Artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are highly specific 21-nucleotide small RNAs designed to recognize and silence complementary target RNAs. Art-sRNAs are extensively used in gene function studies or for improving crops, particularly to protect plants against viruses. Typically, antiviral art-sRNAs are computationally designed to target one or multiple sites in viral RNAs with high specificity, and art-sRNA constructs are generated and introduced into plants that are subsequently challenged with the target virus(es). Numerous studies have reported the successful application of art-sRNAs to induce resistance against a large number of RNA and DNA viruses in model and crop species. However, the application of art-sRNAs as an antiviral tool has limitations, such as the difficulty to predict the efficacy of a particular art-sRNA or the emergence of virus variants with mutated target sites escaping to art-sRNA-mediated degradation. Here, we review the different classes, features, and uses of art-sRNA-based tools to induce antiviral resistance in plants. We also provide strategies for the rational design of antiviral art-sRNAs and discuss the latest advances in developing art-sRNA-based methodologies for enhanced resistance to plant viruses. Caenorabtitis elegans [RNA interference (RNAi) is a biological process conserved in most eukaryotes and characterized by the sequence-specific degradation of target RNA by complementary small RNAs (sRNAs) . RNAi pa elegans  fueled tIn plants, RNAi tools have been extensively used to confer antiviral resistance. Early RNAi approaches, such as virus-induced gene silencing (VIGS) and hairpin (hp)-based silencing, consisted of the expression of dsRNA or hpRNA precursors, respectively, bearing sequences of the target virus (for a recent review see ). AlthouHere, we describe the different classes, features, and uses of art-sRNA-based RNAi (art-sRNAi) tools to target viral RNAs and induce antiviral resistance in plants. We also provide basic rules for art-sRNA design and describe which viral regions are typically targeted. Finally, we discuss the latest art-sRNAi strategies providing enhanced antiviral resistance, such as the high-throughput identification of highly effective antiviral art-sRNAs and their simultaneous co-expression for the multi-targeting of viral RNAs.Artificial microRNAs (amiRNAs) and artificial/synthetic trans-acting small interfering RNAs  are the two main classes of plant art-sRNAs . DespiteMIRNA precursor in which the endogenous miRNA guide/miRNA star sequences are substituted by the amiRNA guide/amiRNA star sequences  and Turnip mosaic virus (TuMV), in transgenic Arabidopsis thaliana (Arabidopsis) plants expressing one or two amiRNA transgenes, respectively [MIRNA precursors in multiple configurations have been designed to target viral RNAs corresponding to VSRs and other key viral proteins  and three syn-tasiRNAs against TuMV from a single TAS3a precursor [TAS1c precursors were used to express five and six syn-tasiRNAs, respectively, against Potato spindle tuber viroid (PSTVd) in Nicotiana benthamiana [Tomato spotted wilt virus (TSWV) in N. benthamiana [Solanum lycopersicum (tomato) [S. lycopersicum reported that syn-tasiRNAs induced higher antiviral resistance most likely because of the simultaneous targeting of multiple viral RNAs [Syn-tasiRNAs were first used to confer multiple virus resistance in transgenic Arabidopsis co-expressing three syn-tasiRNAs against recursor . More rethamiana  and Tomathamiana  and Sola(tomato)  . However, other factors, such as target site accessibility and stability, are much difficult to predict. For instance, target site accessibility may be limited in those sites with high secondary structures or occupied by an RNA-binding protein. Moreover, upon infection some target sites in viral RNAs may accumulate nucleotide substitutions compatible with viral replication but affecting art-sRNA binding and activity. It is also possible that VSRs interfere with art-sRNA biogenesis or action. For all these reasons, it is difficult to predict if a particular art-sRNA will be effective in vivo. However, we provide next a series of basic rules for art-sRNA design. We also describe the diverse viral regions that have been targeted with art-sRNAs and discuss the strategy of targeting conserved nucleotide sequences in viral RNAs.Web MicroRNA Designer 3) [Plant Small RNAMaker Suite) [The two main webtools for the automatized design of art-sRNAs are WMD3 (from igner 3)  and P-SAr Suite) , which wr Suite) ,24. For Tomato leaf curl New Delhi virus (ToLCNDV), HC-Pro of Potato virus Y (PVY), TuMV and Wheat streak mosaic virus (WSMV), NSs of Tomato spotted wild virus (TSWV), P19 of Tomato bush stunt virus (TBSV), p25 of Potato virus X (PVX), P69 of TYMV, Rep and RepA of Wheat dwarf virus (WDV), and V2 of Cotton leaf curl Burewala virus (CLCuBuV) (Cymbidium mosaic virus (CYmMV), Green mottle mosaic virus (CGMMV), Odontoglossum ringspot virus (ORSV), TSWV, Water silver mottle virus (WSMoV), and WDV, or of coat protein (CP) RNAs of Cucumber green mottle mosaic virus (CGMMV), Grapevine fanleaf virus (GFLV), Plum pox virus (PPV), PVY, Rice black streaked dwarf virus (RBSDV), Rice stripe virus (RSV), Tobacco etch virus (TEV), TuMV, and Ugandan cassava brown streak virus (UCBSV)  . VSR RNACLCuBuV) . Other s (UCBSV) . Target N. benthamiana [When selecting the region(s) of the virus to be targeted, a frequent strategy has been the identification of regions with conserved nucleotide sequences. In principle, the targeting of such conserved regions should minimize the possibility of emergence of escape mutants and/or allow the multitargeting of different virus isolates or species. For instance, an amiRNA targeting conserved sequences in the 3′ end of TuMV CP cistron induced high levels of antiviral resistance when stably expressed in Arabidopsis, and no virus variants with mutated target sites were observed . In a dithamiana . Interesthamiana . These mthamiana . Hence, Important improvements in antiviral art-sRNAi methodologies have been reported lately and are presented next. We also discuss the possibility of further improving art-sRNAi based on recent findings in the biogenesis and mode of action of plant miRNAs.BsaI/ccdB “B/c” vectors [N. benthamiana plants, which are subsequently inoculated with the virus of interest. The antiviral activity of each amiRNA construct is assessed by monitoring viral symptom appearance, and through molecular analysis of virus accumulation in plant tissues. This methodology was successfully applied to identify highly effective amiRNAs against RNAs of PSTVd [N. benthamiana. Other amiRNA screening systems, such as ETPamiR screenings in plant protoplasts [As explained above, the antiviral efficacy of a particular art-sRNA is difficult to predict a priori. Thus, systems for the rapid screening of the antiviral activity of large numbers of art-sRNAs are necessary for the identification of effective art-sRNAs prior to the time-consuming generation of stably transformed plants. Recently, a systematic and high-throughput methodology for the simple and fast-forward design, generation, and functional analysis of large numbers of art-sRNA constructs has been described . Briefly vectors , a new g vectors . Each amof PSTVd  and TSWVof PSTVd  in N. betoplasts ,51, haveN. tabacum BY-2 protoplasts expressing TBSV dsRNAs and selected AGO members [N. benthamiana plants by agroinfiltrating each sRNA as an amiRNA, and subsequently inoculating TBSV in the same leaf-sites. Results showed that the functionality of esiRNAs mainly depended on the binding affinity to AGO proteins and the ability to target RNA [Another possibility is to express as art-sRNAs those immunologically effective small interfering RNAs (esiRNAs) that are generated by DCLs from viral dsRNAs produced during viral replication . Because members . First, rget RNA . Thus, tMIRNA precursor and a short hairpin RNA (shRNA) precursor, respectively, in tandem [In addition to the pioneering work by Niu and colleagues , the in n tandem  , TEV, or Pepper mild mottle virus (PMMoV) infection by mechanically co-inoculating N. tabacum leaves with naked dsRNAs of virus sequence and their corresponding target virus [N. tabacum [An alternative to the transgenic expression of antiviral RNAs is their topical delivery into plants. This approach was first used to interfere with et virus . Later, et virus . Since tet virus ,66,67,68et virus ,70,71,72 tabacum , in a no tabacum . Thus, tArt-sRNAi tools have been broadly used in plants to confer antiviral resistance against multiple RNA and DNA viruses, and to viroids as well. Currently, the relative simplicity of the webtool-assisted design of highly specific antiviral art-sRNA, combined with the availability of efficient cloning methods, facilitates the design and generation of antiviral art-sRNA constructs for plant delivery. However, one important drawback in the use of art-sRNAi is the difficulty to predict the effectiveness of a particular art-sRNA. Recently described high-throughput systems for rapid in vitro or in vivo screening of the antiviral activity of virus-derived sRNAs or computationally designed art-sRNAs, respectively, seem to have overcome this limitation. Another drawback is the emergence of resistance-breaking virus variants with mutated target sites when using single amiRNAs targeting single sites in viral RNAs. In this case, the artificial multiplexing of amiRNAs in different precursor configurations or the use of syn-tasiRNA precursors, both allowing the co-expression of multiple art-sRNAs, should circumvent this problem. The synchronized targeting of multiple viral RNAs by co-expressed art-sRNAs may minimize the possibility that the virus simultaneously mutates all different target sites to fully escape each art-sRNA, and thus enhance the antiviral resistance.In the current genome editing era of bacterial CRISPR/Cas-based technologies, we anticipate that art-sRNAi tools will continue to be broadly used to confer antiviral resistance in plants because of their unique features of high simplicity, specificity, and efficacy, as well as for their multiplexing capability and for the availability of high-throughput methodologies for the design, generation, and validation of art-sRNAi constructs. The development of efficient methodologies for the production and topical delivery to plants of art-sRNA precursors, as well as a better knowledge of the basic mechanisms governing art-sRNA biogenesis, mode of action, and viral targeting, are needed to further refine art-sRNAi tools in view of their broader use for enhanced crop protection.


Stomoxys calcitrans flies. The reproductive LSD virus was isolated from organs of dead cattle and insects in the culture of LT and MDBK cells. The virus accumulated in cell cultures of LT and MDBK at the level of the third passage with titers in the range of 5.5–5.75 log 10 TCID50/cm3. Sequencing of the GPCR gene allowed us to identify this virus as a lumpy skin disease virus.This study describes the registration of the first cases of lumpy skin disease in July 2016 in the Republic of Kazakhstan. In the rural district of Makash, Kurmangazinsky district of Atyrau region, 459 cattle fell ill and 34 died . To determine the cause of the disease, samples were taken from sick and dead animals, as well as from insects and ticks. LSDV DNA was detected by PCR in all samples from dead animals and ticks , in 14.29% of samples from horseflies (Tabanus bromius), and in one of the samples from two  Lumpy skin disease (LSD) mainly infects cattle and is characterized by fever, lymphadenitis, edema of subcutaneous cellular tissue and viscera, cutaneous nodules (lumps), ocular discharge, and inflammation of the mucosae , goat pox (GTPV), and lumpy skin disease (LSDV).The causative agent of lumpy skin disease is a DNA-containing virus of the The lumpy skin disease virus (LSDV) was identified as the etiological agent for the condition in the 1940s rather than due to hypersensitivity to insect bites or plant poisoning as previously described , which Kazakhstan protects, and this population constitutes the major part of the remaining global population of this species. Various species of endemic insects and ticks occur in the steppe which might constitute a permanent vector, while the wide temperature variations may restrict the potential for vector competence and completion of the life cycles of the virus in this region.LSD emergence in northwestern Kazakhstan is likely to have serious consequences. The livestock industry is re-emerging including the legalization of pastoral livestock livelihoods once banned during the Soviet period. In addition, the region is inhabited by significant numbers of potentially susceptible wild animals including the critically endangered saiga antelope  sent an investigative mission to the region to further research the disease and samples were collected in July 2016 at a farm of the Kurmaganzy district of Atyrau Province during an outbreak of a disease with unknown etiology.Biological samples from animals with clinical signs of the disease were also collected by state veterinarians as part of routine epidemiological surveillance. RIBSP investigators attended at the sampling. For studies, blood samples were taken from five sick cows and nine samples of internal organs  from two dead cows. In addition to this, 13 ticks were collected from infected cattle and 21 horseflies and two biting flies were collected in the infection locus using Vavoua traps , horseflies (Tabanus bromius), and flies  collected in the field were transported to the laboratory and then were washed one time in ethanol and two times in sterile phosphate-buffered saline (PBS) with . Each insect was transferred to a small chilled porcelain mortar and ground. To prepare the suspension, 0.3 ml of PBS solution with antibiotics  was added. The suspension was frozen and thawed three times, and then clarified by centrifugation at 600g for 10 min. The supernatant was used for the study. Pools were prepared from several individuals of the same arthropod species for virus isolation.Ticks  and 2% fetal calf serum.Virus isolation was performed as described in chapter 1.2 Virus isolation in cell culture, OIE Terrestrial Manual 2018. Primary lamb testicle (LT) cell culture and MDBK cell line were used. One ml of clarified supernatant or blood was inoculated to a confluent monolayer in a 25 cmThe flasks were checked daily during 7–10 days for cytopathic effects (CPE). If no CPE was observed by day 10, the culture was frozen three times, and the clarified supernatant was inoculated into fresh LT or MDBK culture. The specificity of the CPE was confirmed by PCR.DNA was extracted from supernatant, blood, and culture suspension using the “DNeasy® Blood & Tissue Kit (250)”, QIAGEN, following the manufacturer’s protocol.PCR was performed to confirm the presence of LSDV specific nucleic acid using a pair of primers: forward primer, 5′- TTTCCTGATTTTTCTTACTAT-3′ and reverse primer, 5′-AAATTATATACG TAAATAAC-3′  in a 16-channel sequencer Genetic Analyzer 3130 xl, Applied Biosystems (USA). POP-7 was used as a polymer for capillaries. The terminating DNA products were generated by the method of cyclic sequencing.The evolutionary history was inferred using the neighbor-joining method  from cattle and insects were examined. LSDV preparations were assayed in the electron microscope (JEM 100 B) with negative contrasting by 4% solution of phosphate-tungstic acid, pH 6.8.Dermacentor marginatus and Hyalomma asiaticum), in three specimens (14.29%) from horseflies (Tabanus bromius), and in one of the specimens from two flies   and the continuous bovine kidney cell line (MDBK). Both infected cell cultures demonstrated the presence of the cytopathogenic agent.A cytopathic effect was detected at the first passage level in LT cell culture after infection with a suspension from all organs  taken from dead animals.Dermacentor marginatus, horseflies, and flies (Stomoxys spp.) was observed from the 2 passage level after 6–7 days of cultivation. During infection, the same cell culture with suspensions of Hyalomma asiaticum ticks, only one sample of two used, had a cytopathic effect in the second passage, and the second sample in the third passage. The cytopathogenic effect of the virus in the first passage level usually appeared within 7 to 9 days after infection. In the subsequent passages, these terms were reduced, and at the third passage level, CPE was noted for 2–4 days. Distinct CPE with 75–90% affection of the cellular layer was demonstrated in these cultures on the third passage level on the 8th day of cultivation.CPE in cell culture infected with a suspension of Suspensions of the internal organs taken from animals post mortem were used for infecting the MDBK cell culture. The cytopathic effect of the virus in this cell culture was detected in the second passage level after 6–7 days of incubation. In the third passage, the timing of the onset of CPE was reduced and noted on the third day with the defeat of 70–80% of the monolayer cells.50/cm3.The virus accumulated in these cultures  on the third passage level in titers within 5.5–5.75 log 10 TCIDThe specificity of the isolated viruses was confirmed in PCR and by electron microscopy. In all specimens, an amplification product sized 192 bp typical for the LSDV was generated. The study of the morphology and fine structure of the negatively stained preparations showed viral particles typical for LSDV. The size of the virions varied within 300 × 370 nm, while the bulk mass of particles (90%) was sized 300 × 350 nm.A virus isolated from the skin nodules of a dead animal was selected for archiving “Nodulares/Dermatitis/Atyrau-2016” and deposited into the Microbial Collection of the RGE RIBSP, and later this same virus was used to produce an attenuated candidate vaccine strain.Sequencing of the GPCR gene allowed us to identify this virus as a lumpy skin disease virus.2 of land. The remaining live animals were treated with repellents.After the first cases of the disease, in the Kurmangazy region, the veterinary service introduced restrictive measures to localize and eliminate the disease. The movement of all species of domestic animals was prohibited, and a daily farm visit was carried out in order to identify animals with clinical signs of lumpy skin disease. After laboratory confirmation of the disease, the affected area (Kurmangazy region) was quarantined. All 459 animals with clinical signs were culled and the owners were compensated at the market value of the animal. Disinfection of affected sites was completed including 28.116 mAfter confirming the diagnosis and reporting the disease to OIE, all the cattle of the Atyrau Province were vaccinated, as well as animals in the risk zone (threatened zone) of the West Kazakhstan and Aktobe Province. Vaccination was carried out using the lumpivax® vaccine manufactured by the Kenya Veterinary Vaccine Manufacturing Institute (KEVEVAPI).Musca domestica L.  of the samples from sick and dead animals, as well as confirmation of the presence of the virus in the arthropods collected in the epidemic focus. The etiological agent was isolated in the primary lamb testicle cell culture and in the continuous cell culture MDBK. Further sequencing of the gene GPCR and the phylogenetic analysis showed the close genetic relationship of the isolated capripoxvirus with a group of LSD viruses including those circulating in the areas of the Russian Federation adjacent to Kazakhstan. Emergence of the disease in the western regions of Kazakhstan from probable transboundary sources was confirmed. According to the information in Russia during May–June 2016, there were 163 LSD suspected foci including three in the Astrakhan region bordering the Atyrau Province of Kazakhstan (Rosselkhoznadzor Stomoxys and Musca spp), Aedes mosquitoes, in ticks , (Amblyomma hebraeum), and  decolaratus)  can mechanically transmit the LSD virus , horseflies (Tabanus bromius), and other biting flies  collected in the disease focus were assayed as potential transmitters. All tick samples were positive, and a proportion of horse flies and Stomoxys flies (Table Tabanus bromius) collected during the outbreak of the disease. These results support the studies of Sohier et al. (The peak activity of blood-sucking insects and ticks in Kazakhstan falls in April and October Faulde . The emer et al. , which sr et al. . The perStudies have shown that a novel cattle disease in the Atyrau Province of Kazakhstan in 2015 was caused by infection with the lumpy skin disease virus. Virus was also detected among arthropod horseflies, biting houseflies, and or ticks suggesting the possibility of these species as vectors of LSD in this region.


Phytohormones mediate plant development and responses to stresses caused by biotic agents or abiotic factors. The functions of phytohormones in responses to viral infection have been intensively studied, and the emerging picture of complex mechanisms provides insights into the roles that phytohormones play in defense regulation as a whole. These hormone signaling pathways are not simple linear or isolated cascades, but exhibit crosstalk with each other. Here, we summarized the current understanding of recent advances for the classical defense hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) and also the roles of abscisic acid (ABA), auxin, gibberellic acid (GA), cytokinins (CKs), and brassinosteroids (BRs) in modulating plant–virus interactions. During their growth and development, plants are frequently challenged by pathogens such as viruses, bacteria, fungi, oomycetes, nematodes, and insects . Most plDespite having simple molecular structures and low cellular concentrations, phytohormones regulate most plant physiological processes, such as cell growth and division, plant growth and morphogenesis, as well as organogenesis and apoptosis . PhytohoViral infection can disrupt hormonal pathways, which manifests as the simultaneous induction of synergistic or antagonistic hormones and the triggering of defense responses ,15,16,18The ability of plants to successfully resist pathogen invasion relies on crosstalk among different hormone signaling pathways ,5,19. Notomato bushy stunt virus (TBSV) via a competitive interaction with cytosolic glycerol 3-phosphate dehydrogenase (GAPDH)  were susceptible to BaMV [The induction of hormones by virus infection is a common phenomenon during plant–virus interactions; however, the reported effects of viral infection on ABA concentration vary. An increase in ABA content following virus infection has been reported in a number of virus–host interaction systems ,45. For  content , whereas content . In othenfection . Exogenonfection ,48,49, infection ,49,50. A to BaMV . Iriti aR gene–mediated extreme resistance against soybean mosaic virus (SMV) [The intercellular spread of plant viruses involves movement of the viral genome or virion through PD. The PD conductivity is regulated by the controlled buildup of callose at the PD neck. This is mostly mediated via the antagonistic action between callose synthases and β-1,3-glucanases ,51. The us (SMV) . This reus (SMV) . TherefoArabidopsis [aba1-5), in which the expression level of AGO1 was significantly increased, suggesting that ABA regulates the AGO1 protein [MIR168a, a negative regulator of AGO1 that is often induced during virus infection, contains ABA-responsive elements within its promoter [N. benthamiana with potato spindle tuber viroid (PSTVd), and ABA exposure leads to AGO3-mediated resistance to BaMV infection [The RNAi pathway is a well-known major defense mechanism against viruses within plants , and ABAbidopsis , indicat protein . The sampromoter ,57. A stnfection ,58. In vET is the simplest unsaturated hydrocarbon gas molecule and functions in many plant developmental stages, such as senescence and in defense responses against necrotrophic pathogens ,19. ET iArabidopsis mutants that suppress the phenotype induced by the transgenic expression of CaMV P6 are less susceptible to CaMV infection and show reduced ET sensitivity [Arabidopsis to turnip crinkle virus (TCV) was not dependent on ET [Several groups have demonstrated that ET participates in the development of viral symptoms. Using a temperature-induced synchronous lesion formation system, Ohtsubo and colleagues (1999) showed that increased ET production preceded and promoted lesion formation. In addition, inhibitors of ET biosynthesis or action significantly suppressed lesion formation . Anothersitivity . The autsitivity ,70, and nt on ET . Therefoethylene insensitive 2 (ein2) and ethylene response 1 (etr1) showed an enhanced resistance to CaMV infection [1-aminocyclopropane-1-carboxylate synthase (acs1), ethylene-responsive transcription factor 106 (erf106), and ein2, are also resistant to TMV-cg. The ET signaling pathway was also correlated with suppression of the defense response to turnip mosaic virus (TuMV) and enhanced reproduction of the green aphid, Myzus persicae in Arabidopsis [N gene, while JA and ET signaling is essential for systemic resistance to ChiVMV in tobacco [Overwhelming evidence suggests that the ET pathway play a bidirectional role in plant–virus interactions. An experiment using the ET signaling pathway mutants nfection . Other mbidopsis . Howeverbidopsis . Chilli  tobacco . The reaTrichoderma harzianum strain T-22 (T22) led to systemic resistance against CMV infection through JA/ET and SA signaling pathways [NtERF5, an ET-responsive TF from Nicotiana tabacum, caused enhanced resistance of transgenic tobacco plants to TMV [During crosstalk mediated by different phytohormones, ET perception is also required to establish SAR in TMV-infected leaves, which eventually triggers SA accumulation and SAR development in uninfected leaves . Treatmepathways . Alternas to TMV . The ET s to TMV ,68. CleaArabidopsis interaction [Studies on crosstalk have focused on the interaction between sugar signaling mediated by ABA and ET, whereby high ABA concentrations inhibit ET production ,74. Altheraction . These rAlthough it is difficult to formulate general rules about the effects of viruses on phytohormones, viruses clearly affect auxin and GA individually or combinatorially, depending on the virus–host combination. Auxin is an important phytohormone with roles in plant growth and development , but a nMany viral infections result in aberrant phenotypes that parallel the involvement of auxin in virus establishment. Vitti and colleagues (2013) reported that root growth was accompanied by significant changes in the levels of IAA proteins during CMV infection . ARFs arent-kaurene oxidases, which catalyze 1 step in GA biosynthesis [ent-kaurene oxidase-like (KOL) proteins, which have a high similarity in amino acid sequence with ent-kaurene oxidase, and these KOL proteins may be involved in phytoalexin biosynthesis [In addition to disrupting auxin signaling, virus infection also affects GA pathways. GAs are diterpenoid hormones that control various aspects of plant physiological development ,85, but ynthesis . It shouynthesis . In addiynthesis . RBSDV iynthesis . These rynthesis . This revid1) phenotype of turnip vein clearing virus (TVCV), whereas the application of GA had no effect [The functions of auxin and GAs overlap in the regulation of multiple aspects of plant development, most of which relate to cell expansion and differentiation. For example, the level of auxin due to its biosynthesis and transport controls DELLA protein abundance, and DELLA proteins also integrate signals from ET, auxin, and GAs during growth ,90. Altho effect . Zhu ando effect . AlthougNTN infection in soil-grown potato plants, when a shift in the concentration of biologically active free CKs toward inactive 9-glucosides was observed [white clover mosaic virus (WClMV) and showed that production of the inactive 9-glucoside form was a direct response to WClMV infection [Arabidopsis and led to the increased expression of primary CK-responsive genes [CKs act as long-range and local signals and are involved in multiple regulatory processes throughout plant development . CK signobserved . In addinfection . Only feve genes . FurtherPR gene expression, indicating that BL-induced resistance is distinct from SAR [WRKY30 in response to viruses. Components of BR pathways also play important roles during plant–virus interactions. For example, BRASSINOSTEROID-INSENSITIVE 2 (BIN2), a negative regulator of BR signaling, interacted with the geminivirus C4 protein [bak1-4 and bak1-5 mutants than in wild-type plants [BRs are steroid plant hormones that have been extensively studied since their initial isolation and characterization . They refrom SAR . Reportsfrom SAR . This is protein , althouge plants . AdvanceArabidopsis [RCY1 gene, which encodes 104-kDa CC-NBS-LRR type protein. The RCY1-conferred resistance requires both SA and ET signaling but not JA signaling [N. benthamiana [Arabidopsis to TYLCV [Interactions between hosts and their corresponding viruses are extensive and overlapping. To elucidate these affects, the crosstalk among different phytohormones that enables plants to fine-tune energy allocation and minimize fitness costs through flexible complex signaling networks should be characterized. Although antagonistic crosstalk between SA and JA/ET signaling pathways is well known, SA and ET also function synergistically to induce resistance against CMV in bidopsis . Fine maignaling . Crosstathamiana . Furtherto TYLCV . The TMVto TYLCV . The WRKto TYLCV . Very reto TYLCV . Anotherto TYLCV . Infectito TYLCV ; thus, RThe disruption of phytohormone biosynthesis and signaling pathways following virus infection is extremely complex. Significant progress has been made in elucidating the molecular mechanisms that underlie crosstalk among different phytohormones during virus infection. Changes in endogenous phytohormone levels appear to be a direct consequence of virus infection and are tightly coordinated with viral movement, replication, symptom development, and defense responses.


The eHealth self-management application ‘Oncokompas’ was developed to support cancer survivors in monitoring health-related quality of life (HRQOL) and symptoms, and obtaining personalized feedback and options for supportive care. The aim of this study was to assess the cost-utility of Oncokompas compared with care as usual (CAU) among cancer survivors.Survivors were randomly allocated to the intervention or control group. Direct (non-)medical, indirect non-medical costs, and HRQOL were measured at 3- and 6-month follow-up, using iMTA Medical Consumption and Productivity Costs and the EuroQol-5D questionnaires. Mean cumulative costs and quality-adjusted life-years (QALYs) were compared between both groups.n = 320) or control group (n = 305). Base case analysis showed that incremental costs from a societal perspective were − €163 , and incremental QALYs were 0.0017  in the intervention group compared with those in the control group. The probability that, compared with CAU, Oncokompas is more effective was 60%, less costly 73%, and both more effective and less costly 47%. Sensitivity analyses showed that incremental costs vary between − €40 and €69, and incremental QALYs vary between − 0.0023 and − 0.0057.In total, 625 survivors were randomized into intervention effective manner.Oncokompas seems to improve HRQOL and reduces the burden of several tumour-specific symptoms, while costs from a societal perspective are similar to CAU.The online version of this article (10.1007/s11764-020-00912-9) contains supplementary material, which is available to authorized users. Cancer survivorship care includes physical rehabilitation, psychosocial care, lifestyle interventions, existential issues, and the (self-)management of survivors’ health and healthcare. It is, however, challenging to organise cancer survivorship care, because it is difficult to align individual needs and preferences with existing care options, and also to make cancer survivorship care available at acceptable costs , 2.Data from patient-reported outcome measures (PROMs) can be used for optimal referral to supportive care in clinical practice. Behavioural intervention technologies (BITs) are currently often used to collect and process PROM data . Also, eWe developed Oncokompas, a fully automated BIT that supports cancer survivors by monitoring their HRQOL and symptoms; obtaining tailored feedback and advice on their physical, psychological and social functioning, lifestyle, and existential questions; and receiving a personalized overview of recommended supportive care services –13. OncoDuring studies on the feasibility and implementation of Oncokompas , 13, 15,The aim of the present study was to evaluate the cost-utility of Oncokompas compared with care as usual (CAU) among cancer survivors, from a societal and healthcare perspective.A randomised controlled trial was carried out among survivors of head and neck cancer, colorectal cancer, breast cancer, and lymphoma (including high- and low-grade non-Hodgkin lymphoma and Hodgkin lymphoma). These tumour types were chosen to ensure variability regarding age, sex, prevalent and less prevalent tumour types, solid and non-solid tumour types, cancer- and treatment-related symptoms, and the need for various types of supportive care. Other inclusion criteria were the following: age ≥ 18 years (no upper limit) and 3 months to 5 years after treatment with curative intent . Survivors who were still receiving endocrine therapy or immunotherapy, or had a wait-and-see regimen, were included 3 months after their previous treatment or diagnosis, respectively. Exclusion criteria were the following: no access to the Internet or no email address, severe cognitive impairment, insufficient mastery of the Dutch language, physical inability to complete a questionnaire, and male breast cancer survivors , 16.The study protocol was approved by the Medical Ethics Committee of the VU University Medical Center (2015.523), published previously , and regCancer survivors who gave their informed consent were randomly allocated to the intervention group (direct access to Oncokompas) or CAU wait-list control group (access to Oncokompas after 6 months), in a 1:1 ratio. Randomisation was stratified by tumour type, and blocks with a length of 68 were used. Due to the nature of the intervention, participants could not be blinded for the allocated arm.Participants were recruited through The Netherlands Cancer Registry (NCR) in 14 hospitals across The Netherlands, and invited by their (former) treating physician. Data collection was performed using the Patient-Reported Outcomes Following Initial Treatment and Long term Evaluation of Survivorship (PROFILES) registry .Oncokompas supports cancer survivors in self-management, by monitoring (cancer-generic and tumour-specific) symptoms and HRQOL, providing feedback and information on their scores and a personalized overview of supportive care options, with the aim to reduce symptom burden and improve HRQOL , 12, 16.The economic evaluation was conducted from a societal perspective, including direct medical costs , direct non-medical costs , indirect non-medical costs (costs due to absence from paid work or loss of productivity from paid work), and intervention costs. All outcome measures were collected at baseline (time of inclusion), and at a 3-month and 6-month follow-up assessment. Since the follow-up of the study was 6 months, neither costs nor effects were discounted.Direct medical and direct non-medical costs were measured with the Institute for Medical Technology Assessment (iMTA) Medical Consumption Questionnaire (iMCQ) . The iMCIndirect non-medical costs were measured with the iMTA Productivity Costs Questionnaire (iPCQ) . ProductHealth-related quality of life was measured with the EuroQol-5 Dimension (EQ-5D). The utility score was obtained using the Dutch index tariff .Intervention costs of Oncokompas were calculated using a top-down approach. Costs for running Oncokompas  are estimated at €450,000 annually. When reaching 18,000 cancer survivors per year  , 23, theχ2 tests, and independent samples t tests were used to describe and compare baseline characteristics between the intervention and control group. To provide information on types of costs included in the analyses and their relative importance at each time point, data of complete cases (participants who completed baseline and both follow-up measurements) were used.Analyses were performed using SPSS version 25  and STATA version 14 . Descriptive statistics, Was adjusted for baseline EQ-5D scores and baseline total costs,Included varying intervention costs of Oncokompas ,Was performed among survivors with complete data at all time-points,Was performed from a healthcare perspective, including only direct medical costs and intervention costs.To test the cost-utility of Oncokompas compared with CAU, a base case intention-to-treat cost-utility analysis was performed, including all participants, with imputed data for missing time points, and estimated intervention costs of €25 per Oncokompas user. The robustness of this finding was tested by four additional sensitivity analyses in which the base case analysis:In case data was missing on item level , assumptions were made based on means per allocation group and time point. In case data was missing on questionnaire level, missing data was imputed as total costs or utility score per time point per allocation group, using multiple imputations (predictive mean matching) by chained equations. Backward multivariable linear and logistic regression analyses were performed to investigate which variables  were associated with missing data, total costs, or utility scores. A description of these variables is listed in the The total cumulative costs per patient were calculated by summing costs measured with the iMCQ and iPCQ at 3- and 6-month follow-up and intervention costs in the intervention group. Quality-adjusted life-years (QALYs) were calculated as the EQ-5D utility scores per time point, multiplied by the corresponding time period (i.e. 3 months).intervention – mean costscontrol) / (mean QALYsintervention – mean QALYscontrol). Uncertainty around the ICUR was assessed using bootstrapping with 5000 replications and was projected on a cost-utility plane.To obtain costs per QALY gained, an incremental cost-utility ratio (ICUR) was calculated as the incremental costs divided by incremental effects, with the following formula: (mean costsn = 320) or control (n = 305) group. Of them, respectively 205 (64%) and 240 (79%) completed both follow-up questionnaires and were complete cases. Figure In 14 participating hospitals throughout the Netherlands, 2953 cancer survivors were invited to participate between October 12, 2016, and May 24, 2018. In total, 625 (21%) survivors consented to participate, completed the baseline questionnaire at study inclusion, and were randomly allocated to the intervention (p = 0.11). The mean total costs in the previous 3 months at baseline in the intervention group were €1013 (sd 1760), compared with €1158 (sd 1936) in the control group (p = 0.33). The mean costs for survivors with complete data per time point per group are presented in Supplementary Table The mean EQ-5D score of survivors at baseline was 0.89 sd 0.15) in the intervention group and 0.87 (sd 0.17) in the control group , and the probability that costs were lower was 51% , compared with the control group. Because of these results, the subsequent sensitivity analyses were also corrected for baseline EQ-5D and costs.Analyses with intervention costs of €15 and €100 showed that the probability that in the intervention group the QALYs were higher was 13% , and the probability that costs were lower were 52% and 39%  respectively, compared with the control group.Analyses on the complete cases showed that the probability that in the intervention group QALYs were higher was 30% , and the probability that costs were lower was 41% , compared with the control group.Analyses with only direct medical costs taken into account showed that the probability that in the intervention group the QALYs were higher was 20% , and the probability that costs were lower was 57% , compared with the control group.The results of the cost-utility analyses are shown in Table This study investigated the cost-utility of a fully automated BIT ‘Oncokompas’ among cancer survivors, compared with CAU. In the base case analysis, QALYs were similar and costs were non-significantly lower in the intervention group (− €163), compared with those in the control group. When the base case analysis was corrected for baseline EQ-5D utility scores and costs, QALYs were non-significantly lower in the intervention group and costs were similar compared with those in the control group.The finding that Oncokompas is more or less equally effective in terms of utilities and costs as CAU confirms our earlier research on the efficacy of Oncokompas . That stThe previous study also showed that for some tumour-specific symptoms, beneficial effects of Oncokompas occurred at 3- or even 6-month follow-up, and not directly post-intervention . It may With an increasing number of cancer survivors, the costs of cancer survivorship healthcare are growing, together with an increasing healthcare workforce shortage , 27. TheEconomic evaluations of eHealth interventions among patients with chronic diseases are scarce and mostly performed among patients with diabetes and cardiovascular disease , 29. To Potential limitations of this study were that several assumptions were made regarding missing data on healthcare resource use. Missing data was replaced based on assumptions, or imputed using multiple imputation techniques. This might not reflect reality, but since we made similar assumptions and imputations in both groups, it is not expected that this has influenced our findings. Also, since the cost prizes of unit resource and productivity costs in this study are based on the Dutch tariff, the results might not be generalizable to other countries. The Dutch healthcare system and reimbursement of care, and thereby the low barrier to seek care, might also not be representative of other countries. Furthermore, the attrition rate was higher in the intervention group than in the control group, and there were also more complete cases in the control group than in the intervention group. This might be explained by the fact that participants in the wait-list control group obtain access to Oncokompas after the last follow-up measurement, which might have been an extra motivation to complete the follow-up assessments. We cannot be sure whether this has under- or overestimated the results, but since the results from the sensitivity analysis with only taken into account the complete cases did not differ much from the sensitivity analysis with all participants, it is expected that this influence is limited. Finally, the participation rate of the randomized controlled trial was 21%, and participants were mostly long-term survivors and had relatively good baseline scores, which might limit the generalizability of the results. Further research is needed to see whether these results can be confirmed among representative samples of cancer survivors, also diagnosed with other tumour types.In conclusion, results indicate that a fully automated BIT such as Oncokompas is at least as effective as usual cancer survivorship care, and not more expensive. Implementation and upscaling of Oncokompas may help to improve cancer survivorship care in a (cost-)effective manner.ESM 1(DOCX 23 kb).


Virus shedding, transmission and seroconversion were evaluated, and swabs collected at selected time-points were characterized in deep sequencing to assess virus diversity. In general, the virus showed low pathogenicity for the examined bird species, but differences in shedding patterns, seroconversion and clinical outcome were noted. The highest heterogeneity of the virus population as measured by the number of single nucleotide polymorphisms and Shannon entropy was found in oropharyngeal swabs from quail, followed by turkeys and ducks. This suggests a strong bottleneck was imposed on the virus during replication in ducks, which can be explained by its poor adaptation and stronger selection pressure in waterfowl. The high within-host virus diversity in quail with high level of respiratory shedding and asymptomatic course of infection may contribute to our understanding of the role of quail as an intermediate host for adaptation of AIV to other species of poultry. In contrast, low virus complexity was observed in cloacal swabs, mainly from turkeys, showing that the within-host diversity may vary between different replication sites. Consequences of these observations on the virus evolution and adaptation require further investigation.Avian influenza virus (AIV) is a highly diverse and widespread poultry pathogen. Its evolution and adaptation may be affected by multiple host and ecological factors, which are still poorly understood. In the present study, a turkey-origin H9N2 AIV was used as a model to investigate the within-host diversity of the virus in turkeys, quail and ducks in conjunction with the clinical course, shedding and seroconversion. Ten birds were inoculated oculonasally with a dose of 10 Influenza A viruses show the highest genetic diversity and the broadest host spectrum among the four known influenza virus types . ClassifThe high diversity of influenza viruses confers their flexibility, enabling adaptation to novel conditions. The segmented RNA genome may undergo reassortment when two different influenza viruses infect the same cell, potentially generating progeny virions with beneficial gene constellations . Further50) was calculated with the Reed and Muench method.The H9N2 low pathogenic avian influenza virus A/turkey/Poland/14/2013 used in the study was detected in an outbreak in a commercial turkey farm. Phylogenetic and molecular analysis showed that this strain originated from the wild-bird reservoir but acquired markers of adaptation to gallinaceous poultry . The virad libitum. Prior to experimental inoculation, swabs and serum samples were collected and tested by means of real time RT-PCR and ELISA, respectively, in order to exclude any previous AIV infection.Experiments were performed in 3-week-old Japanese quail, meat turkeys and Pekin ducks. Turkeys and ducks were purchased in local hatcheries and reared in the animal facility at the National Veterinary Research Institute (NVRI), Poland. Quail were obtained from a commercial farm at the age of two weeks and were housed for one week to allow acclimatization. The experiment for each species was conducted separately in the animal facility of BSL3+ containment level at NVRI. Birds were randomly allocated to three groups: inoculated (10 birds), contact (2 or 3 birds) and control (5 birds), and housed in separate rooms in metal grid cages. Water and feed were provided 6 EID50 in 0.1 ml divided equally between both infection routes . At 1 day post infection (dpi) three  or two (quail) contact birds of the same species were introduced into the cage with inoculated birds. The control groups consisted of five mock-infected birds of the respective species. Birds were monitored for 14 days for the presence of clinical signs and mortality. At 2, 3, 4, 5, 6, 7, 10 and 14 dpi oropharyngeal and cloacal swabs were collected from all birds using commercial swabs with viral transport medium . Additionally, samples of lungs, trachea, duodenum, cecal tonsils and spleen were collected from a turkey that died during the experiment. At 14 dpi blood samples were collected and birds were humanely euthanized.Groups of ten birds were inoculated intranasally and intraocularly with the virus dose of 10The animal experiments were approved by the Local Ethical Committee . 50 (eqEID50) in 1 mL of swab medium. Samples of organs collected from the dead turkey were homogenized in PBS to 20% (w/v) suspension and centrifuged . The RNA was extracted from the supernatant and the quantity of viral RNA in 0.1 g of organ was determined in real time RT-PCR as described above.Extraction of RNA from swab samples was performed with Viral Mini Kit  according to the manufacturer’s manual. The quantity of viral RNA was tested in one-step real time RT-PCR with primers and probe specific for the influenza M gene . ReactioSera collected from birds at 14 dpi were tested with hemaglutination inhibition (HI) method using A/turkey/Poland/14/2013 antigen according to a standard procedure . The shedding patterns in inoculated birds were characterized by the following parameters: latency period (time from inoculation to shedding), duration of shedding (number of days in which shedding was identified), peak shedding (maximum amount of virus excreted at any sampling day) and mean daily shedding (mean amount of virus excreted during the shedding period). Since some observations were right censored or interval censored, the following assumptions for the assessment of the duration of shedding were made: if shedding was detected at the last sampling day (14 dpi), this day was designated as the last day of shedding; if shedding stopped between two nonconsecutive sampling days, the last day with positive result was defined as the last day of shedding. The data were generated individually for each bird and shedding route, and mean and standard deviations were calculated. To compare the diversity of viral populations generated in the course of infection in the studied poultry species, swabs collected at 2 and 4 dpi, and the virus stock used for inoculation were subjected to deep sequencing. The genome segments were amplified in RT-PCR using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA Polymerase (ThermoFisher Scientific) with primers annealing to the conservative 5’ and 3’ termini  modifiedwww.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapters and low-quality fragments were removed with Trimmomatic [The quality of raw data generated in MiSeq was evaluated with FastQC software .The differences in the shedding parameters between species were assessed using U-Mann-Whitney test. The same test was used to compare shedding from respiratory tract and cloaca in each species separately. Comparison of the number of polymorphic positions and Shannon entropy values was also performed with the U-Mann-Whitney test. To evaluate the association between the quantity of viral RNA and the virus diversity in a sample, the Pearson correlation coefficient was calculated for eqEID5 eqEID50/0.1 g), then in lungs (1.58 × 103 eqEID50/0.1 g), spleen (6.3 × 102 eqEID50/0.1 g), cecal tonsils (63 eqEID50/0.1 g) and trachea (25 eqEID50/0.1 g).No clinical signs or mortality were noted in quail and ducks. In turkeys, only slight apathy was observed from 2 dpi until the end of the experiment. One turkey died on 9 dpi. Slight duodenal congestion and cecum distension were found during necropsy. Viral RNA was detected in all sampled organs of this specimen, with the highest viral load in duodenum (1 × 10p < 0.001) and was shorter in duration (p < 0.01) than oral shedding (p < 0.001). Oral shedding in contact turkeys was observed as soon as at 1 dpc (days post contact) and continued until 13 dpc. Cloacal shedding in contact turkeys was noted at 2–9 dpc.In the challenged turkeys, shedding from respiratory tract was observed for the whole duration of the experiment (2–14 dpi), with all oropharyngeal swabs positive at 5–7 dpi . Sheddinshedding . The ovep < 0.01). The amount of virus excreted from respiratory tract was also higher than from cloaca (p < 0.01 for peak shedding and p < 0.001 for mean shedding). In contact quail, oral and cloacal shedding was demonstrated at 4–5 dpc and 2–3 dpc, respectively.Inoculated quail showed oral shedding between 2 and 6 dpi, and at 2–5 dpi viral RNA was detected in all oropharyngeal swabs . CloacalShedding from respiratory tract was observed in all inoculated ducks and cloacal shedding was found in a total of 8 birds . There wp < 0.01 and p < 0.001, respectively) (p < 0.01) and in ducks (p < 0.001). The statistically significant difference for the oral shedding duration was also noted between quail and ducks (p < 0.001). It was also demonstrated that cloacal shedding in turkeys was longer than in quail (p < 0.025). Comparison of the level of shedding (peak and mean daily shedding) showed no statistically significant differences for turkeys and quail, but both species shed higher amounts of virus via the respiratory route than ducks (p < 0.001). However, cloacal shedding in ducks was higher than in quail (p < 0.025), but it did not differ significantly from cloacal shedding in turkeys. No significant differences in the latency of oropharyngeal shedding were observed between the tested species. However, both quail and ducks showed shorter latency period for cloacal shedding than turkeys (ctively) . The hig2 to 9 log2. Positive results of HI test were also found in 7 out of 10 challenged quail and in both contact quail (titres 4–5 log2). The lowest response was observed in ducks as five challenged and two contact birds seroconverted (titres 4–5 log2).The HI test revealed seroconversion in all challenged and contact turkeys with titres ranging from 5 logIn the case of quail, sequencing results were generated for all oropharyngeal swabs from both sampling points. No sequences from cloacal swabs from quail could be obtained due to very low viral load. Sequences of the excreted virus were obtained also for seven (out of nine) and eleven oropharyngeal swabs collected from turkeys at 2 and 4 dpi, respectively. Viruses from three cloacal swabs collected from turkeys at 4 dpi were also sequenced. Samples from ducks, due to generally low viral load, were most problematic in sequencing. Only one sequence from 2 dpi and eight sequences from 4 dpi (3 OP and 5 CL) of sufficient quality were obtained. Additionally, sequencing of virus in cloacal swabs from ducks and turkeys from 5–7 dpi was also performed. For all of the samples tested, a high coverage was obtained for all segments of the viral genome .p < 0.01) and at 4 dpi . The virus population in the oropharyngeal swab from a duck at 2 dpi contained 20 SNPs and 9–16 SNPs were revealed at 4 dpi, which was significantly lower than in quail at 4 dpi (p < 0.01)  were identified in the virus stock used for inoculation. Most of them were located in the HA gene. The highest number of single-nucleotide polymorphisms in the virus population was observed in quail, as 21–32 and 25–37 SNPs distributed across 172 nucleotide positions were identified at 2 and 4 dpi, respectively . These v < 0.01) .p < 0.05). To clarify whether this trend was maintained later during the course of infection, additional analysis of cloacal swabs collected between 5 and 7 dpi from turkeys (n = 5) and ducks (n = 5) was performed. An increase in the number of polymorphic sites was observed, but these were mainly newly emerged variants with low frequency (<5%)  .p < 0.01 at 2 dpi and 4 dpi) and ducks (p < 0.01 at 4 dpi). The HA gene contributed mostly to the virus diversity in oropharyngeal swabs, while the lowest complexity was found for the PB1 and NS segments (p < 0.01) and ducks (p < 0.05).Based on the frequency of variants in the virus population, the value of Shannon entropy was calculated for each sample and visualized with a heatmap . The ressegments . In contsegments . The scoNonsynonymous variants were present in 77 out of 172 polymorphic positions (44.8%) identified in quail, including seven nonsynonymous mutations  derived from the parental stock. The highest proportion of nonsynonymous substitutions (56.3%) was found in the HA gene. In turkeys the nonsynonymous polymorphisms represented 51.3% (61/119) with seven mutations of inoculum-origin . The highest percentage of nonsynonymous substitutions was shown in ducks , wherein two of them were introduced with the inoculum material . None of the nonsynonymous mutations found in the HA protein have been linked to the increased affinity to mammalian-type sialic acid receptors.The SNPs positions in the original virus stock and in swab samples were compared to differentiate mutations originating from the inoculum from those that arose in birds during infection. Out of 22 minority variants  present in the inoculum isolate, 21, 20 and 9 were detected in quail, turkeys and ducks, respectively. A high variation in terms of the frequency of these variants in individual birds was observed .In quail, changes in the consensus sequences resulted mostly from selection of inoculum-derived mutations, while the vast majority of novel variants were present at low frequencies (<10%) . The con50 in quail , turkeys  and ducks , indicating that the differences in the virus population diversity are independent of the differences in the virus replication level.No significant correlation was observed between the entropy values and eqEIDEvolution of viruses is a multifactorial process depending on the virus mutation rate, within-host replication dynamics, infection and transmission modes and other epidemiological and ecological factors ,37,38,39In the present study, three common poultry species were infected with an H9N2 AIV isolated from an outbreak in turkeys. Each bird was inoculated with the same virus stock, giving an opportunity to study both the pathogenicity of the virus and how its diversity had been fluctuating during the course of infection. Generally, the pathogenicity of the virus for the studied birds was low, although it varied between the species. Transmission occurred in all tested species as evidenced by shedding and seroconversion in contact birds. The levels of shedding in quail and turkeys were similar to those observed for LPAIV in gallinaceous poultry, i.e., higher amounts shed from respiratory tract than from cloaca . There wThe outcome of H9N2 AIV infection in quail, i.e., the lack of clinical signs with high level of respiratory shedding, is in agreement with the results of other experimental studies on the pathogenicity of LPAIV for this species ,12. It wIn contrast to quail and turkeys, there were no differences in the level of shedding by the oral and cloacal route in ducks, which is in agreement with the results of the meta-analysis performed for various species and LPAIV strain . FurtherOther interesting findings of the present study were the differences between the variability of virus population in oropharyngeal and cloacal swabs observed mainly in turkeys. The dominant population in cloacal swabs varied between individual birds, indicating that the low diversity could not be attributed to selection of specific variants conferring the best fitness in the gastrointestinal tract. However, a possible explanation of this phenomenon may lie in a “founder effect”. In turkeys, the oropharyngeal shedding preceded the cloacal shedding, as shown by different latency periods, thus it could be expected that the virus population replicating in the intestines was founded by a small proportion of the population present in the respiratory tract, resulting in lower virus diversity. This observation is consistent with population genetics studies, as a spatial spread of a population results in a loss of genetic diversity, and similar effect could be also noticed in the case of dissemination of a virus within a single host . DistincThe differences in the within-host virus diversity between the studied species could result from the characteristics of the virus strain used, high diversity of the inoculum stock, infection dose and inoculation route. Since no correlation was found between the virus load and virus diversity, it can be assumed that differences in virus complexity between species or sample types were not caused by differences in the replication level. The limited number of studies on the diversity of AIV at the level of individual birds precludes drawing any far-reaching conclusions. Due to the differences in the experimental setup (e.g., infection with HPAI viruses ,52,54,55


High-throughput sequencing (HTS) application in the field of plant virology started in 2009 and has proven very successful for virus discovery and detection of viruses already known. Plant virology is still a developing science in most of Africa; the number of HTS-related studies published in the scientific literature has been increasing over the years as a result of successful collaborations. Studies using HTS to identify plant-infecting viruses have been conducted in 20 African countries, of which Kenya, South Africa and Tanzania share the most published papers. At least 29 host plants, including various agricultural economically important crops, ornamentals and medicinal plants, have been used in viromics analyses and have resulted in the detection of previously known viruses and novel ones from almost any host. Knowing that the effectiveness of any management program requires knowledge on the types, distribution, incidence, and genetic of the virus-causing disease, integrating HTS and efficient bioinformatics tools in plant virology research projects conducted in Africa is a matter of the utmost importance towards achieving and maintaining sustainable food security. This discovery, which also marked the start of the discipline of plant virology, was made from various studies that pinpointed tobacco mosaic virus as the causal agent of a mosaic disease on tobacco plants  Lam) is a dicotyledonous plant that belongs to the family Convolvulaceae. It has become an important staple and co-staple food crop in Africa. Orange-fleshed sweet potato varieties, a rich source of vitamin A especially, are important for infants and young children. Africa was the second-largest producer, producing 21 million tons in 2016, behind Asia’s production that was estimated at more than 78 million in the same year [Sweet potato  and sweet potato badnavirus B (SPBVB), which had never been reported [Virus diseases have been identified as the second-most important biotic constraint to sweet potato production. A viral metagenomic approach was adopted in South Africa to understand the progressive deterioration of the yield and quality of sweet potato crops, usually referred to as “cultivar decline” experienced by farmers across the country. Leaf samples collected from different surveys in the major growing regions were subjected to DNA ,79,80, treported  along wireported ,79,80. Sreported  and theireported . SPBVA, reported .de novo-assembled contigs matched sequences of SPCSV, SPFMV, SPLCSPV, SPPV, and SPSMV-1. The presence of these viruses was confirmed using conventional molecular techniques. SPPV and SPSMV-1 were found widespread and co-infecting sweet potato plants in Tanzania [Tanzania was the second-largest producer of sweet potato in Africa 2016 . Ninety-Tanzania .Manihot esculenta subspecies esculenta Crantz; Euphorbiaceae), which is currently produced in 40 sub-Saharan African countries, was introduced to that region by Portuguese sailors from Brazil in the sixteenth century [Cassava . Blast analysis was performed using blastn. Alternately, metagenomic analysis was performed using MEGAN . As the https://www.geneious). One well-supported contig that matched UCBSV genome sequence was selected and compared with all complete UCBSV genome sequences available in GenBank. This 9070-nucleotide contig constituted the genome sequence of an UCBSV isolate called MW-NB7_2013. This isolate was highly similar to an isolate from Tanzania (93.4% pairwise nucleotide identity) than to those previously reported from Malawi (86.9 to 87.0%) [CBSD re-emerged at the turn of the 21st century. It has since spread through many East and Central African countries, causing considerable yield losses and jeopardizing the food security of subsistence farmers . A cassao 87.0%) . Similaro 87.0%)  and Comoo 87.0%)  and theyThe HTS for viromics studies performed on different host plants in the African continent has led to the detection of known and novel viruses. This observation is consistent in all hosts tested and across all the countries where the technology has been used. Molecular and serological detection methods remain relevant, especially in cases of novel viruses and viruses not previously reported. The unprecedented increase of novel viruses detected using HTS once again accentuates the need for a uniform and practical way of naming plant viruses. This call has been echoed several times in the scientific communities ,137,138.It is apparent that biological characterization of plant-infecting viruses has remained relevant throughout the history of plant virology. This is all the more true in this age of HTS and bioinformatics. In the past virus discovery was symptoms driven thus strongly relying on combinations of biological techniques, morphology and conventional targeted methods. The advent of HTS has resulted in an alteration of this pattern and preeminence has unintentionally been given to the molecular component. It is therefore critical to link molecular observations to their corresponding effects in the host plants. Taking multiple virus infections as an example, follow-up studies should be conducted to understand the host response to single and multiple infections. Plant health risk assessment should actually be carried out for every HTS detected virus more especially that there is evidence of the existence of cryptic ,140,141 The progress made in the field of plant virology throughout the African continent is quite tangible, albeit happening slowly. However, the nescience of the existence of plant-infecting viruses does not in any way prevent their spread. The detection of virus strains infecting maize and common bean in Africa that are similar to Asian strains is an obvious illustration of viruses readily spreading across continents probably through anthropogenic activities. The continual presence of plant-infecting viruses unknown to scientists on the African soil could, in the long term, have devastating consequences for agricultural production. Although the integration of HTS and bioinformatics tools in plant virology research projects in Africa will require substantial financial investments, its implementation and uptake in more countries than the case presently, will go a long way towards the development of effective and sustainable strategies to manage/control viral diseases infecting major crops. This will ensure food security for many food-insecure people on the continent.


This study aimed to investigate the relationship between relative or absolute intensity and changes in range of motion and passive stiffness after static stretching. A total of 65 healthy young adults voluntarily participated in this study and performed static stretching of the plantar flexor-muscle for 120 s. Dorsiflexion range of motion and passive torque during passive dorsiflexion before and after stretching were assessed. We measured the passive torque at a given angle when the minimum angle was recorded before and after stretching. The angle during stretching was defined as the absolute intensity. Dorsiflexion range of motion before stretching was defined as 100%, and the ratio (%) of the angle during stretching was defined as the relative intensity. A significant correlation was found between absolute intensity and change in passive torque at a given angle (r = −0.342), but relative intensity and range of motion (r = 0.444) and passive torque at dorsiflexion range of motion (r = 0.259). A higher absolute intensity of stretching might be effective in changing the passive properties of the muscle-tendon unit. In contrast, a higher relative intensity might be effective in changing the range of motion, which could be contributed by stretch tolerance. Flexibility has been defined as the ability to move a joint with its complete range of motion (ROM). ROM plays an important role in sports performance and the ability to carry out activities of daily living ,2, whichRecently, Apostolopoulos et al. 2015) have reported that the four stretch parameters associated with an increase in flexibility after SS are (1) intensity, (2) duration, (3) position, and (4) frequency  have rep,7. AdditAs described above, the method of setting stretching intensity might be related to why the consensus on the effect of stretching intensity on ROM and passive property was not achieved. A previous study has reported that 120% ROM stretching intensity affects ROM and the passive properties compared to 100% ROM stretching intensity for the same subjects, i.e., relative intensity. Conversely, a previous study has reported that there is no significant difference in ROM change between the stretching intensity defined as the angle when high or low sensation of stretching felt for each subjects as the stretching intensity [This study aimed to investigate the relationship between relative or absolute intensity and the effect of SS on ROM and passive properties. Previous studies have reported that higher-intensity SS affects ROM changes and muscle-tendon stiffness when compared within same subjects ,11. On tA total of 65 healthy young adults  voluntarily participated in this study . Participants who had a history of an operation performed on their back or lower extremity, lower-extremity contracture, neurological disorders, or took hormone or muscle-affecting drugs were excluded. Written informed consent was obtained from all the participants. This study was approved by the ethics committee of our institution (17677).G*Power software  was used to calculate the sample size on the effect size for correlation analysis (effect size = 0.3 (medium), α = 0.05 and power = 0.80), and elicited result have suggested that the minimum required subjects’ number was 64 for this study.A pre- and post-stretching evaluation was conducted. The participants performed 120 s of SS using the plantar flexor muscles . DorsiflThe participants were seated in an isokinetic dynamometer  chair at 0° knee angle  and 70° hip flexion to prevent tension at the back of the knee, with adjustable belts over the trunk and pelvis and the ankle fixed to the footplate . Then, tDF ROM, passive torque at DF ROM, and passive torque at 100% ROM were measured by calculating the torque-angle curve using an isokinetic dynamometer  Figure . DF ROM SS was performed with the plantar flexor muscles in a similar position for the measurement. The participants passively moved the footplate of the dynamometer from the ankle at neutral angle to the maximum dorsiflexion angle without feeling pain  at 5°/s We defined absolute intensity and relative intensity as the stretching intensity. It was defined that absolute intensity reflected the intensity of stretching among subjects. On the other hand, it was defined that relative intensity reflected the intensity of stretching within each subject. Using the dynamometer, we measured the DF angle during SS in a similar position for the static stretching. The participants passively moved the footplate of the dynamometer from the ankle at a neutral angle to the DF angle at the point of feeling of pain or discomfort while their ankles were dorsiflexed. During SS, the DF angle (°) was defined as the absolute intensity. On the other hand, DF ROM at PRE which we measured before stretching was defined as 100% , and durBefore this study, two tests were performed on eight healthy young adults on different days to determine the test-retest reliabilities for DF ROM, passive torque at DF ROM, and passive torque at a given angle. The calculated intraclass correlation coefficients for DF ROM, passive torque at DF ROM, and passive torque at a given angle were 0.94, 0.91, and 0.89, respectively, indicating that the reliability was high for all outcome measures .t-test was used to compare the differences in DF ROM between PRE and POST, passive torque at DF ROM, and passive torque at a given angle. Using the t-value and sample size, the effect size (r) was calculated. The effect size classification was set where r < 0.1 was considered trivial, 0.1–0.3 was considered small, 0.3–0.5 was considered moderate, and >0.5 was considered large. Moreover, using the standard error of the mean, we calculated the minimal detectable change (MDC). The relationship between absolute or relative intensities and changes in DF ROM, passive torque at DF ROM, and passive torque at a given angle was determined using Pearson’s product–moment correlation coefficient. All statistical analyses were performed using R2.8.1 , and significance was set at p < 0.05. All the data are presented as mean ± standard deviation.Paired p < 0.01, r: 0.59, MDC: 4.2°). However, no significant change was observed in passive torque at DF ROM . Passive torque at a given angle significantly decreased after SS .DF ROM significantly increased after SS . Therefore, our results suggest that the DF ROM increase might have resulted from an increase in the stretch tolerance and a change in the passive stiffness of the muscle-tendon unit.This study showed that the DF ROM significantly increased after a 120 s SS. Generally, the mechanism of the increase in DF ROM is related to the increase in the ability to tolerate loading prior to stretch termination (the increase in stretch tolerance)  and the Regarding the relationship between absolute intensity, i.e., DF angle of SS, and changes after SS, our results showed that the absolute intensity was significantly negatively correlated with the change in passive torque at a given angle, suggesting that the higher absolute intensity greatly decreases the passive torque at a given ROM when compared among individuals. The passive torque at a given angle has been shown to indicate the resistance of the muscle-tendon unit , and it On the other hand, a significant positive correlation was observed between the relative intensity, i.e., the angle during SS per DF ROM at PRE (%), and changes in DF ROM and passive torque at DF ROM. These results were consistent with those of the previous study that investigated the relative intensity among same subjects . A previIn this study, the difference in results were found between absolute intensity and relative intensity. There was a significant correlation between absolute intensity and the changes in passive torque at a given angle. Morse et al. (2018) reported that, with increasing dorsiflexion angle, both muscle and tendon elongation increased linearly . BesidesIn previous studies, it has been found that a poor ROM ,26 and iThis study had three main limitations. First, the effect of SS on flexibility may have differed depending on the sex. Miyamoto et al., have suggested that muscular factors associated with ROM are different between men and women . In thisThis study investigated the relationship between absolute and relative intensities of SS and the changes in DF ROM, passive torque at DF ROM, and passive torque at a given ROM after a 120 s SS. The study results showed that there was a significant negative correlation between the absolute intensity and passive torque at a given ROM. Absolute intensity was calculated from the angle of SS and was not different among subjects. Therefore, we suggest that a greater angle during SS is more effective in creating change in the passive stiffness of the muscle-tendon unit. On the other hand, there was a significant positive correlation between the relative intensity and ROM and passive torque at DF ROM. Relative intensity was calculated from the angle during SS per DF ROM at PRE (%) and was influenced by the subject’s sensation. Thus, in an individual, SS of a tolerable maximum angle was more effective in inducing a change in the stretch tolerance, which resulted in an increase in DF ROM.


We detected Crimean-Congo hemorrhagic fever virus infections in 4 provinces of Pakistan during 2017–2018. Overall, seroprevalence was 2.7% in humans and 36.2% in domestic livestock. Antibody prevalence in humans was highest in rural areas, where increased contact with animals is likely. Nairoviridae. The virus is maintained through a tick–vertebrate transmission cycle  because of its potential to cause a public health emergency and the absence of specific treatment and vaccines.Crimean-Congo hemorrhagic fever (CCHF) is caused by CCHF virus (CCHFV), an emerging zoonotic virus belonging to the order Bunyavirales within the family Most human infections occur through the bite of infected ticks. Blood and other bodily fluids of infected animals represent an additional source for human infections. In humans, CCHF is manifested by fever, headache, vomiting, diarrhea, and muscular pain; bleeding diathesis with multiorgan dysfunction is seen in severe cases , sampled from livestock in Punjab Province, for CCHFV antigen by commercial ELISA .During 2017–2018, we tested 3,710 serum samples from 1,872 humans and 1,838 domestic animals  for antibodies against CCHFV . We also>65 years of age  human samples to be positive for CCHF antibodies by using a 2-step approach, ELISA and confirmatory testing by immunofluorescence assay . We obses of age . Of the https://www.id-vet.com). The prevalence of CCHFV antibodies was significantly higher (p<0.01) among camels  than among cattle , sheep , buffalo , and goats   were positive for CCHF by a commercial ELISA  .2 value of 0.277 and the Hosmer-Lemeshow goodness-of-fit test  indicated that this is a poor model for predicting CCHFV exposure in the sampled livestock population.We built a binary logistic regression model to evaluate possible risk factors for CCHFV seropositivity in animals and humans. The final model  Table 6 2 (0.070) and Hosmer-Lemeshow goodness-of-fit test  indicated that our model is a reasonable model for predicting past exposure to CCHFV in the tested human population.In humans, we found the chance of exposure to CCHFV was highest for populations from Balochistan   Table 7 Rhipicephalus tick samples tested positive for CCHFV antigen by ELISA. Of these 8 positive samples, we confirmed 1 tick (T61) and 3 buffalo samples  through partial amplification and sequencing of the small (S) segment (260 bp). The 4 partial sequences of CCHFV S segments (GenBank accession nos. MN135938–MN135941) were 97%–95% identical to virus sequences found in Afghanistan (accession no. JX908640.1), Iran (accession no. KX096702.1), and Oman (accession no. KY362516.1) and clustered together with genotype IV (Asia) (https://www.illumina.com). Phylogenetic trees for the S, M, and L segments showed that the T61 strain clustered with genotype IV (Asia)  . We obtaHyalomma tick positive for CCHFV; CCHFV has been reported from Rhipicephalus ticks from Iran and clustered together with strains from Pakistan and Iran, indicating that Rhipicephalus ticks have been naturally infected with closely related virus in the region  indicates a potential role of these animals in human infections. Demonstration of the virus in animal blood plasma and tick samples by reverse transcription PCR provides strong evidence of active circulation of CCHFV in Pakistan. Furthermore, genetic characterization of the virus reconfirms the circulation of genotype IV in Pakistan (A high proportion of seropositive humans from Balochistan and Khyber Pakhtunkhwa with a history of exposure to animals is in concordance with earlier reports of CCHF in humans from these areas. The rural economy of Balochistan and Khyber Pakhtunkhwa is based on livestock production, and the increased contact with animals may explain the higher antibody prevalence in humans from these areas. Furthermore, the prevalence of antibodies was significantly higher among herdsmen than among the general population.In summary, our results indicate the ongoing circulation of CCHFV among animals and humans in some regions of Pakistan. Longitudinal surveys to identify and define the genomic diversity of CCHFV in Pakistan and investigations to explore the exact role of camels in the ecology of this virus would help clarify the risk to the general population and occupational hazards for livestock farmers and veterinarians.Additional information about surveillance of tickborne Crimean-Congo hemorrhagic fever virus in humans and livestock, Pakistan, 2016–2017.


Serological assays detecting neutralising antibodies are important for determining the immune responses following infection or vaccination and are also often considered a correlate of protection. The target of neutralising antibodies is usually located in the Envelope protein on the viral surface, which mediates cell entry. As such, presentation of the Envelope protein on a lentiviral particle represents a convenient alternative to handling of a potentially high containment virus or for those viruses with no established cell culture system. The flexibility, relative safety and, in most cases, ease of production of lentiviral pseudotypes, have led to their use in serological assays for many applications such as the evaluation of candidate vaccines, screening and characterization of anti-viral therapeutics, and sero-surveillance. Above all, the speed of production of the lentiviral pseudotypes, once the envelope sequence is published, makes them important tools in the response to viral outbreaks, as shown during the COVID-19 pandemic in 2020. In this review, we provide an overview of the landscape of the serological applications of pseudotyped lentiviral vectors, with a brief discussion on their production and batch quality analysis. Finally, we evaluate their role as surrogates for the real virus and possible alternatives. Co-infection of the same cells with ALV allowed the envelope-less RSV to acquire the ALV Env and infect cells, leading to malignancy. The pseudotyped viruses’ tropism at entry level and neutralisation properties are therefore determined by the Env on their surface, and not by the genetic information within the virus particles. Nowadays, this naturally occurring phenomenon has been engineered to serve many purposes such as gene therapy, virus–host interaction and serological studies. Many viruses have been exploited as a basis for a pseudotype [Retroviridae family, genus Lentivirus. The relatively simple genome structure  is convenient for genetic manipulation [The first naturally observed pseudotype was a replication-defective Rous sarcoma virus (RSV) carrying on the particle surface the envelope protein (Env) of avian leukosis virus (ALV) . The RSVeudotype ,3, with eudotype ,5,6. LVspulation . Initialpulation . Howeverpulation . The mospulation  or more pulation . The abipulation ,14.Serological assays are used for the evaluation of the immune response, specifically antibodies, in serum, plasma or other body fluids. Detection of pathogen-specific IgM can aid the diagnosis of acute infection in support of the preferred molecular methods detecting the genetic material . This is particularly important for those infections characterised by a short viremia, such as Zika , or thosDetection and quantification of NAbs are considered the main correlates to evaluate the efficacy of vaccines, determine a protection titre and decipher the immune status of individuals to identify the risk of infection. For enveloped viruses, NAbs are primarily targeted towards the Env, blocking receptor binding and/or fusion and therefore the processes of entry and uncoating within the cell. They are often the best correlates of protection following vaccination . During There are many other examples in the emerging virus field where a pseudotyped LV has been used to investigate NAbs induced by candidate vaccines. Chikungunya virus-pseudotyped LVs have been used to determine the level of NAbs raised in non-human primates by a virus-like particle vaccine candidate and showed good correlation between the pseudotyped LV neutralisation method and the traditional PRNT with the live virus ; similarFollowing a different approach in the case of rabies virus vaccines, pseudotyped LVs have been exploited to evaluate levels of cross-neutralisation afforded by the vaccine to other antigenically diverse species within the Lyssavirus genus—through the screening of sera collected from vaccine recipients . ConstraVaccine development against hepatitis C virus (HCV) is complicated by a high level of genetic diversity, giving rise to multiple mechanisms of immune evasion , coupledEpidemiology studies provide the ability to understand a pathogen’s transmissibility  virulence  impact on the community and to inform guidance for government policies and implementation of countermeasures. Epidemiology studies have focused primarily on the detection of antibodies as they remain in circulation long after the symptomatic stage of infection has occurred. Large serosurveys for emerging viruses have, for example, highlighted the longstanding disease burden of viruses circulating in Sierra Leone and the potentially high prevalence of Ebola virus antibodies in the population ,51. The With many enveloped viruses being zoonotic, a One Health approach is often applied to seroepidemiology studies. The detection of a high prevalence of MERS-CoV NAbs in dromedary camels compared to humans and other domestic livestock helped identify them as the potential reservoir in the Middle East region ,27,28. IFlaviviridae and Togaviridae [Togaviridae family, such as chikungunya virus (CHKV), Semliki Forest virus, Ross River virus and Mayaro virus, cause similar clinical symptoms, usually fever and long-lasting arthralgia. The pseudotyped LV platform not only offers the advantage of a lower containment level  [Ebolavirus. They identified cross-reactive potent NAb epitopes hidden by a glycan shield in the mucin-like domain of the glycoprotein, leading to the suggestion that removal of these regions in a candidate vaccine could increase the induction of NAbs [The outbreak of an arbovirus, Zika virus, in 2015 was recognised by the World Health Organization (WHO) as a Public Health Emergency of International Concern  and has aviridae ,105. Alpgroup 3) ,106,107,group 3) . This ty of NAbs . Viral e of NAbs .A further advantage of the inherent flexibility provided by the pseudotype system has been in the ability to easily manipulate the Env incorporated on the LV, which can be exploited to identify NAb epitopes. For instance, epitope specificity of candidate therapeutic monoclonal antibodies (mAbs) for the post-exposure prophylaxis of rabies virus has been characterised by displaying an Env with mutated antigenic sites on the pseudotyped LV and evaluating the effect on neutralisation ,84. TakiOne of the attractive properties of pseudotypes comes from their amenability to a high-throughput setting, which is particularly useful for the discovery of drugs inhibiting viral entry. In the case of emerging viruses, this has involved screening existing licensed medicines for the potential to be repurposed in the event of an outbreak. A study by Wang et al.  describeDespite many protocols having been published describing the production and titration of pseudotyped LVs ,115,116,Other factors to consider when optimising pseudotyped LV production include the choice of the envelope expression plasmid and the ratio used alongside those containing the packaging and reporter elements, as well as the transfection reagent for delivery to the producer cell. The importance of applying an empirical approach to optimising total plasmid amount and ratios for each specific Env of study has been well demonstrated by Urbanowicz et al. , for hep50) [Reporter genes are incorporated within the LV as an RNA dimer which is reverse transcribed and expressed following integration into the target cell genome post-entry. Multiple reporter genes have been incorporated within the pseudotype platform , yet β-g50) ,131. ThiDetection of the reporter gene signal from pseudotyped LV-infected cells provides a functional titre; however, other non-functional measurements have been used to quantify the LV stock and normalise the viral input in downstream applications. The most commonly used method is determining the amount of the LV core protein by antigen capture ELISA measuring HIV p24 levels. It is accepted in the field that 1 ng of p24 will correspond to 1000–5000 particles ,133. SevQuantification of the infectious viral particles, i.e., transducing particles, and ratio with the total amount of particles in the stock preparation is one of the factors which should be considered when looking at the quality of a batch of LVs, as listed in the guidelines on the development and manufacture of LV published by the European Medicine Agency . This do2 = 0.78 [Optimisation of the production of pseudotyped LVs should also take into account the downstream application of these particles. For serological assays, it is important that the antibody response in a sample is the same, or at least correlates, between assays employing pseudotyped LVs and those using the real virus. There are many examples in the literature showing a good correlation in neutralisation assays. Wright et al. demonstrated that rabies antibody titres in over 190 Tanzanian dogs obtained from a pseudotyped LV- based neutralisation assay correlate strongly with the fluorescent antibody virus neutralisation (FAVN) live virus assay, r = 0.915 . Similar2 = 0.78 ,107. Goo2 = 0.78  and of c2 = 0.78 . Alberin2 = 0.78 . However2 = 0.78 . Moreove2 = 0.78 ,163. A cThere are several examples of viruses whose genome has been manipulated to create pseudotyped vectors. As mentioned in the previous paragraph, the VSV pseudotyping platform has become increasingly popular . A benefFlavivirus genus such as West Nile virus (WNV) and Japanese encephalitis virus [The Envs of some viruses have been difficult to nearly impossible to pseudotype into the above-mentioned platforms—for instance, many members of the is virus . The issis virus . Similaris virus ,172. TheOver the course of the last two decades, pseudotyped LVs have cemented themselves as a robust alternative to handling live virus isolates for serological investigations. Studies have exploited unique flexibilities in their design and construction to advance knowledge in challenging areas, overcoming constraints on resources, the need for high-containment facilities or the lack of cell culture systems. While taking note of where we stand today, it is evident that there is an increasing uptake of pseudotyped LVs; as exemplified by the record number of studies throughout 2020 which have widely reported their use in response to the emergence of SARS-CoV-2. We foresee there will be a benefit and need for enhanced characterisation of pseudotyped LV stocks in support of serological investigations supporting medicinal product licensure. Further, enhanced methods of quantification will play a role in comparisons against other novel vector systems, assisting identification of the most appropriate platform for each Env. LVs will act as a prototype in this continued trend towards pseudotype-based serology.