We conducted a retrospective serosurvey for antibodies against Crimean-Congo hemorrhagic fever virus in wild ungulates along the eastern Mediterranean Coast of Spain. The virus has been endemic in this region since 2010 but is mainly restricted to geographic clusters with extremely high seropositivity associated with high density of bovids. Orthonairovirus, belonging to the family Bunyaviridae. In humans, CCHFV can induce a severe and potentially fatal systemic hemorrhagic disease. CCHFV infections in wildlife and domestic animals are generally subclinical but, in some species, can induce enough viremia to enable virus transmission to uninfected ticks. Moreover, infected animals produce antibodies, enabling the identification of affected areas through retrospective serologic studies  is caused by CCHF virus (CCHFV), a tickborne pathogen of the genus H. lusitanicum ticks from a red deer (Cervus elaphus) in 2010  and of the virus itself were uncertain until recently, when H. lusitanicum ticks were found in wild boars (Sus scrofa) from the metropolitan area of Barcelona  to test for CCHFV antibodies in serum samples collected from 332 wild boars, 126 Iberian ibexes (Capra pyrenaica), and 48 mouflons (Ovis aries musimon). Serum samples were collected during 2010–2021 within the framework of the wildlife surveillance program in the Valencia region. We chose wild boars, Iberian ibexes, and mouflons because they are the main wild ungulate species in the region. Iberian ibexes and mouflons were selected from the 2 areas where they are more abundant. We also selected serum samples taken from boars in the same 2 areas and from areas with low densities of wild ruminants.  had antibodies against CCHFV, which is close to the 100% seroprevalence reported for the same species in the affected neighboring area of Catalonia (CCHFV seropositivity in the Valencia region clustered in 2 areas , panel AWe also detected CCHFV antibodies in a few wild boars outside the 2 main positive areas , panel AA recent study mapped the risk for CCHFV exposure among humans in mainland Spain by using red deer as an indicator of the transmission risk plus environmental variables (H. lusitanicum ticks (Little information is available on the distribution of competent CCHFV vectors in the Valencia region, but a study to the north of the region reported a substantial increase during 2017–2018 in the number of persons receiving tick bites, 85% of which were caused by um ticks , panel BBovidae species. However, as these species move into areas with higher human populations, more human CCHF cases could occur. To protect the population of the region, public health authorities should continue CCHFV surveillance among tick and ungulate species.Our results support an epidemiologic scenario in which CCHFV has been endemic in wild ungulates in different regions of Spain before it was detected in 2010. In eastern Spain, CCHFV circulation mainly occurs in geographic clusters associated with high densities of Additional information on prolonged Crimean-Congo hemorrhagic fever virus endemicity detected by retrospective serosurvey, eastern Spain.


Sexually transmitted infections (STIs) not only have an impact on the health sector but also the private resources of those affected, their families and other sectors of society . This study aimed to i) review and identify economic evaluations of interventions relating to STIs, which aimed to include a societal perspective; ii) analyse the intersectoral costs  included; iii) categorise these costs by sector; and iv) assess the impact of intersectoral costs on the overall study results.Seven databases were searched: MEDLINE (PubMed), EMBASE (Ovid), Web of Science, CINAHL, PsycINFO, EconLit and NHS EED. Key search terms included terms for economic evaluation, STIs and specific infections. This review considered trial- and model-based economic evaluations conducted in an OECD member country. Studies were included that assessed intersectoral costs. Intersectoral costs were extracted and categorised by sector using Drummond’s cost classification scheme . A narrative synthesis was performed.Twenty-nine studies qualified for data extraction and narrative synthesis. Twenty-eight studies applied a societal perspective of which 8 additionally adopted a healthcare or payer perspective, or both. One study used a modified payer perspective. The following sectors were identified: patient/family, informal care, paid labour (productivity), non-paid opportunity costs, education, and consumption. Patient/family costs were captured in 11 studies and included patient time, travel expenses, out-of-pocket costs and premature burial costs. Informal caregiver support (non-family) and unpaid help by family/friends was captured in three studies. Paid labour losses were assessed in all but three studies. Three studies also captured the costs and inability to perform non-paid work. Educational costs and future non-health consumption costs were each captured in one study. The inclusion of intersectoral costs resulted in more favourable cost estimates.This systematic review suggests that economic evaluations of interventions relating to STIs that adopt a societal perspective tend to be limited in scope. There is an urgent need for economic evaluations to be more comprehensive in order to allow policy/decision-makers to make better-informed decisions.The online version contains supplementary material available at 10.1186/s12889-022-14484-z. STIs ansocietal , multisesectoral  or interquences) , 12. ThiInterventions relating to STIs are essential but complex in nature . This complexity can, in part, be explained by the aforementioned wide-ranging, intersectoral impacts of public health programmes, and this can create challenges in adequately capturing them in economic evaluations , 13. TheEven though a societal perspective has been advocated in methodological guidelines for some time , it is nGiven the importance placed on the societal perspective and the potential for suboptimal policy decisions in the absence of a comprehensive evaluation, a systematic review of published economic evaluations which adopted a societal perspective was undertaken. The review aimed to explore the intersectoral costs considered under a societal perspective in economic evaluations of interventions relating to STIs, and the impact of including intersectoral costs on the overall study results. The specific objectives of this review were to i) identify economic evaluations of interventions relating to STIs which aimed to include a societal perspective; ii) analyse the intersectoral costs  included; iii) categorise these costs by sector ; and iv) assess the impact of intersectoral costs on the overall study results.A protocol for this review was published in PROSPERO, the International Prospective Register of Systematic Reviews database CRD42019130940). The review systematically followed the Centre for Review and Dissemination (CRD) guidance for undertaking reviews in health care  and a fi9130940. A search strategy was developed in PubMed for MEDLINE together with an information specialist before adapting for use in other databases  member country. OECD member countries were chosen due to their similarities in terms of health(care) systems and to better compare the methodology of studies concerned with similar health(care) systems. It focused on full economic evaluations that adopted a societal perspective, including cost-effectiveness analyses (CEA), cost-utility analyses (CUA) and cost-benefit analyses (CBA). The methods of CEA, CUA and CBA have varying theoretical foundations and outcomes are expressed differently. A CEA measures assesses outcomes (effects) in natural units, whereas a CUA assesses outcomes in health utilities such as quality-adjusted life years (QALYs). A CBA assesses both costs and outcomes in monetary values. No restrictions were placed on the type of comparator or outcomes. Participants in an intervention had to be at least 10 years of age to reflect international definitions of the start of adolescence   the PIC.Search results were exported into EndNote X9. Citations were de-duplicated following the guidelines by Bramer and colleagues . The stuData were recorded using Microsoft Excel and Word. Intersectoral costs were identified, extracted and categorised by sector using Drummond’s sector-specific cost classification scheme  Table 11. DrummoThe search strategy generated 23,895 studies after duplicates were removed. Studies were further limited to those published from 2009 onwards, excluding 6483. Though this was due to a high number of records identified in the databases, the publication date reflects the period of time during which intersectoral costs and benefits have gained more prominence , 22, 34.n = 11), HIV (n = 8) and chlamydia  (Table n = 1), hepatitis B (n = 1) and hepatitis C (n = 1). The countries of interest in the selected studies included the United States of America (USA) (n = 10), The Netherlands (n = 8), Canada (n = 3), Sweden (n = 3), Germany (n = 2), Australia (n = 1), Austria (n = 1) and Israel (n = 1). The overwhelming approach adopted was a CEA (considering the study authors’ definition of their study as a CEA and CUA). However, not all studies explicitly stated whether they applied a CEA or CUA approach. Only three out of the 29 studies explicitly reported to have undertaken a CUA [Of the 29 studies identified, the majority focused on HPV , non-paid opportunity costs (productivity), education and consumption Table .Table 3CPatient and family costs were captured in 14 studies and included patient time, travel expenses, out-of-pocket costs and premature burial costs. Four studies estimated patient time and travel to seek care as part of healthcare costs  , 54, 60;Caregiver support (non-family) and unpaid help by family/friends was captured in four studies , 52, 56.Productivity costs in terms of paid labour losses were assessed in 24 studies. The majority of studies measured these in terms of absenteeism (time off work). Of those, one study estimated productivity losses in a sensitivity analysis only . Two of Only four studies explicitly reported capturing the costs associated with non-paid work  , 39, 52.School absence was captured in one study only . It refeFuture consumption costs unrelated to health were considered by one study . These cAll studies that applied a societal perspective in addition to a healthcare and/or payer perspective presented more favourable cost-effectiveness results under the societal perspective Table . Four stThis study is the first to systematically review economic evaluations of interventions relating to STIs and explore and categorise the different types of intersectoral costs captured under a societal perspective. It also presents evidence that the inclusion of intersectoral costs has an impact on the overall study results.This review found that the identified studies took a rather narrow approach to the societal perspective and only considered costs relating to a limited range of non-health sectors. For the majority of studies this meant primarily estimating paid labour losses. For others, this meant the inclusion of patient and family costs in their analyses. Very few studies considered informal care costs and other non-paid opportunity costs. Only one study included educational costs and another captured non-medical consumption costs. These findings indicate that even where a societal perspective is adopted, this may often be limited in scope, potentially omitting relevant costs from other sectors. The theoretical definition of a societal perspective, however, does not limit the potential scope to the aforementioned sectors .Even though the inclusion of intersectoral costs was limited to a few cost sectors, where intersectoral costs were accounted for, this resulted in more favourable cost-effectiveness estimates.A primary reason for studies applying a narrow societal perspective could be the methodological challenges associated with capturing these wider costs such as with data collection processes or unavailable data . The ideThis review’s cost classification scheme was established to assess whether (or not) and to what extent intersectoral costs were considered and reported explicitly and transparently. The findings suggest that there is considerable scope for exploring other wider societal costs in relation to interventions addressing STIs. This would help improve understanding of the wider societal impacts of STI-related interventions and inform the design of future, more comprehensive economic evaluations.This review shows that informal care was rarely captured in the evaluations, but where it was considered, it related to chronic conditions including HIV/AIDS, HPV-related cervical cancer and hepatitis B-related cancer. Where (long-term) care is provided informally this makes the inclusion of such costs in economic evaluations crucial. If informal care is not considered (or discussed as a study limitation) this can omit important information and underestimate the total cost burden. Future research is needed to further investigate informal care costs related to STIs, particularly those that can have chronic impacts  and those with severe long-term sequelae .This review also found that the costs associated with unpaid work remained largely excluded from economic evaluations relating to STIs. This was difficult to judge as not all studies were explicit about which cost components/resource items they accounted for when referring to productivity costs – i.e. paid labour, volunteering or household work. A number of study authors were approached to clarify whether productivity losses accounted for paid or unpaid labour, or both. The majority responded that only paid labour losses were included due to missing data or the methodological challenges of including unpaid productivity. These findings suggest that greater transparency is needed when a societal perspective is adopted to clarify which costs and benefits are included/excluded and the justification for these decisions.School absence was only captured in one study. Absence from school due to an STI or seeking treatment for an STI can refer to potential productivity loss or loss of educational attainment. The study that captured absence from school valued each hour missed at school based on the national minimum wage, as informal care and domestic activities. No other costs relating to education were identified.Where future consumption costs unrelated to health were accounted for it was not clear what this involved. Examples of non-medical consumption costs can include travel expenditures or future costs for housing and food . This adOther potentially important distinct health impacts can include costs in the reproductive health and mental health sphere. However, these impacts were not captured in the selected studies. The prevention of STIs can reduce the risk of cervical cancer, pelvic inflammatory disease and infertility among women , which iRelatively few economic evaluations related to STIs have adopted a societal perspective. This is in line with recent findings by Bloch and colleagues  who asseA societal perspective is generally recommended to allow for all relevant costs and benefits to be considered and for an economic evaluation to be as comprehensive as possible. However, where economic studies adopt a societal perspective, but this only includes certain costs in certain sectors, relevant societal implications may be ignored. As a result, decisions based on an analysis with a limited scope might not be optimal . As showThe costs considered and included under a societal perspective differed across studies, resulting in heterogeneity of study results. This highlights that there is a need for a clear understanding of which costs were included and excluded under any perspective when reviewing and synthesising the existing literature, or when combining results from different studies undertaken in different settings. This is particularly important because the different elements of costs  can differ between countries and time points. When researchers adopt data from the existing literature for use in their own work, they need to carefully assess what costs were captured before the results can be relied on and utilised.The main strength of this review is that it followed a structured and rigorous process. To our knowledge, this is the first review in sexual health to apply a cost classification scheme in order to explore and categorise the intersectoral costs considered by sector. The classification scheme provides a valuable foundation for the critical appraisal of economic evaluations, in particular with regard to the consideration and identification of societal costs. The results of this study can inform the design of future, more comprehensive economic evaluations of public health interventions, building on the classification scheme presented. Another key strength of this review is the exhaustive search strategy that was developed in cooperation with an information specialist, searching nine databases and a wide range of key search terms relating to sexual health. It is however possible that some relevant search terms may have been missed. In addition, the update of the review focused on Medline only, which may have resulted in some studies being omitted, although this was mitigated by extensive hand searching. A potential weakness of the review is that because of the high volume of studies identified in the databases, an initial screening was undertaken to exclude studies where the abstract suggested that the study adopted a healthcare perspective only. This means that relevant studies could have been missed. Future research could review economic analyses that adopted a healthcare (system) perspective to assess in detail which costs these studies captured, i.e. direct medical costs, patient costs, or other costs. Another limitation of this review is that it focused on OECD member countries. Reviewing studies in non-OECD member countries could have identified other potentially relevant costs associated with interventions relating to STIs. Further, this review focused on STIs that are sexually transmitted and interventions related to infections transmitted other than sexually, could have revealed additional cost sectors.Further research is needed to investigate wider intersectoral costs related to STIs that were not (sufficiently) captured in this review but that could be important to inform policy/decision-making. Such research could also help explore the intersectoral costs relating to other sexual health aspects beyond disease such as sexuality, sexual behaviour, and related areas. Given the complexity of sexual health future research could explore wider intersectoral costs relating to STIs that have been considered outside of the health economics literature such as in educational journals or journals relating to social services. Furthermore, future research could explore in greater depth the distinction between intersectoral consequences associated with the disease and the intersectoral costs incurred by the intervention being evaluated, as this was not always fully clear.This systematic review suggests that economic evaluations of interventions relating to STIs that adopt a societal perspective tend to be limited in scope. This risks omitting potentially relevant intersectoral costs that could be important information for policy/decision-making. There is an urgent need for economic evaluations to be more comprehensive in order to allow policy/decision-makers to make better informed decisions.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


Hospitalitermes bicolor, Macrotermes carbonarius and Odontotermes wallonensis) collected in the Cat Tien National Park (Vietnam). We assembled four full genomes of novel viruses related to Solemoviridae, Lispiviridae, Polycipiviridae and Kolmioviridae. We also found several contigs with relation to Chuviridae and Deltaflexiviridae that did not correspond to complete virus genomes. All the novel viruses clustered phylogenetically with previously identified viruses of the termites. Deltaflexi-like contigs were identified in the fungi-cultivating M. carbonarius and showed homology with viruses recently discovered in the edible basidiomycete mushrooms.Modern metagenomic approaches enable the effective discovery of novel viruses in previously unexplored organisms. Termites are significant ecosystem converters and influencers. As with the majority of tropical forest insects, termites are studied insufficiently, and termite virome remains especially understudied. Here, we studied the virome of lichenophagous and mycophagous termites ( Modern metagenomic technologies have enabled the discovery of diverse RNA viruses in different species of vertebrates and invertebrates ,2. The sTermites  are keystone species in tropical ecosystems. Being the most important decomposers of dead organic matter, different species of termites exploit a wide range of feeding substrates. Higher termites from the family Termitidae are characterized by highest diet diversity, including wood, grass, lichens or fungi from fungal gardens cultivated in the nest .As with the majority of tropical forest insects, termites are studied insufficiently , while tHospitalitermes bicolor (Haviland), Macrotermes carbonarius (Hagen) and Odontotermes wallonensis (Wasmann). These species represent less virologically studied feeding types of termites, lichenophages and mycophages. H. bicolor (Nasutitermitinae) feed on epiphytes in the forest canopy and forage in large columns, formed by up to 500,000 individuals [M. carbonarius and O. wallonensis (Macrotermitinae) are open-air foraging species that collect plant litter and wood debris to cultivate fungi in the fungal gardens [M. carbonarius can reach more than 60,000 individuals [O. wallonensis can harbor from 45,000 to 75,000 individuals [For our investigation, we chose three species of higher termites: ividuals ,9. M. ca gardens . Colonieividuals , while nividuals .Schedorhinotermes intermedius [Recently, the virome of 50 species of termites from Australia, Kenya, French Guiana, Cameroon and Indonesia was studied, and 67 novel viruses from different phylogenetic groups were identified [ermedius .In the current work, we assembled four full genomes of the novel viruses and obtained data indicating the presence of two more viruses in three hitherto unstudied species of termites from Southeast Asia.H. bicolor and O. wallonensis, and soldier specimens of M. carbonarius  in December 2019. Worker and soldier specimens of bonarius , were taTo avoid contamination with bacterial and fungal cells, insects were treated with nystatin saline solution  and further rinsed with distilled water. Termites were frozen at −20 °C to transport them to the laboratory (which took about 10 days). In the laboratory, insects were stored at −70 °C before molecular analysis.−1. High-throughput sequencing and the data-processing pipeline were the same as described previously [Termite specimens were homogenized in the pools  using Tieviously .RNA was extracted using TRI Reagent LS  according to the manufacturer’s manual. After the extraction, host rRNA was depleted using an NEBNext Globin and rRNA Depletion Kit  according to the manufacturer’s instructions. The obtained RNA was used for library preparation without polyA-enrichment using a NEBNext Ultra II RNA Library Prep Kit for Illumina  according to the manufacturer’s instructions. Final libraries were sequenced  on a HiSeq1500 , and raw reads were deposited under the BioProject accession number PRJNA817887.RNA was extracted using TRI Reagent LS , according to the manufacturer’s manual. Reverse transcription with random hexamer primers (R6) and MMLV RT kit  was performed on an isolated matrix following the manufacturer’s instructions . PCR wasde novo contig assembly by the SPAdes v3.13.0 program with spades pipeline and default parameters [Adaptor sequences, bases with quality lower than Q30 and reads with length less than 35 nt were discarded using Trimmomatic v0.39 . Filtererameters . The resultant contigs were screened for virus-related sequences using the blastn program from BLAST v2.9.0+ package (with default settings) and nt database. Contigs containing virus-related hits were translated using SnapGene Viewer v.5.0.4 (with default settings), and open reading frames (ORF) were visualized and extracted manually. Extracted ORFs were further analyzed with blastp (using default settings) to identify viral proteins. Some contigs with very high identities to known human pathogens were filtered out as possible contaminations. When SPAdes failed to produce contigs resembling full-length genomes, the SeqMan v.7.0.0 program was used to assemble genomes with contigs as an input. Obtained virus sequences and sequence of the deltaflexi-like contig 17_N1 + N237 were deposited in the GenBank database (accession numbers ON082761-ON082766).The protein sequences of the RNA-dependent RNA polymerase, polyprotein or HDV-ag were extracted from the contigs. These sequences, along with homologs, were aligned using MAFFT v7.310 . The aliTrees were visualized with FigTree v.1.4.4. Genome structure of the obtained viruses was visualized using a custom Python script. All post processing of the images was performed with the GIMP v.2.10.24 program.Macrotermes carbonarius, one pool of Hospitalitermes bicolor, and one of Odontotermes wallonensis . The second contig showed homology to the coat protein of the novel solemovirus, with ~47% amino acid identity to the Wifsystermes virus. Based on these data, we concluded that both contigs belonged to the single novel solemoviridae-like virus named Cat Tien Macrotermes solemo-like virus (CMSV).In the current work, we discovered two contigs related to abovementioned novel solemoviruses in the pool of the The genome of the CMSV has two segments, 2809 and 1754 nt in length, respectively B. Both sEpikalotermes kempae, Epikalotermes kempae, Occasitermes sp. 2 and Schedorhinotermes intermedius, respectively) [According to the phylogenetic analysis based on the amino acids sequences of the RdRp A, CMSV gctively) . Lispiviridae  includes one genus composed of six viruses [Lispiviridae (but yet unclassified) viruses are known.According to the tenth release of the ICTV Virus Taxonomy, the family  viruses . Their gHospitalitermes bicolor. The contig was 12323 nt long and contained five ORFs  [Hepatitis delta virus (HDV), of the genus us (HBV) . Whereasus (HBV) .In the current work, we discovered a single contig, 1810 nt in length, with significant similarities (72% identity with 25% query cover) to the previously discovered Termite HDV-like virus (THDV). The contig encoded several ORFs, and one of them showed similarities with hepatitis delta antigen sequences, with 52% identity to the closest relative THDV. Based on these data, we concluded that this contig belonged to the novel HDV-like virus and named it Cat Tien Odontotermes delta-like virus (CODLV). This contig had 123 nt direct repeats on both ends. Since it is known that deltaviruses have a circular genome, we believe that such repeat structure is an artifact of the sequencing and assembling of the circular genome. Thus, we deleted one of the repeating sequences to form a CODLV genome.Dagazvirus genus—detected in the Schedorhinotermes intermedius [Phylogenetic analysis A, based ermedius .The genome of the CODLV was 1687 nt in length B. It shoDuring the work, we also found several contigs with homologies to the viral RdRps  that didMacrotermes carbonarius, we found two contigs that were 4685 and 2470 nt in length, with homology to the Agaricus bisporus virus 9 RdRp-containing polyprotein. Agaricus bisporus virus 9 seemed to be close to the genus Deltaflexivirus. Generally, Deltaflexivirus members have a genome of about 8200 nt, with one large ORF encoding for polyprotein in the 5′end of the genome and 3–4 smaller ORFs close to the 3′end of the genome.In the pool of the Deltaflexivirus members  and encodes only one ORF. Pafsystermes virus is reported to have only one segment with one ORF . While tPolemovirus and Enamovirus (family Solemoviridae)) [Earlier, it was suggested that Wifsystermes virus’ second ORF on the second segment requires the use of a variant genetic code, in which UGA is translated as tryptophan . Here, airidae)) . NeverthH. bicolor, was clustered with Jimsystermes virus, previously identified in xylophagous Occasitermes sp., collected in Australia [Coptotermes sp., collected in Ivindo National Park (Gabon) [CHLV, detected in epiphyte-feeding ustralia . The nex (Gabon) . As all  (Gabon) , we suggPolycipiviridae, forms a monophyletic group with the genus Sopolycivirus  to provide it with envelope protein [O. wallonensis pool; however, we found several chuvirus-like contigs in the O. wallonensis. If these contigs indeed represent a virus, the existence of mixed infection of CODLV with putative chuvirus may be speculated. Nevertheless, proper studies are needed in order to unveil the biology of the HDV-like viruses. In our study, delta-like virus was found in the  protein . Novel H protein . It was  protein ,30. In oCODLV clustered with THDV in the phylogenetic trees. In contrast to HDV, additional ORFs were found in the THDV . WhereasDeltaflexivirus were found in the current study. We assumed that both of the deltaflexi-like contigs belonged to the single virus. However, our attempts to bridge the gap between deltaflexi-like contigs using PCR failed. There are several possible explanations for this. The overall presence of the virus in the pool might have been low, or these two contigs may have belonged to closely related but different deltaflexi-like viruses. It is also possible that these contigs represented endogenous virus-like elements or the virus itself was segmented.Two contigs with the homology to viral polymerases of the genus M. carbonarius (where deltaflexi-like contigs were detected) is a species that cultivates basidiomycete fungi of the genus Termitomyces  as a food source [We performed a phylogenetic analysis on the larger of the two deltaflexi-like contigs. Phylogenetically, it is close to two viruses, Agaricus bisporus virus 9 and Lentinula edodes deltaflexivirus 2, both found in edible basidiomycete mushrooms ,32. Thisd source . If thes


Poxviridae remains elusive. However, emerging sequence data on currently circulating and archived poxviruses, such as the genera orthopoxviruses and capripoxviruses, display a wide degree of divergence. This genetic variability cannot be explained by clonality or genetic drift alone, but are probably a result of significant genomic alterations, such as homologous recombination, gene loss and gain, or gene duplications as the major selection forces acting on viral progeny. The objective of this review is to cross-sectionally overview the currently available findings on natural and laboratory observations of recombination in orthopoxviruses, capripoxviruses, and leporipoxviruses, as well as the possible mechanisms involved. Overall, the reviewed available evidence allows us to conclude that the current state of knowledge is limited in terms of the relevance of genetic variations across even a genus of poxviruses as well as fundamental features governing and precipitating intrinsic gene flow and recombination events.Poxviruses are double-stranded DNA viruses with several members displaying restricted host ranges. They are genetically stable with low nucleotide mutation rates compared to other viruses, due to the poxviral high-fidelity DNA polymerase. Despite the low accumulation of mutations per replication cycle, poxvirus genomes can recombine with each other to generate genetically rearranged viruses through recombination, a process directly associated with replication and the aforementioned DNA polymerase. Orthopoxvirus replication is intimately tethered to high frequencies of homologous recombination between co-infecting viruses, duplicated sequences of the same virus, and plasmid DNA transfected into poxvirus-infected cells. Unfortunately, the effect of these genomic alterations on the cellular context for all poxviruses across the family  In contrast to DNA viruses, RNA viruses have evolved a survival strategy involving high mutation rates, large effective population sizes and short generation times to evade and counter the host immunity . In theiOrthopoxvirus, Parapoxvirus, Leporipoxvirus, and Capripoxvirus genera. Numerous outbreaks in both humans and animal populations have been attributed to these viruses in the past, with the human pathogen variola virus (VARV) being successfully eradicated in the previous century , sheeppox virus (SPPV) and goatpox virus (GTPV) as well as the myxoma virus (MYXV) from the genus Leporipoxvirus. The Capripoxviruses pose a serious threat to the global livestock industry  must be able to replicate in the absence of wild-type virus in the absence of complementation. This requires that the disrupted gene be non-essential. In a laboratory setting, this is observed when an essential gene is disrupted using homologous recombination with selection markers. In this case, an essential gene disrupted virus is generated by visualization of a fluorescent protein, however, this virus cannot be plaque purified without the function of the essential gene provided by wild-type virus or through gene complementation in vitro .It is also worth noting that if two nearly identical viruses recombine, it can be difficult, if not impossible, to identify recombinant viruses as the genome sequences will share significant sequence similarities. In this case bioinformatic tools capable of detecting recombination events need to be used, however their description is beyond the scope of this review.The previously described high recombination rates of poxviruses, allow insights into the recombination process, however these observations have predominantly focused on orthopoxviruses and this can be extrapolated to other poxviruses. Recombination in vaccinia was described through analysis of the marker gene, viral thymidine kinase, during serial passages under different growth conditions . Under sA study by Reversible gene copy number variations as a means of adaptation was also observed at another VACV locus . These sThe processes of replication and recombination in poxviruses are fundamentally linked, either through direct involvement or by utilizing the same enzymes. It has been speculated that recombination could be involved during the initiation of replication, even in the presence of a primase . RecombiAvailable evidence on orthopoxviruses suggests poxvirus replication occurs in membrane-bound virosomes in the cytoplasm of the host cell, called virus factories. Individual infecting particles generate separate factories shortly after virus infection . These sThe genes required to produce all the components involved in viral DNA replication, repair and recombination have been identified. The genomes are large double-stranded linear DNA molecules with terminal repeat regions, that are covalently closed on the ends. DNA replication requires the viral DNA-dependent-DNA-polymerase protein E9, while A20, I3, H6, D4, D5, B1, and A22 are also collectively required to support replication, repair and recombination . The simFor interviral recombination to occur, different viruses must infect the same cell. In cell culture, the likelihood of recombination occurring could increase due to an increase in the likelihood of co-infection with different viruses. Furthermore, during the selection of viruses in cell culture the most important factor is the replication fitness of recombinant viruses. This is due to the lack of an adaptive immune system for host defense in cell culture. It was demonstrated in the 1950’s that recombination can occur between two different vaccinia viruses which differed in five phenotypic characteristics; pock morphology, hemagglutinin production, heat resistance, mouse neuropathogenicity, and skin lesions in rabbits. The characterization of recombinant viruses exhibiting different phenotypes demonstrated that 17 of the 149 new viruses displayed a combination of characteristics . More reIt is likely that many different recombinant viruses could be obtained from co-infection of different lumpy skin disease viruses in cell culture. Unfortunately, the phenotypic properties of the recombinant viruses that were derived from a commercial LSDV vaccine (KEVIVAPI), and known to contain sequence elements derived from the LSDV LW-1959/vaccine and Kenyan sheep and goat pox ovine 240 (KSGPO-240) strains, and from GTPV, have not been evaluated . ExperimOngoing surveillance of circulating viruses enables not only the early detection of infection in the field, but also tracking of the genetic variants evolving under environmental pressures . Orthopo, these viruses are exposed to unusual environmental factors that could impose additional pressure on the evolutionary machinery of the viruses, providing researchers with the unique opportunities to track virus evolution on the genomic level under natural circulation  strains, using the currently available genome sequences of the capripoxvirus members , might be the best reported example of host-virus coevolution in the field, following a new cross-species introduction. There are two examples of this coevolution on two different continents, Australia and Europe.A Oryctolagus cuniculus). This accidentally initiated one of the largest natural experiments in host-pathogen coevolution. The virus strain originally released in Australia, MYXV-SLS , is believed to be derived from an isolate first obtained in Brazil in circa 1910  hosts, it has been highly pathogenic to the naive European rabbit (Oryctolagus) host, making it a classical example of a pathogen that is highly virulent in a new host species with no history of adaptation.The second example is in Europe, where the virus was introduced in 1952 as a means to control wild rabbit population, again using the Brazilian Standard laboratory strain  is the result of recombination between MYXV and Shope fibroma virus SFV; . The autLepus europaeus) and the Iberian hare (Lepus granatensis) with clinical signs suggestive of myxomatosis. Since the rabbit (Oryctolagus cuniculus) was the only target host of the myxoma virus, the observed findings strongly argued for host range expansion within the family Leporidae due to genome alterations. The virus was isolated from a dead female wild hare and designated MYXV-Tol. Analyses of the MYXV-Tol complete genome sequence revealed an insertion of ~2,800 bp in the left terminal region of the genome in comparison to the sequence of MYXV-Lau. This new recombinant region disrupted the M009L gene of MYXV-Lau, without completely deleting the gene, instead regions of the M009L gene were present and flanking the new 2,8 kbp region. Moreover, this recombinant insertion contains at least four ORFs that are predicted to encode four viral proteins homologous, but not identical, to the poxvirus gene families exemplified by the M060R, M061R, M064R, and M065R genes from MYXV. The complete genome of MYXV-Lau contains a locus at ~57,500 bp that includes a set of six ORFs (M060R to M065R) conserved in all MYXVs, including MYXV-Tol . The SNPs assigned as either identical to LSDV/KSGPO-240/Kenya/1958 (gray) or LSDV/LW-1959/Vaccine (black) are presented in The percentage of transition mutations in LSDV parental sequences was 65%, which is comparable with the 72% of SNPs identified in the VACV TP05 and DPP17 clones . Based oRecombination and the generation of novel recombinants have been associated with accumulation of various phenotypic advantages . The comThe majority of the ORFs under selection were involved in either transcription, replication or virion formation . The posRecombination plays an important role in the evolution of viruses including poxviruses. High-throughput sequencing of additional poxvirus genomes is contributing to the elucidation of poxvirus molecular evolution from the fundamental perspective. The apparent low mutation rates are offset by high recombination potential under selective conditions manifested by horizontal gene transfer, gene gain and loss and homologous recombination. The orthopoxviruses have enjoyed the majority of the research attention, since some of its members are deadly human pathogens. In contrast, other members of the Poxviridae family have only recently been gaining research focus, due to their expanded global distribution and a high impact on the economy. Owing to the advent of molecular tools, numerous studies have described novel poxviruses subjected to significant genetic changes and all of these novel viruses were associated with potentially new virulent phenotypes. The unprecedented spread of LSDV from Africa into Europe, Russia and Asia, with particular emphasis on the novel recombinant LSDVs, emphasizes our current lack of knowledge with respect to capripoxviruses. The current state of knowledge is limited in terms of the relevance of genetic variations across even a genus of poxviruses as well as fundamental features governing and precipitating intrinsic gene flow and recombination events. Understanding these mechanisms provides tools for harnessing evolution to the benefits of humankind. The increased amount of genome information available together with improvement of tools for analysis will allow for detailed understanding of the evolutionary history of poxviruses and monitoring of adaptation-driven genetic alterations in poxviral genomes in the field.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the grant no. 075-15-2021-1054 from the Ministry of Education and Science of Russia to implement objectives of the Federal Scientific and Technical Program for the Development of genetic technologies during 2019–2027.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.


Orthomyxoviridae family caused the great influenza pandemic (1918 flu or Spanish flu), the SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) outbreak, and the human immunodeficiency viruses (HIV), lentiviruses of the Retroviridae family, caused worldwide devastation. Rapidly evolving RNA virus populations are a daunting challenge for the designing of effective control measures like vaccines. Developing awareness of the evolutionary dispositions of RNA viral mutant spectra and what influences their adaptation and virulence will help curtail outbreaks of past and future pathogens.Viral quasispecies are distinct but closely related mutants formed by the disparity in viral genomes due to recombination, mutations, competition, and selection pressure. Theoretical derivation for the origin of a quasispecies is owed to the error-prone replication by polymerase and mutants of RNA replicators. Here, we briefly addressed the theoretical and mathematical origin of quasispecies and their dynamics. The impact of quasispecies for major salient human pathogens is reviewed. In the current global scenario, rapid changes in geographical landscapes favor the origin and selection of mutants. It comes as no surprise that a cauldron of mutants poses a significant risk to public health, capable of causing pandemics. Mutation rates in RNA viruses are magnitudes higher than in DNA organisms, explaining their enhanced virulence and evolvability. RNA viruses cause the most devastating pandemics; for example, members of the  Over the period of time, the term quasispecies in itself has evolved with many definitions, often interchangeably used to denote genetic variation within a population. If simplified, the quasispecies can be referred to as the diverse mutants in a population generated as a result of equilibria between mutation and natural selection ,2,3,4. REigen and Schuster, in 1970, explained the “precellular RNA world,” a mathematical framework to formulate the quasispecies theory . Not onlThough the concept of a quasispecies is taken as a single biological entity, its impact becomes more evident when the genome has high mutation rates in limited genome size (viz. RNA viruses) . WhetherFrancis Crick suggested and encouraged Manfred Eigen in a breakfast discussion to work on the first quantitative/mathematical treatment of replication systems, which undergoes a production of regular error copies ,14. ThesBased on population dynamics, quasispecies can be understood by the given diagram: Quasispecies formation depends on the mutation rates or the error rates in replication. If the virus replication rate is accurately perfect, the viral offspring will occupy the same sequence space as shown in In evolutionary terms, all the possible mutations in a gene or amino acid are represented in sequence space. Naturally occurring mutations are present in minimal functional space. So, the theoretically possible region is larger than the practically possible region. Mutations in the sequence space correlate with the expansion of the space depending on the high mutation rate and the selection of the variants  14,16].,16.14,16−4 mutations per nucleotide copied, which is greater than nearly all DNA-dependent DNA polymerases [The ability of viruses to generate and maintain genetic diversity to circumvent rapidly evolving environments and hosts is controlled by various drivers, including mutation, reassortment, and recombination. Random mutations by the RNA-dependent RNA polymerase (RdRps) viral polymerase are the major driving force for genetic diversity in RNA viruses. These low-fidelity RNA polymerases exhibit mutation rates of roughly 10ymerases ,18,19,20ymerases , measlesymerases ,23,24. Vymerases ,25,26. Rymerases . Reassorymerases . The facymerases ,30, causymerases ,32,33, aymerases ,35, and Cumulatively, mutations at the genome level correspond to the changes at the phenotypic level, which are naturally selected in the population towards increased fitness. The two types of natural selection (positive and negative/purifying selection) play an important role in the evolution of viral quasispecies. When a newly derived mutation has an advantage over the other mutations, it is termed as ‘positive selection’, whereas ‘negative selection’ removes the deleterious mutations or variants. Hence negative selection is also sometimes called purifying selection. The positive selection increases the beneficial mutations, and the deleterious mutations are exempted from the population by the negative or purifying selection. In this way, natural selection influences the formation of a new mutation or a quasispecies ,37. DuriRather than genomes acting independently, variants that populate an environment can act cooperatively or in competition, thus dynamically affecting allelic frequencies. Those mutations or its combination that are advantageous in a particular environment can be positively selected (complementing the system), becoming dominant in a mutant spectrum. In contrast, negative selection is the process by which genomes are eliminated from the mutant spectrum or fall in frequency. The result is a fine-tuned ensemble of variants with specific frequencies optimized for that particular environment. Movement into diverse territories, such as an encounter with the immune system or a virus spread to a new tissue type, results in the re-equilibration of alleles to fit that particular environment. Biological constraints are negative interactions imposed on the selection factor to control the over-expansion of the sequence space . A viralThe mutability of RNA viruses is around a million-fold higher than standard mutation rates operating in cellular DNA and is an established facet for RNA viruses. As a result, they are considered the first link between the quasispecies and virology ,15. A neThroughout history, outbreaks of emerging and re-emerging viral diseases have been a threat globally. Pandemics caused by viruses such as Influenza, MERS, Ebola, Polio, and others have been a catastrophe to mankind . A timelAdditionally, mutations in the receptor binding domain (RBD) and N-terminal domain led to an alpha, beta, gamma, delta, and omicron variant . This haParamyxoviridae consists of three genera: Paramyxovirus, which includes the parainfluenza viruses and mumps virus; Pneumovirus, which includes respiratory syncytial virus; and Morbillivirus, which includes the measles virus [The family es virus . Virionses virus . The nuces virus .Paramyxoviridae family are antigenically stable. The reason behind this spectacle can be posited as follows: The genome is non-segmented and thus cannot undergo genetic reassortment. The second reason relates to the idea of antigenic drift. Another major hypothesis behind the stability of the virus is that the mutation leads to a decrease or total loss of function, which would, in turn, cause the new virus to be less efficient. These mutant progenies struggle to survive compared to the wild strains and would eventually die out. A fundamental concept in virus research is that genomes are packaged into particles to spread in the extracellular milieu. In most viruses, individual genomes are packaged. However, the pleomorphic particles of the members of the Paramyxoviridae family often incorporate more than one genome, as deduced initially from particle sedimentation and ultraviolet inactivation studies [Due to the reasons mentioned above, one would expect the virus to be antigenically unstable. However, in contrast to instability in the RNA virus’s genome, members of the  studies , causingPneumoviruses are pleomorphic, enveloped virions ranging from 150 to 200 nm in diameter and capable of producing filaments . Fatal eOrthomyxoviridae family are known as orthomyxoviruses. These are enveloped virions that measure between 80–120 nm in diameter, with nucleocapsids forming a helical symmetry [Orthomyxoviridae family is divided into seven genera: Influenza viruses A, B, C, and D, Quaranjavirus, Thogotovirus, and Isavirus [Viruses belonging to the symmetry . These vIsavirus . InfectiIsavirus . WorldwiIsavirus . The sigIsavirus , due to Isavirus . As per Isavirus . If reasBased on the virulence potential in chickens, avian influenza is divided into two categories: low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI). Due to constant changes in the gene arrangements of influenza viruses in different species, especially in human-animal interaction, many avian influenza viruses are responsible for causing human outbreaks . H5N1 waThe two possible reasons for the influenza viruses’ evolution are antigenic drift and antigenic shift. Mutations in the HA and NA gene of influenza is responsible for antigenic drift. These mutations are ultimately responsible for different HA and NA protein combinations and cause pandemics . This anDifferent combinations of strains are present in the environment, and a pool of viral variants generated from a common species because of mutation or reassortment when undergoing competition and selection gives rise to quasispecies, as mentioned above. This is known to cause pandemics from time to time. So, it becomes challenging to predict what and when will the next combination come into existence ,71. On tThe retrovirus family is widespread and found in varieties of vertebrate hosts. These are enveloped virions that measure between 80–100 nm. The members of this family contain linear, positive sense single-stranded RNA and reverse transcriptase enzyme, which reverse transcribes its genome into DNA. The reverse transcribed DNA is integrated into the host DNA and gives rise to the provirus. If the viral DNA integration occurs into the germline tissue, then it can give rise to a heritable provirus known as an endogenous retrovirus .Retrovirus family virion contains four genes 5′-gag-pro-pol-env-3′, coding for various viral proteins. The env gene of retrovirus encodes for two types of proteins that is a surface protein (SU) and transmembrane protein (TU). The gag gene of retrovirus encodes the matrix protein (MA), capsid protein (CA), and nucleocapsid protein (NA). The pol gene of the retroviral family virion is responsible for synthesizing reverse transcriptase (RT) and integrase . The lacThe retrovirus family virion is responsible for many diseases, such as immunodeficiency (AIDS), autoimmune disease, lower motor neuron diseases, and several acute diseases, including tissue damage. Some of the retroviruses are also associated with malignancies, including lymphoma, sarcoma, and certain leukemia. Retroviruses can be transmitted via both horizontal and direct routes. The horizontal transmission includes transmission via blood, saliva, sexual contact, etc., whereas the direct routes involve the infection of the developing embryos or via milk, etc. . The HIVHIV-1 virus strains are classified into two types that are R5- tropic virus strain and X4-tropic virus strain, depending on the type of coreceptor they used to infect the target cell. Generally, R5- tropic virus strains are found in the early stage of the disease, whereas X4-tropic virus strains are predominant during the late phase of the disease ,82. A clThe capacity of replication or “fitness” is the key parameter of virion that influences HIV-1 behavior and response to selective constraints . MultiplCoronaviridae; they are classified based on their host choice; alpha and beta coronaviruses, infecting mammals, and gamma coronaviruses, infecting birds. These are the largest, positive-sense single-stranded RNA viruses, whose 31 kb genome comprises structural glycoprotein including spike protein (S), an envelope protein (E), glycoprotein (M), and nucleocapsid (N) and accessory regions (ORFs) [Coronaviruses with their signature crown-shaped capsid structure belong to the family s (ORFs) .Coronaviruses were less of a concern until 2002, when the SARS outbreak occurred . The traMERS-CoV has the largest size among all coronaviruses, i.e., ~30.11 kb, compared to SARS-CoV-2 and SARS-CoV, both of ~29 Kb. All coronaviruses mediate entry by attaching the receptor-binding domain to different host surface receptors. For SARS-CoV, angiotensin-converting enzyme 2 (ACE 2) acts as the primary receptor, but it also facilities it’s entry via CD209 and CD209L. MERS-CoV uses CD26 as an entry receptor. SARS-CoV-2 shares ACE 2 with SARS-CoV along with integrins to make an entry in the cell . HoweverSome experimental evidence has shown that the deviation from the wild-type mutation has significantly affected the viral kinetics and its infectivity. As in the case of double mutations in (A9G/R13A) in the non-structural protein 1a (nsp1) of the MERS coronavirus causes lower infectivity and smaller plaque size compared to wild-type viruses . Also, vA variant of wild-type MERS-CoV, comprising an out-of-frame deletion of 530 nucleotides in the spike glycoprotein gene, caused the loss of a large portion of the S2 subunit resulting in the production of defective particles with low infectivity. However, the mutation did provide aid to the wild-type MERS-CoV infection by producing a misfolded S1 protein, which acted as a trap toward the spike-specific neutralizing antibodies .Another study analyzing non-consensus sequences from 24 patients infected with MERS-CoV revealed high-level heterogeneity among samples . After aAnalysis of quasispecies in SARS-CoV-1 from 9 patients showed nine variable sites among 107 variations . The quaPicornaviridae (Polio), Togaviridae (Chikungunya), Flaviviridae (Hepatitis C virus), and Filoviridae (Ebola) have imposed significant mortality in public. The evidence of quasispecies formation in some of the important RNA viruses is reviewed in the following section.Concerns related to global threats due to emerging and remerging viruses are increasing day by day. Apart from the families discussed above, some other families like Picornaviridae family; the Hepatitis C virus (HCV), a single-stranded enveloped RNA virus belonging to Flaviviridae; the Hepatitis D virus (HDV), single-stranded circular RNA virus belonging to Hepadna viruses, and Hepatitis E virus (HEV), another non-enveloped single-stranded RNA virus belonging to Caliciviruses [Flaviviridae family, like Dengue fever virus, Japanese Encephalitis virus, and Classical swine fever virus. All these members have enveloped virions containing positive sense, single-stranded RNA genomes of 9.6 to 12.3 kb. Encoding for structural and non-structural proteins. HCV has a gene NS5B for the RNA-dependent RNA polymerase that lacks proofreading activity. It incorporates nucleotides with a high error rate of 10−3 errors/site, which leads to heterogeneity in the gnome. Another factor responsible for quasispecies formation is the hypervariable region (HVR) in the envelope protein (E2). HVR-1 is located at the N-terminal of E2, while HVR-2 is slightly downstream of HVR-1. HVR-1 acts as a dominant epitope for neutralizing antibodies. Mutations in the HVR-1 are responsible for the formation of escape mutants under host immune selection pressure. HVR-1 and HVR-2 variations are responsible for virus tropism, virulence, and resistance against drugs. During the acute phase of infection, HCV has a higher rate of mismatched substitution per site. One case study suggests that the HVR-1 does not display neutralizing epitopes during infection. The existence of various closely related mutants during the acute phase and their adaptation leads to chronic infection [This class includes a range of unrelated human pathogens like the Hepatitis A virus (HAV), an unenveloped RNA virus belonging to the iviruses . Among tnfection . Other mnfection .Filoviridae family, which includes two other genera: Cuevavirus and Marburgvirus [filoviridae because, in Latin, ‘filum’ means ‘thread’ [The Ebola virus belongs to the urgvirus . When vi‘thread’ . It is a‘thread’ . This ac‘thread’ ,117,118.Picornaviridae family, causes poliomyelitis. It is a non-enveloped, positive-sense single-stranded RNA (+ssRNA) virus [Poliovirus, which belongs to the A) virus . The virA) virus . AnotherA) virus . SequencTogaviridae, is an arthropod-borne virus (arboviruses) that causes high morbidity in humans; it infects humans via mosquitos [Chikungunya (CHIK), which belongs to the family osquitos . It has osquitos . Within osquitos . Both hiosquitos . Small pTill now, we have discussed and analyzed how specific mutations occur in the viral genome and how their selection is influenced by environmental and host-pathogen interactions, which could affect the replication rate in the host. Understanding replication rate, plaque morphology, and sequence analysis could shed light on the intricate phenomenon of quasispecies selection and might provide evidence for resolving obstacles to developing therapies in controlling current and future pandemics.Viral quasispecies under selection pressure led to the formation of escape mutants; these low-frequency strains escape immune targeting and proliferate in the host system. This is the biggest challenge in developing vaccines against RNA viruses ,125,126.In the case of influenza, mutants have shown resistance against administered drugs Amantadine and Rimantadine . Many muRNA interference (RNAi) systems are a promising alternative to antiviral drugs. Synthetic miRNA (siRNA) targeting specific viral sequences  leads toAnother important treatment is lethal mutagenesis, which also holds some promising results. Administration of mutagenic pyrimidine analog in AIDS patients resulted in lethal mutagenesis . When a Even a mutation in a single amino acid could give rise to enough interference to convert a genome into an interfering genome (the reverse is true as well), although the probability of mutation in the sequence space is random. Accordingly, this can be induced in-vitro by increasing the viral load of defective interfering particles in the system ,140. ThiConsequently, we can address the challenge that arose due to the quasispecies selection of the dominant variant by sequential or combination treatment involving alternative inhibitors and mutagenic agents in clinical trials . There aIn experimental virology, the physiochemical milieu affects the frequency of mutants and their nature, while in-vivo selection and biological constraint interfere with the quasispecies formation . Selecti


Phlebovirus named Ntepes virus (NTPV) was recently described and found to infect humans from a wide geographic area in Kenya. However, the entomologic risk factors of this virus such as the potential vectors and the transmission cycles remain poorly defined. This study assessed the ability of the colonized sandfly Phlebotomus duboscqi to transmit NTPV and determined the bloodmeal host sources of field-collected sandflies from the area where NTPV was found in Baringo County, Kenya. Five-day old laboratory-reared P. duboscqi were orally challenged with an infectious dose of NTPV (≈106.0 pfu/ml) and incubated for up to 15 days postinfection. Individual sandflies were dissected into abdomens, legs, and salivary glands and screened for the virus infection by cell culture. Of the 205 virus-exposed sandflies, 19.5% developed non-disseminated infections in the midgut, with no evidence of virus dissemination or transmission in legs and salivary glands, respectively. The midgut infection rates decreased with increasing extrinsic incubation period . Blood-fed specimens analyzed by polymerase chain reaction (PCR) and sequencing of a region of the mitochondrial 12S rRNA, revealed almost exclusive feeding on humans (98%) represented by the sandflies Sergentomyia schwetzi, S. clydei, S. antennata, S. squamipleuris, S. africana, and Phlebotomus martini. One specimen of S. clydei had fed on cattle (2%). These findings suggest P. duboscqi is an incompetent laboratory vector of NTPV. The high human-feeding rate by diverse sandfly species increases the likelihood of human exposure to pathogens associated with these sandflies. Assessment of the susceptibility of Sergentomyia species to NTPV is recommended given their high human-feeding tendency.Phleboviruses transmitted by sandflies are among emerging public health threats. A novel  Phlebovirus infection in humans cause self-limiting acute febrile illness (AFI) collectively known as sandfly fever and infections of the nervous system [Sandfly-associated s system –3. In sus system , 4. In Ks system , 6. In tNtepes phlebovirus species within the genus Phlebovirus, family Phenuiviridae. Serological analysis showed evidence of human infection based on the detection of neutralizing antibodies against NTPV in human serum samples. The virus appears to have a wide distribution in Kenya based on human seroprevalence data in areas beyond its isolation focus. Ntepes virus has repeatedly been detected in sandflies from the ecology where it was first isolated [Sergentomyia have been implicated in the circulation of NTPV based on its detection in naturally infected sandfly pools [Ntepes virus (NTPV) has been isolated from sandflies in 2014 and 2016 in Baringo County, Kenya , 6 and eisolated  indicatily pools , 6. HowePhlebotomus have largely been associated with the transmission of sandfly fever viruses in the Old World and Mediterranean region [Phlebotomus papatasi, P. perfiliwei, andP. pernicious [P. papatasi, P. duboscqi, P. sergenti, and Lutzomyia longipalpis as competent vectors of the mosquito-borne Phlebovirus Rift Valley fever virus [Sergentomyia species can harbor phleboviruses in nature [Sandflies in the genus n region , 5–9 incrnicious . Experimer virus –12. Few Phlebotomus papatasi of the subgenus Phlebotomus rondani is the major incriminated vector of sandfly fever viruses in sub-Saharan Africa [Phlebotomus duboscqi is the only representative of this subgenus in the NTPV focus in Baringo, Kenya [P. duboscqi in the transmission of NTPV could have ramifications for possible epidemiological relationships between NTPV and leishmaniasis in the NTPV foci as has been reported for leishmaniasis and phleboviruses transmitted by Phlebotomus vectors [n Africa . Phleboto, Kenya , and a po, Kenya , 16. A co, Kenya , 18 and  vectors , 20.The transmission of arboviruses in a given ecology is governed by the intricate interactions among the pathogen, competent arthropod vector, and a susceptible vertebrate host in a conducive environment , 22. VecP. duboscqi to transmit NTPV after oral infection through artificial membrane feeding. We also analyzed the blood-host feeding patterns of feral sandfly species in the NTPV foci area to evaluate the potential risk of human exposure to viruses they might transmit.In this study, we sought to understand the entomological risk factors of the transmission of NTPV by investigating the capacity of laboratory colonized P. duboscqi used for the vector competence experiment were obtained from a preestablished colony (in 2010) at the vector biology insectary of the Kenya Medical Research Institute (KEMRI), Nairobi, Kenya. The sandflies were reared on apple slices for sugar sources ad libitum and mouse blood provided thrice a week as anesthetized mice in polycarbonate cages at 26°C and 80% relative humidity with a photoperiod of 12 h:12 h. Archived blood-fed field-collected sandfly specimens preserved at -80°C at the International Centre of Insect Physiology and Ecology (icipe) Duduville Campus, Nairobi, were used for bloodmeal source analysis. The samples were surveyed in December 2018 and January 2020 in Rabai, Baringo County, Kenya; the area where NTPV was isolated [The adult female isolated .2 until the cells attained an 80%-90% confluence on the culture flask. Two hundred microliters (200 μl) of the stock NTPV were inoculated onto the confluent monolayer of Vero cells and then incubated for 1 h with periodic gentle agitation for virus adsorption onto the cells. The infected Vero cells were then maintained for up to 14 days in MEM augmented with 2% heat-inactivated FBS, 2% antibiotic/antimycotic solution, and 2% L-glutamate and observed for cytopathic effects (CPE) daily. Once 80% of the monolayer appeared to show CPE, the flask was frozen overnight at -80°C, thereafter, thawed on wet ice and clarified by centrifugation at 3000 rpm for 10 min. The resulting supernatant was harvested and aliquoted in 1.5 ml cryovials and stored at -80°C for further use.Cryopreserved stock of NTPV (at icipe) isolated from a pool of sandflies in Marigat, Baringo, Kenya in 2014  was usedμl of serial 10-fold dilutions of the harvested virus onto each well of a confluent monolayer of Vero cells in the 12-well culture plate that had been seeded with a growth medium 24 h before virus inoculation. The inoculated virus was incubated for 1 h with periodic agitation for virus adsorption. The NTPV-inoculated cells were maintained on 2% methylcellulose overlay mixed with 2× MEM  and incubated at 37°C with 5% CO2 for 10 days, then fixed for 1 h with 10% formalin and stained for 2 h with 0.5% crystal violet. The plaques were counted and the virus titer was quantified using the formula described by Brady et al. [The amplified virus was quantified by plaque assay by inoculating 100 y et al. ;(1)PFU=P. duboscqi in three replicates were offered an infectious bloodmeal containing 104.3 to 106.6 pfu/ml of NTPV , consisting of MEM, supplemented with 15% FBS, 2% L-Glutamine, and 2% antibiotic/antimycotic solution. Legs and salivary glands of individual sandflies were placed in separate 1.5 ml microcentrifuge tubes containing 300 μl and 200 μl of HM, respectively. The samples were immediately frozen at -80°C until assayed for virus recovery by cell culture.On days 6, 10, and 15 postinfection, approximately 30% of the virus-exposed sandflies from each replicate were analyzed for the virus' presence. Individual sandflies were dissected in a cold chain (wet ice) to separate the legs and the salivary glands from the body containing the midgut. Individual bodies were placed separately in 1.5 ml microcentrifuge tubes containing 500 μl of the supernatant of the clarified body samples onto 12-well plates of confluent Vero cells. The infected cells were incubated at 37°C for 1 h with frequent agitation for virus adsorption. The infected cells were maintained for up to 15 days on MEM as described earlier for virus amplification. For every positive body sample, their corresponding legs were screened for virus infection as evidence of dissemination, as described above for midgut infection in the body. Also, for every positive leg sample, the corresponding salivary glands were screened to ascertain virus transmission, as described above.Individually dissected samples were homogenized using TissueLyserII  at 25 Hz for 3 min with an aid of sterile copper beads. The homogenates were clarified by centrifugation at 12,000 rpm for 10 min at 4°C. For oral susceptibility assessment, evidence of midgut infection was tested by inoculating 100 μl phosphate buffer saline (PBS) with the aid of sterile copper beads using the TissueLyserII  at 25 Hz for 3 min. The homogenates were clarified by centrifugation, and the DNA was extracted using ISOLATE II Genomic DNA Kit  according to the manufacturer's instructions.Engorged sandflies were morphologically identified by dissection and examination of the head and the posterior parts as proposed by Abonnenc and Minter ; detailsμl reaction volume containing 4 μl 1× Mytaq reaction buffer, 2 units of Mytaq DNA Polymerase (Meridian bioscience), 0.5 μM concentration of both 12S-F GGGATTAGATACCCCACTATGC and 12S-R TGCTTACCATGTTACGACTT primers [μl of genomic DNA template (~20 ng). The thermal cycling conditions used were as follows: initial denaturation was done at 95°C for 3 min, followed by 40 cycles of denaturation at 95o for 20 s, annealing at 59°C for 30 s, and extension at 72°C for 30 s followed by a final extension at 72°C for 7 min. The PCR was carried out using a 96-well, 0.2 ml, 3-zoned VeriFlex Block SimplAmp Thermal Cycler . The amplicons were resolved on 1.5% agarose gel electrophoresis stained with ethidium bromide against a 100 bp DNA ladder . The PCR products of amplified samples were purified using the SureClean Plus kit  and Sanger-sequenced using the forward primer only by a commercial firm . Sequences obtained were edited using MEGA v.10 [http://www.ncbi.nlm.nih.gov/blast) search tool. Samples were assigned to bloodmeal host species when sequences had ≥98% identity [A polymerase chain reaction of a 500 bp region of the 12S mitochondrial rRNA was performed in a 20  primers , and 2 μEGA v.10  and queridentity .http://www.nacosti.go.ke) is mandated to accredit research institutions and approve all scientific research in Kenya. The current study complied with NACOSTI guidelines.The study was approved by the Kenya Medical Research Institute Scientific Ethics Review Unit (KEMRI-SERU) (SERU protocol number 3312) and National Commission for Science, Technology and Innovation (NACOSTI) (permit number: NACOSTI/P/22/15808). NACOSTI  of the orally exposed sandflies successfully obtained the viremic bloodmeal . The titρ = 0.61). Only midgut infections were observed in the exposed sandflies, and the rates were 10.7%, 5.4%, and 3.4% on 6-, 10-, and 15-days postinfection, respectively (ρ = −0.71) and regression line equation (y = 15.7 − 0.845x))  survived to the 15 days postinfection and were all dissected or tested . Spearmaectively . The oveectively . That is0.845x)) .S. schwetzi (n = 22), S. clydei (n = 9), S. squamipleuris (n = 6), S. antennata (n = 6), S. africana (n = 2), and Phlebotomus martini (n = 3). Of these, data were successfully generated from 42 specimens (87.5%). The results show that humans and cattle are the most likely only bloodmeal host sources identified by PCR and sequencing. Most of the blood-fed sandflies had fed on humans  and only 2% on cattle by a sample of S. clydei  where about 50% of those orally exposed developed infections in the midgut [L. longipalpis to RVFV, both the virus titers, and the number of infected sandflies reduced with increasing EIP [We report a low level of oral susceptibility  of eriments . Howevere midgut , 12. Thee midgut . Residuasing EIP .Phlebotomus duboscqi might have a strong midgut escape barrier to NTPV likely limiting the possibility of disseminated infection across the midgut. In fact, NTPV failed to disseminate after midgut infections in the fly. The result is consistent with previous findings that reported nondisseminated midgut infections of RVFV in L. longipalpis at comparable or higher ingested titers (104-107.2 pfu/ml) even though 60% of the sandflies were susceptible [Phlebotomus duboscqi likely possesses a strong midgut escape barrier to NTPV potentially conferred by the genetics of the vector [P. pernicious with Toscana virus [P. duboscqi orally infected with RVFV with only 6 of the 145 exposed flies transmitting the infection [P. duboscqi.e vector . Alternana virus . Very lonfection  despite P. duboscqi had no virus infection in the salivary glands reminiscent of the case of RVFV in this same sandfly species with a comparable ingested virus titer [P. duboscqi could equally possess a strong salivary gland infection barrier to NTPV, although this remains to be verified. The dissemination of a virus from the midgut after overcoming the midgut escape barrier is suggested to be enough to guarantee vector competence of P. duboscqi to viruses [in vitro vector competence experiments may poorly represent the natural transmission of a virus. The NTPV dose ingested from a host during natural feeding is not unknown. However, the titers used in this study closely resemble those of RVFV used to assess P. duboscqi vector competence [For a competent vector, the ingested virus infects and replicates in the midgut to higher titers and disseminates progressively to the salivary glands of the vector within the EIP. Sandfly-borne phleboviruses have an EIP of about 7 days after which the sandflies remain infective throughout their lifespan . P. dubous titer . P. dubo viruses . In supp viruses  and alwa viruses , 12. Notmpetence –37. ThisS. schwetzi was predominant among the blood-fed specimens likely reflecting its abundance in the sandfly fauna of this ecology [Sergentomyia species with human feeding [Sergentomyia species (e.g. S. schwetzi) based on DNA barcoding of sandflies in the infected pool [P. martini analyzed all had human-derived bloodmeals agreeing with previous literature regarding its feeding habit [P. duboscqi was represented among captured blood-fed samples perhaps attributed to low fly occurrence at the time of sampling [Phlebotomus species [P. martini in the transmission of the novel phleboviruses including use of recently colonized populations of P. duboscqi. Sandflies exhibiting high feeding on humans have been incriminated as vectors of sandfly-borne pathogens  affecting humans in the same habitat [Sergentomyia species and their detection in positive pools of novel phleboviruses detected in this focus [In this study, a high human feeding rate was observed among several sandfly species collected from Baringo, where NTPV was initially detected. The result is consistent with previous reports describing >90% human feeding rates by feral sandflies in the same ecology , 6. Howe ecology . Despite feeding , 6, they feeding . NTPV wated pool , 6. The ng habit , 39. No sampling . Since t species , further habitat , 40. Theis focus , 6 highlP. duboscqi is an inefficient vector of NTPV, and that several Sergentomyia species show a strong feeding preference to humans as blood-meal sources in Baringo County, Kenya. We recommend the establishment of different sandfly species colonies to allow further studies to examine the role of Sergentomyia species and P. martini in the transmission of NTPV and other novel phleboviruses detected in this study and other arbovirus foci of Kenya.This study demonstrated that


P < 0.001), weekly total steps (P < 0.001), and worse school functioning (P = 0.0211) than control teenagers. No significant difference was found in anthropometric measures and body image between the two groups (P > 0.05). This study implied the needs for teenagers with nonstrabismic amblyopia to enhance physical activities. Teachers and parents are encouraged to pay more attention to teenagers with nonstrabismic amblyopia to improve their school functioning.The aim of this study was to explore the influences of nonstrabismic amblyopia on quality of life in adolescents. Health-related quality of life (HRQoL) scale, a multidimensional construct that indicates a fundamental health outcome, was used to measure physical and psychosocial functioning of the adolescents. Forty teenagers with nonstrabismic amblyopia and 40 control teenagers without nonstrabismic amblyopia were recruited between April 2019 and July 2021. The anthropometric measures, body image, physical activity outcome, and HRQoL scores including physical health, emotional functioning, social functioning, and school functioning were compared between the two groups. The results revealed that teenagers with nonstrabismic amblyopia had less weekly sedentary time ( Amblyopia is a neurodevelopmental disorder, affecting up to 5% of the general population . For patTo assess the physical and social functioning, well-being, and mental health of children and adolescents, health-related quality of life (HRQoL) has aroused much interest . CertainConsidering the importance of studying HRQoL, we performed a study to reveal the effects of nonstrabismic amblyopia on HRQoL in adolescents.The study was conducted following the Declaration of Helsinki and was approved by the ethnical committee of Northern Jiangsu People's Hospital. Participants were recruited by department of ophthalmology in Northern Jiangsu People's Hospital. Written informed consent was gained from a parent or legal guardian before testing their child and after explanation of the nature and possible consequences of the study. Persons who visited the hospital underwent a complete eye examination including ocular motility test, slit lamp examination, refraction, cover-uncover test, and dilated retinal evaluation. The inclusion criteria were set as follows: (1) aged between 12 and 18 and (2) diagnosed with amblyopia , (2) witThe Pediatric Quality of Life Inventory (PedsQL) , recommeParents were invited to identify the most similar image with body of their children among various silhouettes to evaluate the parent's body image perception about their children . There a2, which was used to assess the weight status of each participant according to Cole cut-off values by sex and age [BMI was calculated as weight/height and age . The uni and age , 25.Sedentary time was monitored using ActiGraph accelerometry monitors  and was analyzed using the ActiLife 6.13.3 software (ActiGraph) . AccelerN (%) or mean ± SD. All analyses were carried out using SPSS, version 22 . Chi-square test and unpaired t test were applied. The significance threshold was set as P ≤ 0.05.Data are expressed as P > 0.05).General information including age, gender, and parents' education of teenagers in the healthy control and nonstrabismic amblyopia groups was collected. As revealed in P > 0.05, Teenagers in the healthy control and nonstrabismic amblyopia groups had no significant differences in BMI and WHTR . However, the nonstrabismic amblyopia group showed longer weekly sedentary time and less weekly total steps than the control group . However, the nonstrabismic amblyopia group showed less school functioning scores than the control group . However, the nonstrabismic amblyopia group showed less school functioning scores than the control group (P = 0.0017).As revealed in P > 0.05). In addition, body image is a multifaceted mental representation of our bodies and their emotional experiences that is constantly updated. During adolescence, due to the constant physical and cognitive changes occurring, body image concerns occur frequently, thus increasing the evaluation and attention to the body and appearance. Sociocultural influences and social comparisons internalize the ideal of beauty, which leads to negative body image and dissatisfaction with appearance [P > 0.05).The subtypes of amblyopia (strabismic and nonstrabismic amblyopia) are not only disturbance of the development of the visual system at different points but also primarily different pathologic processes . Some prpearance . TherefoP = 0.0211), which might be associated with the vision loss in leaning period. Besides school functioning, physical health, emotional functioning, social functioning are also important for adolescents. It was reported that the nonamblyopia group had significantly better motor competence on the physical activity than either the corrected amblyopia group or the noncorrected amblyopia group [P > 0.05).Amblyopia is associated with academic scores in mathematics, reading, social science, and science . Consistia group . Moreoveia group . Previouia group . HoweverP < 0.001). Considering that adolescents with nonstrabismic amblyopia also had lower school functioning in HRQoL (P = 0.0211), the positive association of physical activity and HRQoL in nonstrabismic amblyopia was identified.Moderate physical activity has positive association with high HRQoL  in children , 39. AccP > 0.05), which is similar to a previous study [P = 0.0017), while there are no differences in physical health, emotional functioning, and social functioning (P > 0.05).Moreover, HRQoL perceptions between children or adolescents and their parents are likely to be different with increasing age because the child may have a more complicated and distinct understanding of the world instead of simply accepting opinions from parents. Thus, Tsiros et al. made suggestions to attach importance to the parent's perceived HRQoL . Williamus study . Our stuThere are also some limitations in our study. First, participants were recruited from a small geographical area. Further studies are needed to determine whether the identified HRQoL is present nationwide. Moreover, many of the effects of amblyopia and/or its treatment on HRQoL are experienced by the children; however, parents' opinions and perspectives on treatment may directly or indirectly influence children's beliefs.After controlling for gender, age, and education of parents, there is a significant difference in physical activities and social functioning between adolescents with or without nonstrabismic amblyopia, which highlights the needs for teenagers with nonstrabismic amblyopia to increase physical activities for improving physical health. Teachers and parents should pay more attention to teenagers with nonstrabismic amblyopia to enhance their school functioning.


Polydnaviriformidae (former Polydnaviridae) have thus far been found exclusively in the genomes of braconid and ichneumonid parasitoid wasps, whereas the completely unrelated gene transfer agents (GTAs) are widely distributed among prokaryotes. In addition, recent discoveries likely extend viriforms to mammalian genomes. Here, we briefly outline the properties of these viriform groups and the first accepted and proposed ICTV frameworks for viriform classification.The International Committee on Taxonomy of Viruses (ICTV) recently accepted viriforms as a new polyphyletic category of classifiable virus-derived genetic elements, juxtaposed to the polyphyletic virus, viroid, and satellite nucleic acid categories. Viriforms are endogenized former viruses that have been exapted by their cellular hosts to fulfill functions important for the host’s life cycle. While morphologically resembling virions, particles made by viriforms do not package the viriform genomes but instead transport host genetic material. Known viriforms are highly diverse: members of family  In 2021, Koonin et al. outlined a definition of the term “virus” and assigned all mobile genetic elements (MGEs) that fit that definition sensu stricto into the orthovirosphere, and virus-like MGEs that possess some but not all distinctive features of viruses into the perivirosphere  and subs“…a type of MGEs that encode at least one protein that is a major component of the virion encasing the nucleic acid of the respective MGE and therefore the gene encoding the major virion protein itself; or MGEs that are clearly demonstrable to be members of a line of evolutionary descent of such major virion protein-encoding entities” (ICVCN Rule 3.3) .Polydnaviridae; conversely, MGEs not considered viruses but classified by the ICTV into separate categories (viroids and satellite nucleic acids) also kept their statuses, as they did not fit the new virus definition. This result enabled the formal definition of viroids and satellite nucleic acids for the ICVCN:This definition was created on operational/practical, rather than philosophical grounds; tobacco mosaic virus, the first virus described ,6, was ccis-acting hammerhead ribozymes; or MGEs that are derived from a viroid in the course of evolution” (ICVCN Rule 3.3);“Viroids are defined operationally by the ICTV as a type of MGEs that are uncoated, small, circular, single-stranded RNAs that do not encode proteins and do not depend on viruses for transmission, and that replicate autonomously through an RNA-RNA rolling-circle mechanism mediated by host enzymes and, in some cases, by “Satellite nucleic acids are defined operationally by the ICTV as a type of non-viroid MGEs, which are dependent on viruses for replication and transmission; or MGEs that are derived from such entities in the course of evolution. (ICVCN Rule 3.3)” ,3,4.Polydnavirids, however, could not be added to these two categories. Because they did not fit within any of the definitions, a novel ICTV classification category was deemed to be required.Polydnaviridae, including a single genus Polydnavirus, was established by the ICTV in 1984 for a group of virus-like entities related to Campoletis sonorensis virus (CsV) ).The first GTA, now known as Rhodobacter capsulatus gene transfer agent (RcGTA), was described in 1974 as an uncharacterized agent facilitating “genetic exchange” among apsulata  …are considered to be viriforms in classification and nomenclature” .gtaviriformidae”) for the thus-far best-characterized GTAs [Subsequently, Kogay et al. outlined and officially proposed a taxonomic framework for GTAs in 2022 ,32. Thiszed GTAs ,36,37,38Spirochaetota, but also in bacteria of the phylum Thermodesulfobacteriota and even in euryarchaea [Caudoviricetes, such as the recently described candidate class “Mirusviricota” [Varidnaviricota infecting prokaryotes (“tailless phages”).However, similar to the current taxonomy of bracoviriforms and ichnoviriforms, the new framework for GTAs can only be seen as a first step in the systematic classification of prokaryotic virifoms. First, although all GTAs listed in the table are derived from caudoviricete ancestors, the viriforms of the three proposed families are not directly related to each other . Second,yarchaea ,44,45,46yarchaea . Fourth,iricota” , and/or “Viriforms” is an umbrella term for a variety of unrelated virus-derived genetic elements that is envisioned to be used similarly to the terms “viruses”, “viroids”, and “satellite nucleic acids”. All of these terms denote a range of polyphyletic entities and hence do not imply common evolutionary origins but rather reflect particular “lifestyles” of their members and are therefore of practical use.The GTA-type viriforms resemble, and superficially could be confused with, caudoviricete prophages (a form of EVE), and likewise, bracoviriforms resemble nudivirid EVEs. However, whereas prophages are dormant (latent) viruses that can be reactivated , GTA-type viriforms are complex virus-like systems that have, however, been fully domesticated. They do not incorporate full sets of genes encoding GTA components and therefore never complete an infectious cycle, thereby never producing progeny particles. Furthermore, GTAs evolved small particle heads that can only incorporate segments of host DNA about 5 kilobases in length, much shorter than a typical caudoviricete genome. Similarly, EVEs are only fragments of endogenized virus nucleic acids that may or may not be functional, whereas bracoviriforms and ichnoviriforms maintain most viral lifecycle features but do not incorporate (most of the) genes for the components of their particles that consequently remain non-infectious.The similarity of EVEs and viriforms implies that many viriforms might have been overlooked. A particularly interesting question is whether there are viriforms in animals beyond hymenopteran insects. The genomes of many animals, in particular vertebrates including mammals, are replete with retrovirid EVEs . Strikin