Oryctolagus cuniculus) as a model species to compare virome compositions in a vertebrate host and known associated ectoparasite mechanical vectors, in this case, fleas and blowflies. In particular, we aimed to infer the extent of viral transfer between these distinct types of host. Our analysis revealed that despite extensive viral diversity in both rabbits and associated ectoparasites, and the close interaction of these vertebrate and invertebrate species, biological viral transmission from ectoparasites to vertebrate species is rare. We did, however, find evidence to support the idea of a role of blowflies in transmitting viruses without active replication in the insect.Ectoparasites play an important role in the transmission of many vertebrate-infecting viruses, including Zika and dengue viruses. Although it is becoming increasingly clear that invertebrate species harbor substantial virus diversity, it is unclear how many of the viruses carried by invertebrates have the potential to infect vertebrate species. We used the European rabbit  and myxoma virus. We compared virome compositions in rabbits and these ectoparasites, sequencing total RNA from multiple tissues and gut contents of wild rabbits, fleas collected from these rabbits, and flies trapped sympatrically. Meta-transcriptomic analyses identified 50 novel viruses from multiple RNA virus families. Rabbits and their ectoparasites were characterized by markedly different viromes, with virus abundance greatest in flies. Although viral contigs from six virus families/groups were found in both rabbits and ectoparasites, they clustered in distinct host-dependent lineages. A novel calicivirus and a picornavirus detected in rabbit cecal content were vertebrate specific; the newly detected calicivirus was distinct from known rabbit caliciviruses, while the picornavirus clustered with sapeloviruses. Several picobirnaviruses were also identified that fell in diverse phylogenetic positions, compatible with the idea that they are associated with bacteria. Further comparative analysis revealed that the remaining viruses found in rabbits, and all those from ectoparasites, were likely associated with invertebrates, plants, and coinfecting endosymbionts. While no full genomes of vertebrate-associated viruses were detected in ectoparasites, small numbers of reads from rabbit astrovirus, RHDV, and other lagoviruses were present in flies. This supports a role for flies in the mechanical transmission of RHDV, while their involvement in astrovirus transmission merits additional exploration.IMPORTANCE Ectoparasites play an important role in the transmission of many vertebrate-infecting viruses, including Zika and dengue viruses. Although it is becoming increasingly clear that invertebrate species harbor substantial virus diversity, it is unclear how many of the viruses carried by invertebrates have the potential to infect vertebrate species. We used the European rabbit (Oryctolagus cuniculus) as a model species to compare virome compositions in a vertebrate host and known associated ectoparasite mechanical vectors, in this case, fleas and blowflies. In particular, we aimed to infer the extent of viral transfer between these distinct types of host. Our analysis revealed that despite extensive viral diversity in both rabbits and associated ectoparasites, and the close interaction of these vertebrate and invertebrate species, biological viral transmission from ectoparasites to vertebrate species is rare. We did, however, find evidence to support the idea of a role of blowflies in transmitting viruses without active replication in the insect. Ectoparasites act as vectors for many notable viral pathogens of vertebrates –3. Trans–4–Oryctolagus cuniculus) has been profoundly impacted by ectoparasite-mediated viral transmission. As rabbits are a pest species in Australia, two virus biological controls—rabbit hemorrhagic disease virus  and myxoma virus —were deliberately introduced to control wild rabbit populations in the 1950s and 1990s, respectively (Spilopsyllus cuniculi and Xenopsylla cunicularis) aid MYXV transmission  (9The European rabbit (iruses) 913. As viiruses) 9.Despite the importance of the ectoparasite-vector system in virus transmission and evolution, little is known about the composition of virus communities in vertebrates and their associated ectoparasites and particularly how commonly viruses are able to infect both host types. Metagenomic studies of arthropod vector species such as mosquitoes and ticks have revealed an unexpectedly rich diversity of viruses, most of which likely do not infect vertebrates , 16. HowThe advent of bulk RNA sequencing (“meta-transcriptomics”) has revolutionized our perception of viral diversity and host range , 18, revRNA sequencing of 10 rabbit sample libraries and 9 invertebrate sample libraries generated a total of 200,290,927 paired-end (PE) reads (40 Gbp) and 260,721,139 PE reads (52 Gbp), respectively, with rabbit library size ranges of 19,127,004 to 21,164,747 PE reads and invertebrate library size ranges of 27,455,130 to 30,538,954 PE reads. Of the reads from the rabbit libraries, 85% did not map to host rRNA, while 96% of invertebrate reads did not map to published host rRNA.Spilopsyllus cuniculi (rabbit fleas) based on the presence of several highly abundant contigs of Spilopsyllus cuniculi rRNA and EF1a genes and the absence of any other Spilopsyllus species genes. A library of unidentified Chrysomya species (GUNChsp library) was determined to represent Chrysomya rufifacies or Chrysomya albiceps  based on EF1a and rRNA gene data. An unknown Sarcophaga species was most likely Sarcophaga impatiens based on 28S rRNA identity.The majority of arthropods analyzed in this study were identified to the species level through visual inspection. The remainder were characterized using the transcriptome sequencing (RNA-Seq) data. Fleas were confirmed to be n = 5 species), than at site 2 (Gudgenby) in Namadgi National Park (n = 2 species) , and Sarcophaga (Sarcophagidae) were collected from Gungahlin, while species from Calliphora and Musca (Muscidae) were isolated from Gudgenby. Calliphora augur was the only species trapped at both sites , a suburb of Canberra (species) . Speciesth sites . While iustralia .Calliphora vicina had the highest virus abundance (as a proportion of non-rRNA reads), with almost 2% of non-rRNA reads of viral origin, while Chrysomya species had viral abundances of only 0.012% to 0.018% , and the Chrysomya and Musca libraries were the least diverse, with 4 to 5 viral families per library. Although only fleas and Calliphora augur were sampled from both sites, the viral diversity of these two species at each site suggests that viral composition was associated with host species rather than collection location , with decreasing similarity in viral composition at the family level and beyond , and unclassified Bunyavirales groups. Additionally, many of the viruses found in insects, particularly fleas, were potentially viruses of fungi, protozoa, or algae, being present in the Hypoviridae, Narnaviridae, Partitiviridae groups, the Totiviridae-Chrysoviridae group, and certain Phenuiviridae . The Bromoviridae virus identified in Gungahlin fleas clusters firmly among plant viruses, and with an abundance of only 0.002%, it likely represents a plant virus incidentally carried by one or more fleas in the library rather than a virus that replicates in fleas. Indeed, care must be taken in assigning viruses to hosts on the basis of metagenomic data alone. The Iflaviridae flea viruses found in this study clustered most closely with Watson virus (Pygiopsylla) from Australian marsupials (sharing 68.6% amino acid identity in the RdRp).To establish the diversity and potential hosts of newly defined viruses, family-level  phylogenetic trees were estimated using the virus RNA-dependent RNA polymerase (RdRp) , and 5. on virus , detecteNo viral contigs could be assembled from the rabbit liver, duodenum, or lung libraries. A small number of viral contigs were found in the Gudgenby blood library, but these represented potential contaminants since (i) the rabbits from Gudgenby had been shot, occasionally resulting in perforation of the cecum, which would contaminate blood in the body cavity; (ii) all viruses detected in the blood were also detected in the cecum (including plant viruses unlikely to be in blood); and (iii) no viruses were found in the blood of rabbits from the Gungahlin site where there was no body cavity contamination.Narnaviridae and Virgaviridae were both highly abundant in the cecal content of rabbits from both locations, while Tombusviridae was a major component of the cecal virome of Gudgenby rabbits , the first encoding a polyprotein that included RdRp and capsid domains and the second encoding a small protein of unknown function . Ac. AcPicorSince RNA sequencing was conducted on pools of 18 to 20 samples, specific reverse transcriptase PCRs (RT-PCRs) for the novel racaecavirus and orycavirus identified here were designed to determine their frequency in individual animals. In the Gungahlin cecal content samples, racaecavirus was detected in 4 of 20 samples tested, while orycavirus was detected in 10 of the 20 samples. Despite our finding no racaecavirus or orycavirus contigs in the Gudgenby cecal content library, one sample from this library had a weakly RT-PCR-positive result for both viruses. Subsequent mapping of reads from this library to racaecavirus and orycavirus contigs resulted in 8 and 7 reads mapping to each, respectively. Therefore, these viruses may also occur at a low frequency in Gudgenby.Rabbit picobirnavirus 1 to 9. Importantly, these viruses did not form a monophyletic group but were distributed throughout genogroup 1 among picobirnaviruses from different hosts. This pattern is typical of the Picobirnaviridae, i.e., showing limited host structure in the RdRp phylogeny , Solemoviridae/Sobemo-like (plant/invertebrate-associated), Narnaviridae (fungus/parasite/invertebrate-associated), Partitiviridae (plant/invertebrate/fungus/vertebrate feces-associated), Tombusviridae (plant/invertebrate-associated), and Toti-Chryso (parasite/invertebrate/fungus-associated) groups were assembled from both the rabbit cecal content and the ectoparasites , and the benign rabbit calicivirus Australia-1 (RCV-A1). Because of recombination between RHDV, RHDV2, and RCV-A1 . This is consistent with the absence of visible clinical signs of myxomatosis in the sampled rabbits. In addition, no viruses with known pathogenic potential in humans were detected in fleas or flies.Importantly, these viral reads were unlikely to represent contaminants from other libraries since they were not highly abundant in any library and were not present in every library. However, since some viruses were represented by as little as a single read per library, we confirmed the presence of RHDV-like viruses in invertebrates by RT-PCR . NotablyEctoparasites are rich in virus diversity , 24, andNarnaviridae, Solemoviridae, Bromoviridae, Virgaviridae, Partitiviridae, Tombusviridae, and Toti-Chryso. Those in the Narnavirdae, Partitiviridae, and Tombusviridae likely constitute new viral species   , suggestctively) , althougctively) , 31. As Caliciviridae and Picornaviridae are frequently detected in vertebrates  and Marmot norovirus  likely belongs to that genus  were discovered in fleas collected from rabbits and Calliphoridae, Sarcophagidae, and Muscidae flies trapped sympatrically  and 8. Viviridae . Generaliviridae  to 5, wiarsupial . Virusesarsupial , althougarsupial  and 5. TCaliciviridae) in three Calliphoridae fly species , Australia. Site 1 was at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Crace , Gungahlin (GUN), a suburb of Canberra, while site 2 was at Gudgenby Valley  in Namadgi National Park (Gudg). At Gungahlin, rabbits were trapped in carrot-baited cages and killed by cervical dislocation. Trapping occurred over 3 to 5 consecutive nights for two separate weeks of the 2016/2017 Southern Hemisphere summer (18 to 22 December 2016 and 8 to 11 January 2017). A total of 20 rabbits were sampled , with weights ranging between 0.27 kg and 1.95 kg . At Gudgenby, rabbits were killed by shooting on 2 February 2017. Eighteen rabbits were collected , weighing between 0.52 kg and 2.2 kg . Blood (in EDTA tubes), lung, liver, duodenum, and cecal content were collected from each rabbit. Where fleas were present on rabbits, they were collected and grouped by rabbit. Tissues and fleas were stored below –80°C immediately after collection. Sampled rabbits displayed no obvious signs of serious pathology.Commercially available fly traps (Envirosafe) were placed at the same locations in the same weeks as the rabbit sampling. Traps were baited with rabbit tissue/gut content and/or chicken necks, and bait was physically separated from flies to prevent contamination. Fly traps were left out for periods of up to 24 h. To ensure fresh samples, only live flies were taken from traps. Live flies were chilled at 4°C or –20°C for periods of 5 to 10 min to allow initial visual identification of fly species before they were frozen at –80°C. A total of 149 flies representing 5 species were collected from Gungahlin, while 22 flies from 2 species were collected from Gudgenby . While tAll work was carried out according to the Australian Code for the Care and Use of Animals for Scientific Purposes with approval from the institutional animal ethics committee (permit CWLA-AEC#16-02).RNA was extracted separately for each sample from 20 mg of rabbit tissue or bone marrow, 75 μl of rabbit blood, individual whole flies, or groups of at least 5 fleas from individual rabbits. RNA was extracted using a Maxwell 16 LEV simplyRNA tissue kit in combination with a Maxwell nucleic acid extraction robot , according to manufacturer’s instruction, including DNase treatment.Rabbit RNA was pooled by tissue type and collection site, with up to 20 individuals per pool, while insect RNA was pooled by species and collection site, with pool sizes ranging from 2 to 10 individuals . Where lDe novo assembly of reads into contigs was performed using Trinity (−10) and nonredundant protein (nr) database , respectively. Results were filtered so that only contigs that had a viral hit (excluding endogenous viruses/retroviruses) from each BLAST search were retained. Trinity  followin Trinity . The RSE Trinity  in Trini−4 for BLASTx and 1 × 10−10 for BLASTn. A conservative approach was taken such that only those reads that had a virus result in both the BLASTn and BLASTx analyses were considered legitimate hits. Ectoparasite library read-mapping to specific virus reference sequences or rabbit viral contigs was conducted using Bowtie2  and a near-complete O. cuniculus 28S rRNA sequence obtained from the SILVA high-quality ribosomal database (GBCA01000314). The arthropod rRNA target index was generated from 18S and 28S GenBank sequences from Spilopsyllus cuniculi and multiple Chrysomya, Calliphora, Sarcophaga, and Musca species. The total number of reads that did not map to host rRNA for each library was used as the denominator to calculate the percentage of reads mapped to viral contigs.To quantify the amount of residual host rRNA sequences in each data set, all reads were mapped to host rRNA using Bowtie2 . The rabdatabase   the virus was detected in at least a third of the libraries sequenced on the same sequencing lane, (ii) the read count representing the abundance of the virus was less than 0.1% of that representing the highest count for that virus among the other libraries, and (iii) the virus shared >99% nucleotide sequence identity with a virus from another library. These criteria were defined on the basis of the observations that barcode hopping usually results in contamination of most libraries on a lane, the index hopping rate is usually about 0.01% to 0.1%, and viral sequences from barcode hopping would be genetically identical to a virus from another library .Reference RNA-dependent RNA polymerase (RdRp) amino acid sequences for each virus family were downloaded from NCBI and aligned with viral contigs using MAFFT v7.271 . Where nPrimer sets were designed to amplify a small region of each of the novel rabbit calicivirus and picornavirus genomes for the detection of these viruses in individual cecal content samples. Calicivirus primer set GRC_F5.6  and GRC_R5.9  amplified a 278-bp region, while picornavirus primer set GRP_F6.5  and GRP_R6.9  amplified a 401-bp region. RT-PCRs were conducted using a Qiagen OneStep Ahead RT-PCR kit according to the manufacturer’s directions with 1 μl of RNA (diluted 1:10 in nuclease-free water) in a 10-μl reaction volume with a 0.25 μM concentration of each primer. PCR conditions included 10 cycles of touchdown PCR, with the annealing temperature decreasing by 0.5°C each cycle from a starting temperature of 60°C and a further 30 cycles with annealing temperature at 55°C. Representative amplicons were subjected to Sanger sequencing to confirm their legitimacy.Taq Polymerase High Fidelity kit according to the manufacturer’s protocol with specifically designed forward primer GRC_F6.2 (5′-CAG AGA ATG AGC TCA ACC GAC A-3′) and reverse primer GV271 , with 5 μl of RNA and 0.5 μM GV270 gene-specific primer  in a 20-er GV271 . ReactioRNAs from individual flies and from the bone marrow of rabbit carcasses found near Gungahlin fly traps were screened for the presence of pathogenic lagoviruses using a multiplex RT-PCR method described previously .PRJNA594431 and BioSample accession numbers SAMN13518403 to SAMN13518421. The new virus nucleotide (consensus) sequences presented in phylogenies are available in NCBI/GenBank under accession numbers MT129676 to MT129780.All raw data (fastq files) generated for this study are available in the NCBI SRA database under BioProject accession number


Changes in stoichiometry, temperature, strain and other parameters dramatically alter properties of LSMO perovskite. Thus, the sensitivity of LSMO may enable control of the magnetic properties of the film. This work demonstrates the capabilities of interface engineering to achieve the desired effects. Three methods of preparing STO substrates were conducted, i.e., using acid, buffer solution, and deionized water. The occurrence of terraces and their morphology depend on the preparation treatment. Terraces propagate on deposited layers and influence LSMO properties. The measurements show that anisotropy depends on the roughness of the substrate, the method of preparing the substrate, and oxygen treatment. The collected results suggest that the dipolar mechanism may be the source of LSMO anisotropy. For thi3 (LSMO) ,19,20.3 (STO). Its structural complementarity with LSMO  allows epitaxial growth of a multilayer when the pulsed laser deposition (PLD) method is applied [3 [An ideal substrate for the LSMO layer is monocrystalline perovskite SrTiO applied ,13. Therplied [3 ,22,23 anplied [3 ,25, haveplied [3 ,28,29,30Currently, there are several unresolved problems of a fundamental nature that at least hinder the formation of multiferroic junctions with parameters allowing for their practical applications. One of these problems is too low magnetocrystalline anisotropy of LSMO layers, resulting in a lack of two distinctly different magnetization configurations/settings in one of the electrodes. There are also other sources of anisotropy, e.g., magnetoelastic ,32,33. CThe aim of this article is to show how to control the magnetic anisotropy of LSMO. It can be done by introducing disturbance sources of the atomic arrangement symmetry, such as steps on the STO substrate and thus on the deposited LSMO layer. Different STO substrate preparation methods lead to different roughnesses of the surface, and thus different magnetic anisotropies. Magnetic anisotropy also depends on the amount of oxygen during and after deposition.3 (3:1), deionized water, acetone and methanol, followed by annealing at 1000 °C in an oxygen atmosphere for an hour [4F: HF (7:1), acetone, methanol and deionized water, followed by annealing at 1000 °C in an oxygen atmosphere for an hour [Three preparation methods of a single-crystal STO (100) substrate were conducted. The selected substrates showed a similar miscut effect (measured using the XRD measurement). The miscut is the effect of a slight mismatch between the polishing direction and the atomic plane. It results in a stepped surface with a random terrace direction and the width of the terraces corresponding to the miscut angle. The first method involves successive ultrasonic rinses in HCl-HNO an hour . For sim an hour , hereaft an hour , called −2, with the distance between the sample and the target equal to 6.5 cm, pulse width of 20 ns, repetition rate of 10 Hz, and 1200 as the number of pulses. The aim was to obtain stoichiometric LSMO. Laser deposition was performed under 200 mTorr partial oxygen pressure and a substrate temperature of 750 °C. The thicknesses of the deposited LSMO layers are about 20 nm.Thin LSMO films were grown onto the prepared STO by the pulsed laser deposition (PLD) technique, using a 248 nm excimer laser system  operated at an energy density of ~2 J·cmThe surface morphology and the grain size dependence on the buffer were examined with an atomic force microscope (AFM) using the NTEGRA Aura—NT-MDT system . The crystallographic structure of the samples was investigated by x-ray diffraction (XRD) measurements, using an X’Pert MPD diffractometer . The hysteresis loops were measured by a vibrating sample magnetometer (VSM) LakeShore 7407  at room temperature in an external magnetic field applied at different angles in the plane of the sample.The proper preparation of the STO substrate is crucial for obtaining high quality magnetic properties of the deposited layers. The substrate must have sufficient smoothness to allow epitaxial growth. Three different substrate preparation methods were tested, followed by atomic force microscopy (AFM) measurements. The roughness is calculated from the height profile of the samples measured perpendicular to the terraces. It is calculated as the average absolute value of the height. In the ideal case, the maximum value would be half the unit cell , and the roughness would be a quarter, i.e., 0.97 Å. The measured roughness for all samples is less than half of the height of the unit cell, which means that they all have an atomically smooth surface. However, the roughness at this level should be understood as a higher number of defects, which will directly affect the deposited layers on such a substrate. In the real case, it is often difficult to distinguish where the terrace ends and where it begins, and the model terrace rarely takes place.2 termination), whereas the “buffer” method results in a surface rougher than that obtained with the “acid” one but smoother than in the case of the “DIW” method. Sometimes even larger defects appear. Generally, a good measure of these effects is roughness, which even for clearly stepped surfaces may have different values  are shown in Thin ferromagnetic films grown on stepped surfaces show complex magnetic anisotropy due to their reduced symmetry ,41. The M—magnetization, D—thickness of the layer, In the lowest order of the perturbation theory, we have a simple formula for approximation :(1)ΔEΔE0relation :(2)HA~ααL is the average width of the terrace.In their model, Arias and Mills assumed the ideal form of terraces. As shown in Thus, we get:For our sample, we are in the regime of low roughness, so we should see a simple linear dependence of magnetic anisotropy as a function of roughness3+ ions because of missing O-Mn bonds. The oxygen vacancy thus leads to the destruction of Mn3+–Mn4+ chains, in which hopping electrons in the double exchange mechanism lead to ferromagnetic ordering. In turn, the Mn3+–Mn3+ ion chains in the superexchange mechanism may lead to segregation into a form of larger antiferromagnetic clusters [All the previously shown results concerned quickly cooled (after deposition) LSMO samples without adding additional oxygen after deposition. clusters . This reclusters . Oxygen Three methods of STO substrate preparation were tested to obtain atomically flat surfaces. All these methods resulted in visible surface terraces with heights of about a unit cell and a roughness of less than 2 Å. LSMO layers were deposited on engineered STO substrates using PLD method. The propagation of the terraces on the deposited layers, with the same width and direction as those on the substrate, was confirmed even in the case of several dozen nm of LSMO. The use of appropriate deposition conditions  and the existence of terraces causes defects and oxygen vacancies, and thus magnetic anisotropy. The existing correlations between terraces and magnetic anisotropy have been confirmed in the samples by VSM, XRD and AFM measurements. The magnetic hard axis is perpendicular to the terraces and the magnetic easy axis is along the terraces. The correlation between the preparation of the substrate and its roughness as well as between its roughness and magnetic anisotropy has been shown. The most invasive methods, such as the use of acids and buffers, give the smoothest surface and lowest magnetic anisotropy. The highest anisotropy occurs for rougher surfaces obtained, for example, by only annealing and ultrasonic bath in deionized water. The dependence of the anisotropy field on roughness can be explained by the dipolar mechanism proposed by Arias and Mills . It occu


Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins  in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today. Viruses are infectious agents associated with many diseases in the human population, some of which can lead to epidemics and pandemics. To prevent such events, both the quick detection of agents isolated from patient and natural samples and the study of the properties of isolates are necessary. Currently, there are several approaches for virus detection. There are detection methods associated with PCR, CRISPR/Cas technology, NGS (next generation sequencing), immunoassays, and cell-based assays ,5,6,7,8 Another popular method for virus detection is to use antigen-based assays. This approach can be used both in research and in clinical diagnostics. The standard methods using antibodies against viruses include virus neutralization tests, hemagglutination inhibition, complement fixation, and indirect immunofluorescence. These methods are used only for diagnosing already known viruses whose capsid structure has been studied. The evaluation of viral titers using end-point dilution analysis is based on a hemagglutination test of the cell culture supernatant. However, as observed for influenza virus due to the rapid accumulation of mutations in the viral genome sequence encoding hemagglutinin leading to amino acid replacements, the investigation of the antigenic characteristics of viruses using this method is complicated . One of There is a group of methods for detecting viable viruses using cell culture. The development of these methods for plaque detection began in 1950 ,22. MethIn summary, cell-based methods are one of the important categories of viral detection and characterization approaches that are currently available. All of these strategies have advantages and disadvantages, which are described in this section, but the main distinguishing characteristic is that cell-based methods can be used to isolate and study living viral particles; however, there is a problem in the application of these methods related to low susceptibility (virus ability to enter cells) and permissivity (virus ability to replicate in cells) in cell cultures. Since these two properties of cell lines make a shared contribution to the development of cellular methods, they can be generally referred to with the term “sensitivity” unless specific differences are mentioned in a report. To solve this problem, new approaches are being developed. The sensitivity of cell lines to specific viruses depends on both the virus attaching to cell membrane receptors and the host genes associated with the cellular antiviral response. Modern methods of molecular biology have allowed the development of genetically modified cells in which viral production is increased ,26,27. TTechnologies for increasing the sensitivity of cell cultures are developed to improve approaches for the study of viable viral particles. To study the biology of viruses, such as the factors determining their host species specificity and proliferation rate and the production of viable viral particles, cell lines that allow virus propagation are needed. One of the essential properties of a virus is its ability to enter the cell and to produce viral progeny. Viral replication is followed by morphological and biochemical host cell alterations, which ultimately lead to cell destruction. There are several forms of these changes depending on the virus species, such as syncytium formation, cell rounding, or cell lysis. Such visible cellular changes are commonly referred to as cytopathic effects . Based oIn another study related to the influenza virus, the MDCK cell line was modified by the overexpression of human airway transmembrane proteases (TMPRSS2 or MSPL). In the absence of trypsin, these cells supported the growth of three recombinant vaccines based on pathogenic avian influenza viruses . The folIt can be concluded that virus isolation by cell culture followed by CPE determination is the most commonly used method for virus identification in biological samples , and modTo make cell culture-based methods simpler, more informative, and more convenient, reporter cell lines have been developed. A reporter cell line comprises specific cells that produce a reporter protein in response to virus infection. The reporter should be very specific to the virus of interest to allow researchers to detect a certain virus. Upon viral infection, a viral protein recognizes a reporter construct as a viral genome or chimeric protein and produces a signal that can be detected. Reporter cell lines can be very helpful for virological studies and vaccine manufacture. Although reporter-based methods can require the growth live pathogenic viruses and therefore be confined to laboratories with certain biosafety levels, they are easy to scale up and less laborious than standard virological methods, thus greatly increasing infectious virus detection. Such methods have led to rapid, objective virus quantitation studies, including the assessment of viral titers, antiviral susceptibility, and neutralization titers ,46. The The key feature of a reporter construct is the reporter gene. Fluorescent proteins and enzymes such as beta-galactosidase or luciferase can serve as reporter genes. It should be noted that there are various elements used for detecting reporter activity in studies of different virus families depending on their genome structure and life cycle. Negative-strand RNA viruses  showing replication in the host nucleus have been detected using a construct known as a minigenome ,8. The rBelow, we will consider the development of some reporter cell lines that are currently used in virology for the detection of different virus families. These studies can provide a basis for the development of detection systems for novel viruses based on similarity of the design approaches used to assemble the reporter constructs for previously characterized viruses belonging to the same class.Influenza A and B are widespread viruses that are responsible for annual epidemics in humans and are associated with large economic losses. Influenza is categorized into different subtypes and strains depending on its neuraminidase and hemagglutinin types . The accEbolavirus is a filovirus that causes hemorrhagic fever that has been widespread in Central Africa and is now emerging in Western Africa and Europe. Ebolavirus disease takes the form of an acute fever and results in approximately 50% mortality . As mentReporter cell lines for ebolavirus detection were developed based on the negative-sense minigenome, which is regulated by the constitutive cytomegalovirus (CMV) promoter for transcription by cellular RNA polymerase II  Figure . The addThe subfamily of alphaviruses includes the Sindbis virus, which causes severe fever in humans. The nonsegmented genome of the virus consists of two functional regions. The direct translation of the polyprotein, which is further processed by the viral protease, begins from the first part of the ssRNA(+). These proteins are required for the transcription of late proteins and the replication of the viral genome. Late or structural proteins are encoded by subgenomic RNA transcribed from ssRNA(−). The cDNA cloning of the Sindbis virus genome was the first step toward reporter cell line development . The repRubella is a severe contagious viral infection. Although the disease caused by this virus is mostly mild in children and adults, during pregnancy, it can lead to stillbirth or fetal malformations. Thus, an accurate diagnosis of rubella is necessary when infection is suspected ,62. CurrHepatitis C virus (HCV) causes severe acute or chronic hepatic injuries. HCV infections progress to chronic hepatitis, which is highly associated with hepatocellular carcinoma. An estimated 71 million people exhibit chronic hepatitis C virus infection . HepatitThe other reporter cell-based method involves the construction of a minigenome mimicking the viral genome that is recognized by NS5B RNA-dependent RNA-polymerase (RdRP), which is a common target antiviral therapy . The repDengue virus (DENV) and Zika virus ZIKV are dangerous human pathogens capable of causing massive outbreaks . The mecEnterovirus is a genus that includes many species of viruses, such as Coxsackie A and B viruses, echovirus (enterovirus A and B), poliovirus (enterovirus C), and enterovirus 68 or 72. These viruses are the most pervasive human viral pathogens . EnterovClassical enterovirus detection and quantification approaches are based on CPE observation in cell cultures. However, this type of assay is very laborious and cannot produce rapid results; therefore, it is not convenient for routine applications. Molecular methods relying on reverse transcription PCR are much more rapid and accurate for the detection of viral genome equivalents. The limitation of this method is that it is considered to provide an indirect assessment of virus infectious ability .The development of a cell-based reporter system represents a compromise between rapid experimental procedures and studies revealing the viral nature and the details of viral infection. Enteroviruses as well as other viruses with an ssRNA (+) genome produce specific proteases necessary for mature viral proteins processing . One of The obtained reporter cell line showed ECFP fluorescence in response to infection by three enteroviruses and was not activated by hepatitis A virus (HAV), which does not produce 2Apro. Presumably, such a reporter system can be used in research devoted to the development of antiviral drugs based on antiprotease activity.The main infectious agents in the lentivirus genus are bovine immunodeficiency virus (BIV) and human immunodeficiency virus (HIV). HIV causes acquired immunodeficiency syndrome (AIDS) . AIDS leThe ability to determine the number of infectious particles in a viral stock is essential for studying viral life cycle stages, developing viral drug therapy, and searching for mutations in the virus genome. Quantitative titers of HIV stocks can be determined from end-point dilutions of the virus, but these assays are labor-intensive for a large number of samples. Therefore, investigations focused on reporter cell line development have been performed ,86.In HIV research, cell susceptibility is conferred by the presence of CD4 (cluster of differentiation 4) receptors on the cell surface. The first reporter cell line for HIV was developed in the late 1980s and was based on transactivation in transfected lymphoid cell lines. The CAT reporter gene was cloned under the control of the LTR promoter of HIV-1 and can be induced by the HIV Tat transactivator . These rHowever, an HIV LTR-based reporter system presents some disadvantages. First, this type of system exhibits a certain basal expression level in the absence of Tat. Second, it can be nonspecifically activated by cellular transcription factors, cytokines, mitogens, or even viral envelope proteins ,91. It wA BIV detection method based on RT-PCR has been introduced . AlthougAnother retrovirus that is currently being studied with a cell-line-based approach is simian foamy virus (SFV). A reporter cell line for SFV was constructed on the basis of BHK-21 cells. A reporter cell line with the beta-galactosidase gene driven by the LTR SFV was obtained. In this study, several SFV strains were used as laboratory-adapted strains, and natural strains from the blood of two chimpanzees, two gorillas, and one Cercopithecus individual were also used. Reporter gene expression was initiated within 72 h after infection by an autologous strain. The same result was obtained with another strain of SFV. The cells were shown to be an effective tool for the quantification of different strains. The authors showed that the replication rate of different strains of SFV is different among different cells .Human infections involving HSV type 1 and type 2 are common throughout the world. Morbidity from herpesvirus infection is an important issue . HSV infAlthough HSV is well propagated in mammalian cell cultures, the detection of virus infection is a laborious procedure, since the immunostaining of infected cells for antigen identification must be performed for accurate diagnosis. Using this approach, a result can be obtained in 24 h to 48 h in the majority of cases ,100.A reporter cell line allowing the more rapid detection of this virus was developed in the early 1990s . The repOne of the most important species of gammaherpesviruses is Kaposi’s sarcoma-associated herpesvirus (KSHV or human herpesvirus 8 (HHV8)). This virus can cause Kaposi’s sarcoma, a cancer that often develops in AIDS patients. A barrier to studying viral life cycle features by cell-based methods is related to the fact that the virus is difficult to cultivate in most cell lines. Therefore, the use of such standard methods as determining the titer of the virus is challenging. Thus, the development of reporter cell lines is based on cells that are permissive, at least at a low level  . As noteFor indicator cell line development the following approach was used. The HH8 genome encodes the major regulator Rta, which activates a range of host and viral promoters and regulates lytic program activation in the viral life cycle ,103,104.Varicella-zoster virus (VZV) is a cause of varicella, which may present severe complications due to primary infection or virus reactivation in infected cells . The avaThe reporter cell line for VZV was developed based on the specific activation of the viral promoter . SeveralVirus detection methods include a range of molecular and cellular approaches. Cell-based methods account for a large portion of virology research. Any cell line used for virus detection must be characterized by sensitivity, which refers to both susceptibility and permissivity. In general, sensitivity is the ability of the cell to be infected by a small amount of the virus independent of the species. In the laboratory, permissivity can be achieved by knocking out cell antiviral response genes or by the simple selection of sensitive cell lines among different cultures from a laboratory collection. Susceptibility is a property of the cell line to be infected by certain virus species. In the laboratory, susceptibility can be increased by increasing the expression of virus-binding cell receptors. The improvement of these two properties makes cell lines a convenient tool for virus isolation and study. The study of virus infection has traditionally been carried out on the basis of CPE, but this approach does not work for every virus, and additional time-consuming studies are necessary in such cases. This problem has been solved by the use of cell lines carrying molecular sensor systems that allow the detection of infectious virus agents by the presence of easily detected reporter proteins. Such an approach allows the differentiation of a specific virus species or family. This method can be used for quantitative studies as a faster analog of CPE approaches. The optimization of cell-based methods in virology can be achieved by integrating molecular techniques to obtain increased sensitivity in molecular reporter construction.


Knowledge of service use and costs and wider societal costs in Europe is limited; (2) Method: Using an international sample, we analysed services and costs in 357 children (4–6.99 years) with ASD based on parent reports. Costs were transformed into EU-28 average using purchasing power parity; (3) Results: 122 children (34%) received specialist autism services; 149 (42%) received sensory/motor therapy; 205 (57%) received speech/language therapy; 35 (10%) received play therapy; 55 (15%) received behavioural interventions; 31 (9%) received social skills training; 47 (13%) participated in therapeutic recreational activities; and 59 (17%) received other services. The total number of hours for these services combined over two months was M = 34 . Estimated total costs of health-related services were M = 1210 EUR (SD = 2160 EUR); indirect societal costs were M = 1624 EUR (SD = 1317 EUR). Regression analyses suggested that costs rise with age and presence of intellectual disabilities, but not with severity of autism; (4) Conclusions: The high extent of community-based services indicates good accessibility but also considerable variation in the receipt of services. The costs of autism services are considerable. Further research is needed to investigate whether services received match individual needs. Autism spectrum disorder (ASD) refers to a range of lifelong neurodevelopmental conditions characterized by the presence of early appearing symptoms in two core domains: impairments in reciprocal social communication and the presence of restricted interests and repetitive behaviours DSM-5; ICD-11) . ASD aff; ICD-11 https://www.who.int/news-room/fact-sheets/detail/autism-spectrum-disorders, accessed on 1 April 2020) recommend evidence-based psychosocial interventions including behavioural treatment and parent skills training programmes which can minimalize limitation in communication skills and social behavioral skills with improving well-being and quality of life for both individuals with ASD and their parents. Based on current best evidence, guidelines developed by Autism-Europe [It is important to provide individuals with ASD and their families with support to live their best possible life. There is no cure for autism nor one care package that suits everyone. People with autism require different support at each age of their lives, and it is therefore essential to ensure the right type of evidence-based therapy at the right time. Relevant guidelines often emphasize general principles in the absence of more specific evidence. One relevant policy overview by World Health Organization  , we analDetails on study procedures were described separately , while aType of therapy or intervention ;Institutional stay ;Outpatient treatment ;Dietary supplement and medication ;Special diet ;Working hours spent on treatment as described above, including driving, of all private care givers (weekly average in hours);Work situation (working or not working);Reduced hours due to the child’s needs (in percentage);Employing anybody privately to help care for the child ;Any additional assistant/aide in school/at home for the child ;Care giver investment:TIME-A used assessments by blinded clinicians as well as reports by parents/caregivers collected at several time points. For the current study purpose, only data collected at baseline on scores of the ADOS and ADI-R, IQ level, as well as costs and concomitant treatments were included. Parents answered a detailed questionnaire regarding services received and out-of-pocket expenses during the past two months. The following data were collected:Respondents were asked to reply with regard to the last 2 months. The limited recall time frame was used to minimalize the recall bias following other economic evaluations e.g., ,36. The https://data.oecd.org/conversion/purchasing-power-parities-ppp.htm, for 2018, accessed on 4 March 2021). All health services costs  were sourced from personal communication and represent typical costs in Austria, a Central European country and one of the countries where data were collected. Heath services costs were set up to be 84 EUR per 60 min (one session) based on 1:1 setting, and discounted for group settings, assuming a group size of 5. Costs of institutional stay (310.08 EUR per day) and outpatient treatment (167.10 EUR per contact/visit) were retrieved from a report of Unit Costs of Health and Social Care [https://ec.europa.eu/eurostat/en/web/products-datasets/-/ILC_DI08, accessed on 4 March 2021), the following unit costs were obtained: 1946.08 EUR per month for full-time work where both parents had a university degree; 1391.42 EUR per month where both parents had 12 or more years of education; and 1129.42 EUR per month where at least one parent had less than 12 years of education or education level was unknown. No unit costs were imputed for medication and special diets because these costs were found to be negligible in children with ASD [https://ec.europa.eu/eurostat/tgm/table.do?tab=table&init=1&language=en&pcode=tec00118&plugin=1, accessed on 4 March 2021).Unit costs were inserted where a service was used but parents reported no expense for the service. Costs were transformed into EU-28 average using purchasing power parity , and intellectual disability (below-average intelligence) and health services costs and societal costs. Following literature that the age and severity of autism symptoms are significant predictors of costs for children at preschool age  and in an = 140).A sensitivity analysis was used to determine how data retrieved from non-European countries affected the obtained health services costs and societal costs. In order to determine the robustness of our results by examining the extent to which results of health services costs and societal costs are affected by country of origin, we calculated costs including only 217 children from Europe  and excluding individuals from other non-European countries  and clinical judgement (n = 140), the results of which indicate that 47% of children (n = 163) had IQ below 70. The majority of individuals attended to school (62%), while 22% to full-time care and 11% to part-time care. More details can be found in the top half of The total of 357 children (83% boys) were included with a mean of age 5.4 years (SD = 0.9). As can be seen from In regard to parents’ situation of work, the majority of mothers were homemakers (34%) followed by 26% being part-time employed, while the majority of fathers were working full-time (74%). Regarding education, many parents had a university degree , followed by education equivalent to high school , and less than high school . The bottom half of Of 357 children, 122 (34%) received specialist autism services; 149 (42%) received sensory/motor therapy; 205 (57%) received speech/language therapy; 35 (10%) received play therapy; 55 (15%) received behavioural interventions; 31 (9%) received social skills training; 47 (13%) participated in therapeutic recreational activities; and 59 (17%) received other services . The totThe sum of indirect societal costs was M = 1624 EUR  over the last 2 months with a wide range . It can be seen that the SDs are relatively high compared to the means; sometimes higher than the means. This reflects the skewed nature of these data, which is a common phenomenon in health economic data ,41.p < 0.048) and intellectual disability (p < 0.005) on health costs  attended school, 91 of included children (25%) attended kindergarten ; while 5 (1%) attended none of aforementioned. Regarding other services, 10 children (3%) had an institutional stay over the last 2 months; 24 (7%) received outpatient treatment; 55 (15%) received medication; and 38 (11%) a special diet. It can be seen from the data in n = 85; 24%); 111 had reduction in working hours (31%) due to a child care assist  and required an additional aide at school or home .The results about schooling indicate that the majority of individuals from European counties . It may be explained by the fact that communication problems belong to the core autism symptoms; thus, children may benefit the most from this kind of treatment at this early stage of life. The second and most frequently chosen form of therapy was sensory and motor therapy . High use (42%) of this intervention might be due to the core nature of sensory symptoms in individuals with ASD, particularly to sensory over-responsivity . On the Estimated total costs of health-related services were M = 1210 EUR for a 2-month period, while specialist autism services contributed to these costs the most (315 EUR) followed by behavioural intervention (284 EUR) and speech/language therapy (251 EUR).Roggie and Janssen  in theirThe present results indicated a high diversity among children’s needs and therefore health care services used. Institutional admissions occurred in 12 cases (3% of cohort), while visits to psychiatric, somatic, or other specialized outpatient services occurred in 37 cases (10%). Even though our data indicated a relatively low mean cost of institutional stay (3 EUR) and outpatient services (1 EUR) over a period of two months when averaging across the whole sample, the wide range of costs show that there were families who incurred very high costs  related to these services, whereas other families did not have these costs at all. A large and growing body of literature has investigated healthcare utilization expenditures for individuals with autism. Other research in medical and healthcare costs has emphasized the higher costs for individuals with autism compared to individuals without ,19,21,22One third (31%) of the included families in our sample experienced some costs related to medication. Even though the present data indicate a relatively low cost of medication (approx. 6 EUR) over a period of two months, the range of costs (from 0 to 290 EUR) emphasizes that there were families who incurred very high costs related to these services, and families who did not have these costs at all. There is a large volume of published studies e.g.,  describiSeventeen percent of the included children in our study experienced costs associated with a special diet; across all included children, the cost of special diet was on average 7 EUR over a period of two months. The broad range of these costs from 0 to 455 EUR indicates that some families have had a very high cost associated with a special diet. These results are likely to be related to a possible relationship between nutritional status and general condition of ASD children. Peretti and colleagues  emphasizResults of the present study indicated the indirect societal costs to be around 1624 EUR over a period of two months. In terms of societal costs, an additional aide at school or home contributed the highest costs (621 EUR), followed by reduction in working hours (M = 524 EUR), childcare/school (M = 461 EUR), and child care help (M = 18 EUR).Our findings seem to be consistent with other research which found a high economic burden linked with ASD on society . For insFamilies of individuals with ASD face significant economic burden due to reduction or interruption of employment . The resData from our study indicate that the reduction in working hours was linked with costs of 524 EUR; child care help with additional costs of 18 EUR over a period of two months. Our findings have confirmed previous results ,30 that It was hypothesized that younger participants with a diagnosis of autism with intellectual disability will bear higher costs compared to those with older age, with broader ASD category and with intellectual disability. Regression calculation of costs confirmed that health costs change with age for children with ASD during ages 4–7 and with presence of intellectual disabilities, but not with societal costs. Our results indicated that health costs increased by 213 EUR per additional year of child’s age and by 566 EUR for children with intellectual disability, indicating that both age and intellectual disability are significant factors related to total health costs. However, such a relationship was not observed for the social costs.Our results are in line with earlier findings. Research by Järbrink  found thThe results of our study did not confirm the predictive role of symptoms severity in the health costs and societal costs measured by ADOS total score, which is consistent with another study . HoweverLittle is known about autism-related costs in Europe. Total costs including health services costs and societal costs were estimated to be around 2834 EUR for the whole sample size compared to 2489 EUR for individuals from Europe. The results obtained from the sensitivity analysis showed that for children from European countries the pattern of services used is similar (but not exactly the same) for those from non-European countries. Children outside Europe attended more often health care services in the pre-school period, which has a direct impact of health services costs (1210 EUR vs. 740 EUR for the 2 months). Speech/language therapy; behavioural intervention; and sensory/motor therapy were associated with the highest costs for children from Austria, Israel, Italy, Norway, UK; while for the whole sample the main cost drivers were specialist autism services followed by behavioural intervention and speech/language therapy. Estimated societal costs  were relatively close both in Europe and outside  with the same key cost driver .The generalisability of these results is subject to certain limitations. For instance, the most important limitation lies in the fact that the study used self-reported data of costs threatened by self-reporting bias as: social desirability and recall bias , but theThe present research explores, for the first time, the costs of services received and associated costs for children with ASD bason on 5 European countries  and 4 non-European countries . Total costs including health services costs and societal costs, were estimated to be around 2834 EUR over last 2 months with a wide range from 84 EUR to 26,051 EUR. Estimated total costs of health-related services were around 1210 EUR  and indirect societal costs were 1624 EUR (with a wide range from 0 to 6263 EUR). Specialist autism services; behavioural intervention; and speech/language therapy were key cost driver in this group  followed by sensory/motor therapy (213 EUR) play therapy (32 EUR), therapeutic recreational activities (23 EUR) and social skills training (12 EUR). Among indirect societal costs the important cost driver was an additional aide at school or home (621 EUR) followed by reduction in working hours (524 EUR), childcare/school (461 EUR) and child care help (18 EUR).The current findings add to a growing body of literature on services and costs for children with ASD by confirming that families of children with ASD bear higher costs related to the child’s age  and child’s intellectual functioning . This study has also shown that the diversity of approaches recommended in guidelines across countries. The pattern of services used by children with ASD from European countries is similar (but not exactly the same) for those from non-European countries. The high extent of community-based services indicate good accessibility but also considerable variation in the receipt of these services. Notwithstanding the limitations, these results suggest that the costs of autism services are considerable. There is still abundant room for further progress in evaluating data on services received by individuals with ASD and associated costs for Europe. To develop a full picture of costs additional studies targeting adolescents and adults with ASD is recommended. Further research is needed also to investigate whether services received match individual needs.


Γ) to quantify the degree of self-similarity of a shape using branch length similarity (BLS) entropy which is defined on a simple network consisting of a single node and its branches. To investigate the properties of this measure, we computed the Γ values for 70 object groups (20 shapes in each group) in the MPEG-7 shape database and performed grouping on the values. With relatively high Γ values, identical groups had visually similar shapes. On the other hand, the identical groups with low Γ values had visually different shapes. However, the aspect of topological similarity of the shapes also warrants consideration. The shapes of statistically different groups exhibited significant visual difference from each other. Also, in order to show that the Γ can have a wide variety of applicability when properly used with other variables, we showed that the finger gestures in the  space are successfully classified. Here, the Z means a correlation coefficient value between entropy profiles for gesture shapes. As shown in the applications, Γ has a strong advantage over conventional geometric measures in that it captures the geometrical and topological properties of a shape together. If we could define the BLS entropy for color, Γ could be used to characterize images expressed in RGB. We briefly discussed the problems to be solved before the applicability of Γ can be expanded to various fields.We propose a new measure ( Image processing refers to the process of converting an actual physical image into a digital image and applying a variety of algorithms to distinguish between the target object we are interested in and other parts (background). Over the last few decades, the development of these algorithms has been cost-effective and rapid, and image processing has now become a technology used throughout the modern information society, consisting of multimedia systems. Most of these algorithms regard the target object as a shape (silhouette) ,2 for thThis investigation can be generally divided into three types according to the approach used: skeleton-based, region-based and contour-based methods .The skeleton-based method uses the area center axis of a shape to extract its features, which has the advantage of robustness in the case of occlusions and joints ,5. This Canorhabditis elegans in response to very low toxic substances. The authors set 10 equal coordinate points on the body of the worm and calculated the BLS entropy values for the angle and distance between the coordinate points. This study showed that the two entropy values could be very good indices for describing the movement of worms with elongated bodies such as C. elegans. Jeong et al. (2019)  −1. When calculating the number of similar windows, we exclude the case of comparison to itself, i.e., in the j-th row, the number of similar windows (hj) can be written as follows: Here, we denote the number of similar windows as , …, M0K B where KTherefore, the ratio of the number of similar windows in the k= 1, 2, …, K and j = 0, 1, 2, … θ. The highest γ value best reflects the self-similarity of the entropy profile obtained for the pentagonal shape. Thus, the self-similarity can be finally written as follows:Here, Γ value with the node density. To this end, we constructed a single straight line by connecting two straight lines of the same length with different node densities to intuitively understand the density effect  on each side may be slightly different. The border of a horizontal or vertical side has a higher pixel density than oblique lines. This is due to the limitations of shape resolution. Therefore, it is necessary to understand the variation of y effect A. The nuN1 = N2, the entropy profile was exactly symmetric. However, in the case of N1:N2 = 120:100, the entropy profile was relatively high in the region of low node density. For the case of N1:N2 = 140:100, the entropy profile tended to rise further in regions with lower node density. This can be illustrated through the example shown in a and b had 100 nodes, and the line consisting of points b and c and a and c had 120 nodes, respectively. The entropy profile was clearly higher in straight lines a–b with lower node density.When Γ values from a geometric point of view, we computed the Γ values for 1400 shapes in the shape database (MPEG-7) [N, M, and G denote object groups, the average value of Γ, and statistical similarity indication, respectively. Considering the values of M and G, there were groups that belong to the same set with high M values, while there were also sets that are bound with low M values and sets that are loosely bound . The loosely bound groups appeared at levels where the M value was not too high or too low. This indirectly means that the shape classification from the Γ point of view is somewhat different from the existing method of simply classifying from the geometric point of view. Groups 19, 56 and 70 were statistically identical to each other with relatively high Γ values. The shapes belonging to these groups had the characteristic of being long in one direction and were visually similar  . The com similar . ConversΓ values. However, these shapes had a common topological structure in which several arms were attached to a circular body. This means that lower Γ values reflect topological attributes than the geometric attributes of the shapes. For the groups 24, 45, and 60 with low and statistically different Γ values, the shapes belonging to each group were completely different visually.This was caused by the low Γ) to the finger classification problem and showed that the Γ works well for this problem. It took about 5 min to solve this problem using MATLAB 2019a on the same computer used to analyze the MPEG-7 shape database. For this purpose, we obtained finger gesture shapes representing the numbers 1 to 10 from five different adults [Γ value for MPEG-7 image shapes, we reasonably inferred that for hand gesture shapes with a low Γ value, it would be difficult to distinguish the shapes from each other with only one Γ value. We, therefore, introduced Z, a new quantity based on the BLS entropy profile to improve discrimination between finger gesture shapes.In this study, we applied the self-similarity . The length of the entropy profile for each shape is 1,000; max {} represents the maximum value among the correlation coefficient values obtained for Z as the BLS entropy value for the network as follows:Here, BLS” represents the entropy value for the mean values of j = 1, 2, …, 10.Here, the notation, “Γ and Z values of the shapes that belong to each number group. Gesture shapes that symbolize the numbers 1 to 5 are made by showing the back of the hand, while gestures that represent the numbers from 6 to 10 are implemented by showing the palm. Using loci coordinates of the little finger (pinky) and the longest finger (middle finger), we can easily distinguish between 1–5 and 6–10. In the lower figures, the center coordinate values of the square are the mean values of Γ and Z, respectively, while the width and height of the square represent the standard deviation values of Z and Γ, respectively. The lower left figure shows the results for a finger shape representing the numbers 1 to 5 (see the number next to each square), while the lower right figure shows the results for a finger shape representing the numbers from 6 to 10. In each figure, the squares were well separated from each other. This means that Z and Γ can be useful for resolving finger gesture recognition challenges.Γ), which characterizes shapes based on the BLS entropy profile. This characterization allows direct comparison of the degree of differences between shapes, e.g., judging which two shapes among three given shapes , we can clearly affirm that the shapes in the middle and right are more similar to each other, and even estimate that these two shapes are roughly 3% different from the shapes in the left. This shape comparison approach could be used to solve shape problems such as image retrieval. In this study, we proposed a new measure, the self-similarity of a shape (apes see  are moreIn Γ value. As shown in Γ values had visually similar characteristics, while shapes belonging to statistically identical groups with a low Γ value were geometrically similar or different. However, they were all somewhat similar topologically. In this respect, the existing method and our method cannot be directly compared in terms of the classification performance accuracy. Grouping shapes with a certain level of Γ value would allow us to select shapes that appropriately contain both geometric and topology information. From this point of view, it would be mathematically interesting to explore the relationship between the information and Γ values. For Γ and Z based on BLS entropy, yielded approximately 92–95% classification accuracy. We photographed the hands of five people several times to get the shape of the hand. Among the obtained images, there were cases where finger gestures were made well, and cases were not. The results obtained from a well-made gesture shape yielded higher accuracy, and a relatively poorer gesture shape resulted in lower accuracy. Also, the intrinsic property of Γ contributed to the degradation of our method. The Γ cannot distinguish between symmetrical shapes. In this application example, Γ could not distinguish between the right hand and the left hand, and also could not clearly distinguish between the gesture shape when one index finger was incompletely straightened and the gesture shape when one little finger was completely straightened. To improve our method, it is necessary to introduce an additional variable that breaks the symmetry. In terms of shape classification accuracy, our method is inferior to the existing method. However, this comparison does not devalue the results in Γ and Z. For Γ values calculated from the profiles could characterize the color shape. It would be very interesting to explore this problem. One more issue that needs to be considered to be a follow-up study of this paper is to measure the self-similarity of shapes with colors or gray levels. One idea is to represent a shape with RGB channels as two BLS entropy profiles. One is an entropy profile that defines the distance between nodes as an edge, and the other is an entropy profile that defines a color value of nodes as an edge. Since two BLS entropy profiles can be created for each channel, a shape with RGB colors can be characterized by six BLS entropy profiles. The six Consequently, we believe that this study is not only meaningful in defining and introducing a new concept of self-similarity based on BLS entropy, but also showing its applicability. In addition, we consider it worthwhile to propose problems that need to be addressed to broaden the applicability of self-similarity.


Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to -4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to -4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups , together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids. Rhabdoviridae) are transmitted by aphids through the circulative (persistent) manner and replicate within their vectors (Aphids (order Hemiptera: superfamily Aphidoidae) are phloem feeders and important insect pests that affect a wide range of crops. Aphid infestation can reduce both the yield and quality of crops by direct feeding and transmitting symptomatic plant viruses . Many ap vectors .Triticum aestivum L.) and barley (Hordeum vulgare L.), aphids can cause great yield losses by directly feeding on the phloem sap and more significantly by transmitting several yellow dwarf disease-causing viruses  , corn leaf aphid (R. maidis), greenbugs (Schizaphis graminum), English grain aphid (Sitobion avenae), and rose-grain aphid (Metopolophium dirhodum) , such as wheat  . These virhodum) .Dicistroviridae, and the Brevicoryne brassicae virus (BrBV) of the family Iflaviridae (Acyrthosiphon pisum virus (APV) (distantly related to the members of the family Solinviviridae)  and Dysaphis plantaginea densovirus (DplDNV)]  and posiaviridae ; and twoviridae) . Many ofviridae) . More reviridae) , a tetraviridae)  and a soviridae) ; a negatviridae) ; and a Dviridae) . NeverthFlaviviridae), chikungunya virus (family Togaviridae) and Rift Valley fever virus (family Phenuiviridae)  . These viruses have a host range that is restricted to insects and are related to human arthropod-borne viral pathogens , such as dengue, Zika and West Nile viruses (family viridae) . One of viridae) . Negevirviridae) . Their gviridae) . Based oviridae) . Negevirnervirus . These gaviridae . Kitaviraviridae , and somachnida) . Recentlachnida)  and otheachnida) . NeverthIn this study, a meta-transcriptomic approach was used to investigate the viromes of aphid populations collected from a barley field for three consecutive years, 2016–2018. We identified at least 60 virus-like sequence contigs related to RNA viruses such as nege/kita-, flavi/jingmen-, tombus-, phenui- and luteoviruses, including eight putative novel RNA viruses. In particular, our study reveals the new nege/kita-like virus lineages with the members that are associated with aphid species and, thus, enhances our knowledge on the diversity and evolution of insect viruses that are related to plant viral pathogens.Collection of aphids were performed in the experimental field at the Institute of Plant Science and Resources (IPSR) in Okayama University, Kurashiki, Japan (34°59’ N and 133°77’ E) on April and May in 2016–2018. Aphids  were obtained from the colonies that infest leaves and ears of barley cultivar “Golden Promise”, “Morex”, “Kikai Hadaka”, “Minori Mugi”, “Ryofu”, “Kobin Katagi”, and three landraces , and then aphids samples were stored at −80°C until further analysis.To determine the aphid species, a 658-bp fragment of mitochondrial DNA from the 5′ region of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene, was analyzed . Total gAcyrthosiphon pisum  (this study) and the 16S rRNA gene of a putative secondary parasitoid species   was extracted using TaKaRa RNAiso Plus Reagent , following the manufacturer’s instructions. The total RNA fraction was analyzed by electrophoresis in a 1% (W/V) agarose gel in 1 × TAE buffer. For reverse transcription (RT)-PCR to detect virus-like sequences, cDNAs were synthesized using MMLV reverse transcriptase  with random hexamers, following the manufacturer’s instructions. The cDNAs were then used as templates for PCR amplification with QuickTaq HS Dye Mix. The primer sets used to amplify virus-like sequences are provided in on pisum . To confpilidae) . PCR conTo determine the major uncertain or unknown regions of virus-like sequences, RT-PCR was performed using the specific primer sets, and PCR fragments were then sequenced using the Sanger method. Sequences of the primers used in the RT-PCR are provided listed in de novo assembled using the CLC Genomics Workbench version 11 . The assembled-contigs derived from each sample pool (over 1.0 kb contigs) were subjected to local Blast searches against the viral reference sequence (RefSeq) dataset of NCBI1 . To roughly estimate the other sequence read origins for each NGS data set, we also conducted local Blast analyses against the following genome sequences: (1) aphid (R. padi) transcript data obtained from AphidBase2; (2) insect parasitoid wasp  transcript data from NCBI; (3) an aphid endosymbiotic bacterium  genome sequences; and (4) R. padi mitochondrion (accession no. KT447631) genome sequences. Mapping of sequence reads to each virus-like contigs or gap-filling virus-like sequences determined using RT-PCR was done using the Read Mapping algorithm of the CLC Genomics Workbench.Total RNA fractions were prepared separately from 15 aphid samples (each population from a single colony) and according to the year of aphid collection, pooled into three groups: pool-1  consisted of samples from 2016 , pool-2  consisted of samples from 2017  and pool-3  consisted of samples from 2018  . Each RN3. Pairwise sequence comparisons were performed using the Sequence Demarcation Tool version 1.2, with the MUSCLE alignment  tree construction was carried out as described previously with minor modifications , 2016. Mrameters . Ambiguorameters . ML treeelection . The NJ election  were conR. padi (the bird cherry-oat aphid), R. maidis (the corn leaf aphid), and S. avenae (the English grain aphid)   . Neverthde novo assembled contigs that were larger than 1.0 kb  were subjected to local Blast (tBlastX) analyses (L. testaceipes compared with those of other pools (7% or 3%)  and subjected to transcriptomic analysis using NGS. The analyses . Most of% or 3%) .Kitaviridae (or proposed Nelorpivirus and Sandewavirus), Flaviviridae, Tombusviridae, Phenuiviridae, Narnaviridae, Chrysoviridae, Partitiviridae, Rhabdoviridae, and Luteoviridae (Luteoviridae). The detailed analysis of luteovirus, or luteo-like virus sequences identified in the current study will be reported elsewhere as a part of the ongoing barley virome project conducted in this barley field.Local Blast analyses revealed that the virus-like contigs present in the aphid transcriptomes have sequence similarity to relatives or members of the virus families, the oviridae . Notablyoviridae . The thrWe performed RT-PCR to verify the presence of virus-related RNAs in the aphid samples using the specific primer sets . The virWe identified four contigs that resembled almost complete (coding complete) nege/kitavirus-like genome sequences  with 18,472–328,424 mapped reads . Some otR. padi and S. avenae) that infested barley plants. The two contigs BaA3_c89 and BaA1_c637  showed 97% nucleotide identity with each other and 71% nucleotide identity with Hubei virga-like virus 4 (HVLV-4) (R. padi and S. avenae (BaA1_c637) or R. maidis (BaA3_c89) [our tentative cut-off value for the species classification criteria for nege-like viruses is <90% amino acid identities of RNA-dependent RNA polymerase (RdRp) sequences], rather than being a strain of HVLV-4; therefore we tentatively designated the virus as barley aphid RNA virus 1 (BARV-1).The nucleotide sequence and encoded proteins of BaA1_c12 are almost identical (>99% nucleotide and 99–100% amino acid identities) to those of the Wuhan house centipede virus 1 (WHCV-1) , suggest(HVLV-4) . A Blast(HVLV-4) . AlthougR. maidis, appeared to be a novel nege/kita-like virus related to Wuhan insect virus 9 (WhIV-9) because they share moderate levels of identities at the nucleotide (72%) and amino acid (66%–80%) sequence ; R. maidis ; or R. padi, R. maidis, and S. avenae   .Paulownia tomentosa (family Paulowniaceae)  , most liniaceae) .Aphis glycines (the soybean aphid) (referred to as “group 2”) (centipede virus 1) and “Aphiglyvirus” (after Aphis glycines virus 3), respectively. Members of these two proposed genera, together with two proposed taxa of arthropod-restrictive viruses, “Nelorpivirus” and “Sandewavirus,” which all have non-segmented genomes, could be classified into a novel viral family or be assigned to the family Kitaviridae. However, it is to too early to establish a family for these viruses or to assign them into the Kitaviridae, because there is no reliable statistical support for these lineages within the currently available phylogenetic trees , at least in the case of the cile/higreviruses lineage.To understand the relationships between newly discovered nege/kita-like viruses, viral-like TSAs and reported nege/kitaviruses, we conducted ML phylogenetic analysis based on amino acid alignment of their replicase and replicase-like sequences. The ML tree showed that the BARV-1 variants clustered with HVLV-4 and its relatives, WHCV-1 and Wuhan insect virus 8 (WhIV-8) (referred to as “group 1”); whereas the BARV-2, -3, and -4 form a well-supported clade with the relatives of WhIV-9, and HWhIV-9, and ApGIV-3 from roup 2”) . These troup 2”) . Our phyic trees . The resic trees , suggestic trees , the genScutigeridae sp., phylum Arthropoda) and also from three other arthropods, spiders, and insects  , a parasitoid wasp , and some other insects such as dipterans, an orthopteran, and a coleopteran   or the pomegranate  , maize and sea barley (WHCV-1 (a centivirus) was identified from the house centipede (t mix 4) . Becauseeopteran , WHCV-1 eopteran . WHCV-1 eopteran . Notably in 2016 . Intrigu in 2016 . WHCV-1-lonicum) . WhIV-8-hraceae) , and soma barley . Thus, tOdonata sp., order Odonata) , but not a secondary parasitoid (D. carpenteri), was detected in the aphid samples using RT-PCR (Lysiphlebus fabarum) (Vespa velutina (Hymenoptera)  , whereasoid wasp . The prehe field . In our he field . A primag RT-PCR . It has fabarum) . Its clofabarum) . An aphinoptera) . TherefoR. maidis from the 2018 sample (R. padi colony from the 2016 sample (BaA1_c139) and in two aphid colonies (R. padi and R. maidis) from the 2016 sample (BaA1_c13), respectively  (In addition to the nege/kita-like viruses (centi- and aphiglyviruses), we identified several contigs related to other RNA viruses, such as flavi/jingmen- and tombus-like virus sequences . WhAV-1,8 sample . In contectively . In the ectively . WhAV-1 Ziebell) , raisingZiebell) .M. euphorbiae populations (R. padi and S. avenae) from the 2016 sample and SmFLV-1 probably also in a R. maidis colony from the 2018 sample in which low levels of viral RNA accumulation was detected using RT-PCR, whereas no virus reads were found in the NGS data, thus expanding its aphid host range. BARV-9-like TSA sequences were also found in S. avenae (R. maidis colonies from the 2016 and 2018 samples (BaA1_c326) or an R. padi colony from the 2016 sample (BaA1_c33)  . SimilaraA1_c33) .R. padi and S. avenae aphids from the 2016 sample, but not from R. maidis   from the 2016 sample, whereas the BaA2_c4524 sequence was only detected in R. maidis aphids from the 2017 sample (R. maidis aphids from the 2017 sample (Linepithema humile rhabdo-like virus from the queen samples of the Argentine ant (Linepithema humile) , together with Hubei rhabdo-like virus 3 and Tacheng tick virus 6  represent phenuiviruses (family viridae) . These tviridae) . We tent7 sample . In the 7 sample . Similar7 sample . This co humile)  and form virus 6   and the 2018 sample  (Narnaviridae) and many invertebrate-associated narna-like viruses (Two positive sense RNA virus-like sequences (BaA2_c1487 and BaA3_c16152) were identified in  2.0 kb) . Both se 2.0 kb) . We name 2.0 kb) . In the  viruses .R. maidis colony from the 2016 sample (Alphachrysovirus and Betachrysovirus (R. maidis and probably R. padi) from the 2016 sample, we also identified another dsRNA virus-like sequence  (Two dsRNA virus-like contigs (BaA2_c19028 and BaA2_c18060) were derived from an 6 sample  (Supplem6 sample . Howeverysovirus . BARV-8 ysovirus . In addiragment)  whose Rdragment)  and alphR. padi, R. maidis, and S. avenae) that were collected from a barley field in the spring of 2016, 2017, and 2018. From these sequences we identified eight potentially novel RNA viruses belonging to nege/kita-, flavi-, phenui-, mononega-, narna- and chrysovirus lineages, as well as some previously described RNA viruses. Moreover, based on the phylogenetic analyses, we proposed novel genera, Centivirus and Aphiglyvirus, for aphid associated nege/kita-like viruses and their relatives. Our data provide novel information on the diversity of aphid-associated viruses in aphid populations infesting the barley field. Our study, along with others, has discovered that some aphid-associated viruses are present in different aphid species and in plant-derived samples. This raises the speculation that aphid-associated viruses may be distributed across varieties of aphid species, with plants being the reservoirs. Since aphid virus-like sequences are also present in the hyperparasitoid wasp, it would also be interesting to explore the possible horizontal transmission of viruses between aphids and primary parasitoids and/or hyperparasitoids. To deepen our understanding on the population dynamics and spread of aphid viruses in the field, investigation of horizontal virus transmissions between insects and plants as well as between insects and their parasitoids is an important topic for future research. Some aphid viruses cause diseases in their hosts, for example, rapid population decline and phenotype alternations of insect performances (In the current study, we discovered at least 60 virus-like sequences related to nege/kita-, flavi/jingmen-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses from 15 aphid population samples (ormances , implyinormances . On the ormances  is regarormances , 2015. Mormances . Our datLC516834 – LC516856.The virus and virus-like sequences derived from this study can be found in GenBank under the accession numbers HK designed the experiments and analyzed the data and wrote the manuscript. HH and HK collected the samples. HK, MF, HH, KH, IA, and NS performed the experimental work. IA and NS were involved in discussion and manuscript revision. All authors have given approval to the final version of the manuscript.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.


Otosclerosis is a common cause of conductive hearing loss which is an autoimmune inflammatory disorder related to abnormal bone remodeling of the human otic capsule that has complex etiopathogenesis attributed to genetics, autoimmunity, viral infection, inflammation, hormonal factor, environmental factor, and disturbed bone metabolism. It has a prevalence of 0.3%-0.4% in Caucasians, which makes up 5-9% of all hearing loss and 18-22% of all conductive hearing loss. This review article aims to study the postulated role of sustained measles virus infection in the etiopathogenesis of otosclerosis, among others. A PubMed search of the related topic identified 97,990 articles. After we applied the inclusion and exclusion criteria, it was determined that 52 articles were relevant, which included 38 observational studies, 13 review articles, and a systematic review. Among them, 33 observational studies, 13 review articles and a systematic review spotted a positive association between persistent measles virus infection and otosclerosis. On the contrary, five observational studies reported no evidence of the association. The majority of the current literature supported the presence of the measles virus component in the otosclerotic stapes samples and its role in the etiopathogenesis of otosclerosis. Measles virus infection may have the amplitude to initiate a pathological process, which in the presence of other factors like autoimmunity and genes plays a significant role in causing otosclerosis. However, other studies have failed to show the presence of the measles virus component in otosclerotic stapes. So, more studies are needed to probe the role of persistent measles virus infection in the etiopathogenesis of otosclerosis. The prevalence of otosclerosis is 0.3%-0.4% in Caucasians, which makes up 5-9% of all hearing loss and 18-22% of all conductive hearing loss. However, this disease affects only 0.03%-0.1% of Africans and Asians . HistoloOtosclerosis is an autoimmune inflammatory disorder related to abnormal bone remodeling of the human otic capsule . It causThe role of sustained measles virus infection is postulated in the etiopathogenesis of otosclerosis . Many stThis review article aims to determine the relationship between persistent measles virus infection and otosclerosis by reviewing most of the relevant articles on PubMed. This article also focuses to differentiate whether the relationship exhibited in different articles is actually present or merely coincidental by comparing and contrasting the majority of the available articles on this subject.MethodLiterature was searched in PubMed using regular keywords and Medical Subject Headings (MeSH) keywords and data collection was done. Table Studies were selected after applying the following inclusion criterion: human studies only. No exclusion criteria were set.ResultsTable A total of 62 articles were found with keyword otosclerosis and measles virus by regular search in PubMed. Out of which 60 articles fulfilled the inclusion criteria of human studies. Among the 60 articles that were reviewed, 52 articles were relevant for this research topic.Finally, these 52 articles were selected for review, out of which 38 articles were observational studies, 13 articles were review articles, and one was a systematic review. The maximum sample size in a study was 1,351 and the aggregate sample size was 5,863.DiscussionHistopathology of otosclerosisOtosclerosis is related to abnormal bone remodeling of the human otic capsule. Normal bone remodeling is a naturally occurring process in all bones where there is an equilibrium between bone resorption and bone formation by osteoclasts and osteoblasts, respectively . AbnormaGenetics in otosclerosisThere is a greater incidence of otosclerosis in certain families than in the general population which suggests the role of genes and heredity in its etiopathogenesis . AutosomAutoimmunity and inflammationA study showed the presence of an autoantibody against type II collagen in the serum of otosclerotic patients . TGF-β1 Role of hormones in otosclerosisOtosclerosis occurs two to three times more frequently in females than in males, and symptoms manifestation occurs more frequently during or after pregnancy, which indicates endocrinal factors in its pathogenesis . AngioteViral infection: role of persistent measles virus infectionThe measles virus is from the Paramyxoviridae family, which has a non-segmented, negative-sense RNA genome. Measles virus infection presents as fever and maculopapular rash. Other clinical manifestations include cough, coryza, and conjunctivitis. The widespread use of measles vaccines has decreased its incidence, morbidity, and mortality . ComplicWe found out that the majority of observational studies detected measles virus RNA in otosclerotic stapes by different methods. Several observational studies used various methodologies like reverse transcription polymerase chain reaction (RTPCR), reverse transcription-quantitative polymerase chain reaction (RTQPCR), and glyceraldehyde 3-phosphate (GADP) for detecting messenger RNA (mRNA) of measles virus from otosclerotic stapes sample and control samples , 11, 12.In our study, we figured four major groups of researchers who contributed studies on measles virus and otosclerosis. Karosi T, along with his colleagues, performed 11 observational studies, one review article, and one systematic review. All of his 11 observational studies detected measles virus mRNA in most clinical and histological otosclerotic stapes footplate. His notable observational findings in which he detected measles virus mRNA were in 62 out of 102, 175 out of 261, 99 out of 154, 20 out of 34 otosclerotic stapes samples -28. His Table Despite all this evidence of the association, few observational studies have failed to obtain the association between persistent measles virus infection and otosclerosis. A recent observational study by Crompton M et al. in 2019 did not show any relationship. Another observational study by Singh MP et al. (2005) could only detect immunoglobulin M (IgM) antibodies against measles in 18.1% of his sample study, IgM antibodies against varicella zoster virus (VZV) could be detected in 4.5% of the same sample . With thTable One large observational study by Niedermeyer HP et al. with a sample size of 1351 showed strong support for the involvement of the measles virus in the causation of otosclerosis while another sizeable observational study by Crompton M et al. in 2019 with a sample size of 657 did not show any relationship , 48.Persistent infection by the measles virus has been postulated in many chronic diseases with an inflammatory component, such as otosclerosis, multiple sclerosis, Paget’s disease, and Crohn’s disease . In thisSeveral research articles describe chronic measles virus infection as one of the main critical factors in the development of otosclerosis in an individual. A total of 33 observational studies from 1986 to 2015, 13 review articles, and a systematic review found the same. On the contrary, a total of five observational studies from 2000 to 2019 reports no evidence of the association.We analyzed that even with highly sensitive methods, the measles virus is detected in a large number of otosclerotic samples. However, few other studies have failed to show their presence. This observation questions about the hypothesis however these conflicting results make the role of measles virus in otosclerosis a big mystery. Since so many studies favour the association, it can be hypothesized that persistent measles virus infection has to be among one trigger for otosclerosis.Treatment discussionSurgery, either stapedotomy or stapedectomy, is the chief curative approach in the management of otosclerosis to cure hearing loss. Sodium fluoride has also been tried to reduce the severity of the disease. However, its use in the clinical scenario is still lacking. Few studies show that it can reduce the degree of hearing loss while other studies do not support this association . Due to The objective of our review article was to study the etiopathogenesis of otosclerosis and its association with persistent measles virus infection. The goal was to study the impact of persistent measles virus infection on triggering the disease process and therapeutic consequences if any. Otosclerosis is an autoimmune inflammatory disorder related to abnormal bone remodeling of the human otic capsule, which has complex etiopathogenesis attributed to genetics, autoimmunity, viral infection, inflammation, hormonal factor, environmental factor, and disturbed bone metabolism. The majority of the literature review supported the evidence of the presence of the measles virus component in the otosclerotic stapes samples even after the elimination of measles virus infection. Measles virus infection may have the amplitude to initiate a pathological process, which in the presence of other factors like autoimmunity and genes implicated to play a significant role, can lead to the onset of otosclerosis. Due to this association, the widespread use of a vaccine to prevent measles has decreased the incidence of otosclerosis fortuitously. This relationship also opens up a scope for the research in related clinical pharmacotherapy for useful treatment advances. Though it is hard to say that the mere existence of the measles virus part in otosclerotic samples can lead to otosclerosis as other studies contradict this theory. Nevertheless, more studies are needed to probe the role of persistent measles virus infection in the etiopathogenesis of otosclerosis.