furthermore, there is also a limited availability of vaccine provisions and therapeutics due to the low possibility of African countries producing their own supplies .7.Public health awareness from all over the world could be improved and coordinated efforts on an international level should be made in order to take proactive measures regarding the prevention of even more cases of mpox than the current situation, thus not allowing it to become the next pandemic .Information sharing among countries worldwide, disease control via personal and collective hygiene, and monitoring new cases are important rules when it comes to global prevention. It is especially important to start all these measures in the early stages of an infection, and immediate action is necessary for controlling further outbreaks. Another effective measure is vaccination. The US FDA approved two vaccines that may provide protection against mpox disease, ACAM2000 and JYNNEOS . These vIt is of high importance that groups of individuals at risk, such as men having sex with men , are infThe result of vaccination is an improved clinical response to the infection, milder symptoms and faster recovery . The bes8.Since smallpox was eradicated, the most common Orthopoxvirus infection in humans known today is the infection with the mpox virus . As mentCurrently, there is a low immunity , 2023 inAt the moment, there are only three available vaccines against mpox: ACAM2000, JYNNEOS, and LC-16, the first one being a second-generation vaccine and the latter two being third-generation vaccines.LC-16 contains the Lister strain of Vaccinia and is partially replicating as it is an attenuated strain. It can be administered in a single dose. It is a vaccine that lacks severe adverse reactions, and the local and systemic adverse events are mild and easily manageable . The vacThe only vaccine approved by the US FDA, JYNNEOS, is also used for the prevention of smallpox, since it is based on a harmless vaccinia virus, with a decreased viral load . It is a9.Since there is no approved drug and current specific treatment for the mpox virus infection and because there is a high structural resemblance between the mpox virus and the smallpox virus, both being part of the Poxviridae family, Orthopoxvirus genus, antiviral drugs used, and approved for the infection with smallpox virus may potentially be used in treating mpox virus infections as well. The main purpose of the clinical treatment is to improve the quality of life by reducing the chances of developing long-term sequelae. The drugs approved by the US FDA are tecovirimat, brincidofovir, and cidofovir in adults and children . These din vitro, by targeting the variola virus VP37 protein related to the release of enveloped virions , either used alone or as an adjuvant to vaccination, is fully protective against an mpox infection, as opposed to vaccination alone. Moreover, antiviral drugs do not interfere with the protective immunity induced by vaccination, by not compromising it, as shown in both mice and monkeys .It is of high importance to know how antiviral drugs and vaccines interact in order to trigger the best immune response in individuals. As mentioned before, tecovirimat is of first choice when it comes to antiviral therapy. Thus, the interaction of this drug with vaccines was intensely studied .via aerosol challenge, in these non-human primates. All in all, 100% survival was reported when this drug was administered before the emergence of clinical symptoms , are useful against mpox for both infection prevention and treatment. In addition, the use of the tecovirimat antiviral drug is highly effective against an infection with the mpox virus. The vaccinia immune globulin, a hyperimmune globulin, could be used as an adjuvant to antivirals. This hyperimmune globulin could prevent, or even treat, if there is the case, complications and adverse reactions to the vaccines. However, further studies are required, because there are not enough data and research about this globulin and its use as a vaccine could be considered unsafe so far.Moreover, the wisdom  and awarRIS, AMS, and OB designed and organized the review. RIS and AMS analyzed and summarized the information, and wrote the manuscript. OB, ALS, and AT supervised the work and contributed to the writing and improvement of the manuscript. All authors contributed to the article and approved the submitted version.ALS was funded by a T32 grant from the US NIH. OB is funded by a grant of the Romanian Ministry of Education and Research, CNCS-UEFISCDI, project number PN-III-P4-ID-PCE-2020-2027, within PNCDI III. The authors would like to acknowledge the funding from the Ministry of Research, Innovation, and Digitization in Romania, under Program 1—The Improvement of the National System of Research and Development, Subprogram 1.2—Institutional Excellence—Projects of Excellence Funding in RDI, Contract No. 31PFE/30.12.2021.The authors declare that the manuscript was developed 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.


A similar pattern was observed in an additional analysis using fluorescence microscopy. However, measurement of the free aptamer concentration after the aptamer-bead conjugation reaction revealed a large aptamer loss compared to the 1.28 × 105 aptamer-bead ratio. In addition, the binding efficiency of the aptamer/bead to the target was highest at the aptamer-to-bead ratio of 1.28 × 105. Taken together, our data suggest that the proposed method is the best and easiest for determining the optimal aptamer-to-bead ratio.Research on the effective attachment of aptamers to beads, which is essential for using aptamers, has made relatively little progress. Here, we demonstrate a new method based on flow cytometry to determine the optimal aptamer-to-bead ratio for aptamer immobilization. The fluorescence intensity increased with a gradual two-fold increase in the aptamer fluorescence concentration, peaked at an aptamer-to-bead ratio of 2.56 × 10 Flow cytometry is a popular cell analysis technique used in various fields. It was first used in the 1950s to measure the volume of cells in a rapidly flowing fluid stream and was then developed to analyze cell characteristics . The greThree independent groups of aptamers have been identified. The term “aptamer” refers to a short single-strand nucleotide (DNA or RNA) or peptide that can specifically bind to the desired target –9. OwingHowever, comprehensive studies to determine the optimal aptamer-to-bead ratio have not yet been reported. This motivated our study, in which we used flow cytometry to determine the optimal amount of aptamer to form the aptamer-bead complex.The aforementioned complex is essential for aptamer studies. Some papers provide information about the reagents, but the molar ratio bead-aptamer is ill-defined . Further2-5′-GGGAGAGGAUACUACACGUGAUAGUCAGGGAACAUG ACAAACACAGGGACUUGCGAAAAUCAGUGUUUUGCCAUUGCAUGUAGCAGAAGCUUCCG-3′) (Supplementary Table) with or without FAM (5′-Fluorescein phosphoramidite), selected as a specific aptamer binding Ni2+/Co2+ [2+/Co2+ bound to the aptamer. The amine group at the 5′-terminus was designed to allow covalent binding of the aptamer to the carboxyl groups on the CLB9 beads (Sigma), which were used to immobilize the aptamers. MES buffer  was used as the solvent in which to couple the aptamer to the beads and to subsequently wash the beads. For coupling, the carboxyl groups on the surface of the beads were activated using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) (Sigma) and N-hydroxysulfosuccinimide (sulfo-NHS) (G-Biosciences). Cobalt (II) chloride was purchased from Sigma–Aldrich .RNA aptamer N1  aliquot of beads was transferred to a microtube and centrifuged for 4 min at 13,000×g. The supernatant was carefully removed and discarded. The bead pellet was resuspended in 200 μL MES buffer and vortexed thoroughly for 10 s. To activate the bead surface, 40 μL of 0.3 M EDC and 40 μL of sulfo-NHS 0.4 M solution were added, followed by mixing. After activation, different concentrations of the aptamer were added to the microtubes and the total volume was adjusted to 500 μL. The coupling reaction was performed for 2 h in the dark at room temperature with rotation . The tubes were centrifuged for 4 min at 13,000×g. The supernatant was used to measure the amount of free aptamer, and the pellet was resuspended in 500 μL PBS-T. This washing step was repeated thrice.To prepare the aptamer-bead complex, a 50-The fluorescence of the supernatants was measured using a Tristar 5 plate reader . Absorbance data were collected and processed using the MikroWin2010 program (Labsis Laborsysteme GmbH). The manufacturer's instructions were followed for operating the machine.μL PBS, followed by analysis using the FACS Canto II flow cytometer . Data were collected using FACSDiva software  and subsequently analyzed using FlowJo software .Beads with or without aptamer labeled with FAM were suspended in a total volume of 300 μL aliquot of beads was transferred onto a slide glass and covered with the cover glass. The microscope was controlled by a computer with the CellSens Entry software (Olympus) at 200× magnification. Photographic images were captured in a completely darkened room, and the entire process was completed within 10 minutes.The fluorescently labeled aptamer-bead complex was analyzed using a CKX53 microscope . After aptamer-bead coupling, a 5 The fluorescence intensity on the images was measured using ImageJ software . First, Photoshop  was used to select regions of interest (ROIs), measuring 100 × 100 pixels from the images. ROIs of the same size were selected and cut with Photoshop in the same positions in all images to maintain a standard in the analysis of the measurements. Next, ImageJ software was used to extract the average brightness value from the ROI. Each pixel was assigned a numerical value that represented its brightness on the grayscale 22, ranging from 0 (black pixels) to 255 (white pixels).μL of 10 μM CoCl2 was transferred to a microtube containing the aptamer–bead complex and the mixture was incubated for 10 min at room temperature with rotation (Roto-Bot) [g, and the concentration of Co2+ in the supernatant was measured by ICP-OES using a PlasmaQuant PQ 9000 spectrometer  equipped with a Standard Kit. The data were collected and processed using Aspect PQ 1.2.4.0 software. The manufacturer's instructions were followed for operating the machine.After aptamer–bead coupling, 300 oto-Bot) . After iAll statistical analyses were conducted using SPSS software (IBM® SPSS® Statistics Ver. 23). For multiple comparisons, the data were analyzed using one-way analysis of variance (ANOVA). Tukey's posthoc test was used to identify significant differences among groups.Flow cytometry, in which the laser passes through the cells and is reflected, uses pattern analysis to acquire information about the characteristics of cells. To explore whether flow cytometry could recognize polystyrene beads, we started our investigation by analyzing the beads without and with aptamers attached. Flow cytometry did not recognize the beads without aptamers; however, those with aptamers were detected . SpecifiBecause various types of beads are used in research with aptamers, it is inefficient to use the same aptamer-to-bead ratio for all beads. Therefore, determining the most effective ratio for each type of bead is essential. In our work, the aptamer-to-bead ratio was gradually increased from 5 × 102 to 5.12 × 105 by doubling the aptamer concentration incrementally while the concentration of beads was kept constant. As expected, the fluorescence intensity increased approximately 2-folds with a 2-fold increase in the aptamer concentration Figures . This anTo confirm the aptamer-to-bead ratio we determined by flow cytometry, we used fluorescence microscopy to analyze complexes containing fluorescently labeled aptamers and beads. The fluorescence intensity gradually increased under the same conditions as those in The results from the flow cytometry and fluorescence analysis suggested that it is not effective to continue increasing the aptamer-to-bead ratio beyond a certain value. To confirm this hypothesis, we analyzed the concentration of free aptamer that remained after exposure of the aptamer to the beads for complexation. The concentration of the free aptamer gradually increased depending on the initial amount of aptamer . At low 2+ [2+ remaining after Co2+ binding to the aptamer-bead complex. The rate at which Co2+ was removed by the aptamer-bead complex was the highest at the aptamer-to-bead ratio of 1.28 × 105 , has the effect of decreasing the binding efficiency between the aptamer and the target. This experimental method is expected to be useful for determining the combination of various aptamers and beads in the future.


In the hospital-based Nigerian cohort, HepB LiveTest performed optimally with an AUROC of 0.94 . The model provided tailored predictions that ensured most cases of HBV infection did not go undetected. However, its discriminatory measure dropped to 0.60  in the Australian cohort. These findings indicate that HepB LiveTest exhibits adequate cross-site transportability and clinical validity in the hospital-based Nigerian patient cohort but shows limited performance in the Australian cohort. Whilst HepB LiveTest holds promise for reducing HBV prevalence in underserved populations, caution is warranted when implementing the model in older populations, particularly in regions with low incidence of HBV infection.HepB LiveTest is a machine learning decision support system developed for the early detection of hepatitis B virus (HBV). However, there is a lack of evidence on its generalisability. In this study, we aimed to externally assess the clinical validity and portability of HepB LiveTest in predicting HBV infection among independent patient cohorts from Nigeria and Australia. The performance of HepB LiveTest was evaluated by constructing receiver operating characteristic curves and estimating the area under the curve. Delong’s method was used to estimate the 95% confidence interval (CI) of the area under the receiver-operating characteristic curve (AUROC). Compared to the Australian cohort, patients in the derivation cohort of HepB LiveTest and the hospital-based Nigerian cohort were younger (mean age, 45.5 years vs. 38.8 years vs. 40.8 years, respectively;  Hepatitis B virus (HBV) is a significant public health concern, causing liver infection and leading to substantial morbidity and mortality worldwide. With over 296 million people living with HBV globally, 90% of infected individuals are unaware of their infection status, missing out on essential clinical care ,2. In 20Our prior investigation into HBV infection levels in Nigeria revealed a prevalence of 9.5% , highligTo support the World Health Organization’s goal of eliminating viral hepatitis by 2030 , there hIn a recent study, we developed HepB LiveTest, a machine learning decision support system for early detection of HBV infection based on routine blood test data, including hepatitis B surface antigen (HBsAg) immunoassay results . The modConducting external validation separately from the model development has been recommended to ensure methodological rigor, reduce biases, and increase the transparency of performance evaluation and generalisability in new and diverse patient populations. This approach enhances the credibility and practical utility of clinical prediction models in a real-world setting ,11,12,13Predictor and outcome measurements may vary for various reasons, thus distorting the performance of a prediction model. Variability in measurements can arise from differences in equipment specifications, timing of data collection, subjectivity in interpretation, and nuances in biomarker quantification. These factors can introduce heterogeneity in predictive modelling studies, based on electronic health records. Such variations in measurement procedures may significantly impact the discriminative performance of the prediction model and also compromise its clinical validity in different patient populations and settings—and seemingly “better” measurements at validation may also not necessarily lead to improved model performance ,19. ThisThe main objective of this study is to independently validate HepB LiveTest in two external patient cohorts from Nigeria and Australia and evaluate the case-mix variability on performance drift. This geographic validation is critical in determining whether HepB LiveTest accurately predicts HBV infection in patients from diverse populations/settings, providing insights into the model’s generalisability and potential clinical utility. By evaluating the performance of HepB LiveTest in independent patient cohorts, we aim to contribute valuable evidence to inform the adoption and appropriate use of this machine learning decision support system for early detection of HBV infection.The study protocol was approved by the Institutional Review Board of the University of Ilorin Teaching Hospital (ERC PAN/2020/06/0022) and the Human Research Ethics Committee of the Australian National University (2019/803) as minimal-risk research that used retrospective patient data collected from routine clinical care and, as such, the requirements for informed consent were waived.The study was reported in accordance with the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis (TRIPOD) guideline for prediction model validation .To assess the clinical validity and cross-site transportability of HepB LiveTest model, it was externally validated in independent Australian and Nigerian patient cohorts, with datasets collected from the Sullivan Nicolaides Pathology  and the University of Ilorin Teaching Hospital , respectively, across two different time periods.SNP is Australia’s largest private pathology referral laboratory, regarded for its expertise in routine pathology testing. It delivers comprehensive laboratory services to hospitals in Queensland, northern New South Wales, and the Northern Territory, with the central laboratory in Brisbane designed to foster interdisciplinary collaboration between specialist pathologists and clinical scientists, while UITH is one of the major Federal Teaching Hospitals in Nigeria, located within the North Central Geopolitical Zone of Nigeria at latitude 8°30′ N and longitude 4°33′ E. The hospital provides care to a large number of patients from Kwara State and equally serves other neighbouring states, including Oyo, Niger, Kogi, and Ekiti.The two validation datasets from SNP and UITH included patients suspected of HBV infection and who had undergone HBsAg immunoassay testing. Patient samples to produce the SNP validation dataset were collected between 1 June 2011 and 31 May 2012, and samples to produce the UITH validation dataset were collected between 1 April 2018 and July 2021. All patient records were anonymised and de-identified. Patients with a definitive HBsAg immunoassay result and routine blood test values measured during pathology examination were considered. Patients with incomplete data profiles were excluded.Patients were first evaluated using routine clinical chemistry and haematology blood tests, and those suspected of HBV infection were referred for enzyme immunoassay testing. A suspected HBV case is defined as a case that was compatible with standard clinical description . The priHepB LiveTest is a machine learning model for early detection of HBV infection, translated into a publicly available web app . The modt-test if two groups), and the distribution of the categorical variables was compared using Pearson’s chi-square test.Patient baseline characteristics were compared between the NIMR derivation cohort and the UITH Nigerian and SNP Australian validation cohorts. Baseline characteristics were presented as mean (±SD) for continuous variables, while categorical variables were summarised by the number of subjects (with percentages). Parametric tests were applied, since the population data have a normal distribution. The baseline characteristics between HepB LiveTest derivation cohort and the external validation cohorts were compared using the one-way ANOVA test  curve ,22. The Patient characteristics in the UITH-Nigerian and SNP-Australian validation cohorts in comparison with the original HepB LiveTest derivation cohort.p < 0.001). In addition, the SNP-Australian validation cohort had a lower baseline ALT level (57.9 U/L vs. 101 U/L vs. 182.5 U/L) and lower incidence of HBsAg positivity (1.9% vs. 69.4% vs. 57.3%) than those in the derivation and the UITH-Nigerian validation cohort, respectively. The reference interval of the pathology markers contained in the dataset are presented in The final SNP-Australian sample size was 9102, while the UITH-Nigerian sample size was 258. Current evidence suggests a minimum effective sample size of 100 for external validation [The performance of HepB LiveTest is summarised into a single measure of AUC , as obseFor the UITH-Nigerian patients, HepB LiveTest correctly identified at least 9 out of every 10 HBV patients. To put this result in context, that is an estimated 91% in sensitivity performance. However, when HepB LiveTest was tested on the SNP-Australian validation cohort, its performance dropped to 66%. The performance measures in Dataset shift in terms of the difference in the mean of predictors was observed in individual features from HepB LiveTest derivation cohort to the validation cohorts. Notable feature mean differences were found between the HepB LiveTest derivation cohort and the SNP-Australian validation cohort, and this could influence performance owing to case-mix effect. The largest differences were in GGT with 202.5% increase and ALT with −42% decrease. Many of the features between HepB LiveTest derivation cohort and the UITH-Nigerian validation cohort had similar distributions in mean, as shown in We present the external validation results of HepB LiveTest, a machine learning decision support system designed for early detection of HBV using routine pathology markers. Our findings demonstrate that HepB LiveTest performs optimally in the UITH-Nigerian patient cohort but exhibits limited clinical validity in the SNP-Australian patient cohort. This suggests the need for caution when adopting the model on older populations in settings with low incidence of HBV infection and those with similar predictor value distribution observed in the Australian cohort.The variability in prediction model performance across different settings and populations is widely recognised ,26,27. TIn our study, the substantial deviation in HBV incidence between the original HepB LiveTest derivation cohort and the UITH-Nigerian validation cohort (69.4% vs. 57.3%) from the low incidence observed in the Australian patient cohort (1.9%) may, in part, explain the observed performance drift. Therefore, recalibration of the model, considering changes in infection rates/outcome incidence, may be necessary when applying the model in settings with low levels of HBV infection.The presence of heterogeneity in measurement procedures can also significantly impact the performance of prediction models ,19,33,34In our study, we recognise that the performance drift observed in the Australian cohort compared to the Nigerian cohort may have been influenced by differences in the distribution of predictor values . The significant differences in certain feature distributions, such as GGT (202.5% increase) and ALT (−42% decrease), between the HepB LiveTest derivation cohort and Australian validation cohort may have contributed to the observed limited clinical validity. Whilst the elevated baseline serum GGT level in the Australian population might be a reflection of alcohol misuse, the normal baseline ALT level was expected for a population with low levels of HBV infection. Additionally, the lack of AST data in the Australian validation cohort, which is an important predictive marker of HBV infection required by HepB LiveTest, may have influenced the model’s performance. These findings highlight the potential impact of case-mix variability on the performance of HepB LiveTest. More broadly, the findings also suggest that multicentre external validation studies offer the potential to capture heterogeneity across different populations and settings, thus providing evidence on the appropriate level of model generalisability within specific contexts.The incorporation of routine laboratory blood test markers in HepB LiveTest that are readily available in many outpatient and inpatient clinical settings, along with its user-friendly interface, makes it potentially deployable for early detection of HBV in Nigerian patients, without resorting to expensive second-tier immunoassay testing. However, during the clinical deployment phase, the model would need to be closely monitored for necessary updates, particularly when patient demographics and local practice patterns/norms inevitably shift. Continuous monitoring and updates will ensure that the model remains adaptable and effective in capturing evolving epidemiological trends and clinical practice patterns.The strength of this work lies in the universal availability of the required predictive pathology markers in the majority of healthcare settings and the validation using data from external patient cohorts in two different population settings. Three studies have created a machine learning model to predict HBV infection ,7,8. TheNonetheless, this study has limitations. It is challenging to fully understand how ethnic variability and HBV genotypic variation between Nigerian and Australian populations collectively and independently impact the performance drift of HepB LiveTest. For example, HBV genotype C is the most frequent genotype in the Australian population, while genotype E exclusively predominates Nigeria . GenotypIn conclusion, HepB LiveTest demonstrates adequate geographic validation and generalisability beyond the development cohort, with optimal performance in the hospital-based Nigerian patient cohort. Future works will be required to assess the interface integration and implementation of HepB LiveTest within the clinical workflow. It may also be necessary to evaluate the adoption of HepB LiveTest in real-world clinical settings, preferably through randomised clinical trials, to inform evidence for improved patient outcomes and process optimisation. As the first, to the best of our knowledge, externally validated machine learning decision support system for early detection of HBV, HepB LiveTest provides a platform to drive a reduction in HBV prevalence through timely linkage to care and optimise the quality of life for millions of HBV patients, particularly in underserved populations such as Nigeria. Fostering collaboration between population health scientists, clinicians and software developers will facilitate seamless integration and optimisation of HepB LiveTest into routine healthcare workflows, streamlining the clinical diagnostic process and ultimately enhancing patient outcomes.