Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia ringspot virus are unusual in encoding an enzyme not yet found in any other virus, the “house‐cleaning” enzyme inosine triphosphatase. Inosine triphosphatases (ITPases) are highly conserved enzymes that occur in all kingdoms of life and perform a house‐cleaning function by hydrolysing the noncanonical nucleotide inosine triphosphate to inosine monophosphate. The ITPases encoded by cassava brown streak virus and Ugandan cassava brown streak virus have been characterized biochemically and are shown to have typical ITPase activity. However, their biological role in virus infection has yet to be elucidated. Here we review what is known of viral‐encoded ITPases and speculate on potential roles in infection with the aim of generating a greater understanding of cassava brown streak viruses, a group of the world's most devastating viruses.Plant viruses typically have highly condensed genomes, yet the plant‐pathogenic viruses  What we know and do not know about the inosine triphosphatase Ham1 found in the viruses causing cassava brown streak disease, namely cassava brown streak virus and Ugandan cassava brown streak virus. The viral‐encoded Ham1 shows sequence motifs typical of ITPase enzymes involved in dephosphorylation of noncanonical nucleotides, atypical nucleotides that arise from oxidative damage or stresses within the purine salvage pathway.2Escherichia coli and purification of the ITPases encoded by CBSV and UCBSV has shown that both are indeed functional ITPase enzymes with typical activity towards deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP)  can build in the cells through normal metabolic processes or via exposure to external mutagens that result in modification of canonical nucleotides. If incorporated into DNA or RNA, noncanonical nucleotides can result in nonspecific base pairing and random mutagenesis or lead to chromosomal aberrations through error‐prone repair mechanisms. A number of different enzymes function in house‐cleaning roles to reduce the pool of noncanonical NTPs by hydrolysing pyrophosphate to give nucleotide monophosphates. There are four structurally distinct families each defined by a strictly conserved substrate specificity domain that is essential to exclude canonical nucleotides. The families are Nudix hydrolases, dUTPase, all‐α NTP pyrophosphatases (MazG), and ITPase (Maf/Ham1) , human (ITPA), and yeast (Ham1). The yeast ITPase is named for its mutant phenotype that shows sensitivity to the mutagenic purine analog, 6‐N‐hydroxylaminopurine (HAP). This mutant phenotype was also seen in ITPA knockdown HeLa cells and E. coli ‐causal viruses. These studies showed no change in expression of either ITPase‐like gene in response to UCBSV CBSV by cassava whiteflies and EuRSV by aphids. Despite the similarities between the viruses, the genes encoding ITPase are not considered to be homologs. Although it is likely that the genes encoding ITPase in CBSV and UCBSV are evolutionarily related due to their close phylogenetic relationship, the EuRSV Ham1 is distinct. The latter, when coupled with the absence of Ham1 in other members of the It is possible that the viruses acquired the ITPase gene from a host plant by horizontal gene transfer (HGT). HGT between host and virus is predicted to have protective functions through either production of a viral antigen by the host or mimicry of a component of the host immune signalling pathways by the virus CBSV infection with increased expression of genes belonging to the pathogenesis‐related (PR) gene family  has been shown to increase expression of PR genes in 7.3E. coli ITPase, YjjX, has been shown to interact with the GTPase, elongation factor Tu (EF Tu), which is involved in peptide elongation CBSV and EuRSV: prevention of mutagenic effects of an increased pool of ITP, disruption of plant signalling following infection, and disruption of other potential cellular roles of ITP. Further research should target these as well as the function and expression profiles of plant‐encoded ITPase, particularly in the host plants for (U)CBSV and EuRSV. It is unclear if viral ITPase expression is important as a result of reduced expression of endogenous ITPase, potentially resulting in increased pools of cellular ITP. Viral sequences do not appear to show increased levels of mutation; however, this has yet to be shown in the host plant. Potentially, these viruses, which infect members of the Euphorbiaceae family, have acquired ITPase in response to a host‐specific regulation of ITP pools. A greater understanding of the host's maintenance of cellular ITP will help to elucidate a role for viral Ham1.


H9N2 avian influenza virus is one of the most widely circulating viruses in poultry and poses a huge potential threat to human health due to its frequent gene reassortment with other influenza viruses. In this study, we generated a series of H9N2-H7N9 reassortant viruses and examined their pathogenicity in a mouse model. We found that HA or combined HA and NA replacement on the H9N2 background led to no substantial change in the virus-induced pathogenicity, whereas H9N2 virus containing H7N9 internal genes had significantly higher virulence in comparison to the parental H9N2 virus. This increased pathogenicity is associated with enhanced viral replication both in mice and in MDCK cells. We further demonstrated that the viral ribonucleoprotein complex from H7N9 virus possessed higher activity than that from its H9N2 counterpart. Collectively, our data demonstrated that genetic compatibility between H9N2 and H7N9 viruses facilitated the reassortment between H7N9 and H9N2 viruses co-circulated in poultry and that internal gene replacement would convert H9N2 virus into a novel threat to human health. Moreover, viral genome analysis revealed that HA genes of the human H9N2 isolates belonged to the G1-W, G1-E, or BJ94 lineages and were related closely to the viruses isolated from local poultry (H9N2 avian influenza virus (AIV) is the most widely circulating AIV in poultry populations globally. H9N2 viruses were first isolated from turkeys in American in 1966 . In the  poultry .H9N2 viruses are often found to be co-circulated among poultry with other AIV subtypes, increasing the rates of reassortment between these viruses and thus posing a huge threat to public health. H9N2 AIV could serve as gene donor to supply internal genes to other viruses, including the H7N9, H10N8, and H5N6 influenza viruses that recently caused human infection . ReciproH9N2 infections, distinct from H7N9 infections, normally only cause mild symptoms in human, and therefore, the potential risk of enhanced virulence and/or transmissibility resulting from reassortment with other viruses is easy ignore. In this study, we aimed to address the risk that H7N9 human influenza virus will provide internal genes to H9N2 AIV.AY253750.1-AY253756.1, AY743216.1) isolated from chicken in China was conserved at our laboratory. A/Shanghai/4664T/2013(SH/4664) H7N9 virus (Genbank accession number: KC853225.1-KC853232.1) was conserved at a Biosafety Level 3 (BSL3) lab at Shanghai Public Clinical Center. For the generation of recombinant virus by reverse genetics, the cDNA of genes from CK/F98 or SH/4664 was PCR-amplified and inserted into pHW2000 vector through BsmBI cutting sites. Viruses were rescued by using an eight-plasmid reverse genetics system A/Chicken/Shanghai/F/98 (CK/F98) H9N2 virus (Genbank accession number: 6EID50 per 50 μl virus (eight mice per group). Additionally, five mice were inoculated with 50 μl PBS buffer, serving as negative controls. Among the experimental group, five mice were monitored daily for 14 days post-inoculation (dpi) for body weight and mortality. Mice with 30% body weight loss or more were considered dead and were euthanized. The other three mice were sacrificed at 4 dpi to determine virus titers in the lung, nasal turbinate, liver, spleen, kidney, and brain. For virus titration, tissues were weighed and homogenized using the TissueLyser II (Qiagen) to produce a concentration of 0.1 g/ml (wt/vol) homogenate in 0.01 M PBS. After centrifugation at 12,000 rpm for 10 min, 100-μl aliquots of the supernatants were collected, and a series of dilutions were made for inoculating 9-day-old embryonated eggs. Virus titers were subsequently determined by the Reed–Muench method.Six-week-old female BALB/c mice  were maintained under specific pathogen-free conditions at the animal facilities of Shanghai Public Health Clinical Center. All animal studies were performed in strict accordance with the Home Office regulations and were approved by the Shanghai Public Health Center Local Ethical Committee. The experiments involving live viruses were carried out in a BSL3 facility at Shanghai Public Clinical Center conforming to the institutional biosafety manual. For virus challenge, mice anesthetized with 5% pentobarbital were intranasally inoculated with a dose of 1 × 10A minigenome assay was used to measure the activity of reconstituted viral ribonucleoprotein (vRNP), as reported previously . BrieflyL-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK). Infected cells were further incubated at 37°C, 5% CO2 in a humidified incubator. Aliquots of cell-culture supernatants were collected at indicated time points post-infection and used for virus titration in MDCK cells.Confluent MDCK cells in a 25-cm flask were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.001. The viral inoculums were removed after 1 h of adsorption at 37°C and washed three times with PBS, followed by the addition of serum-free Dulbecco’s Modified Eagle Medium supplemented with 1 μg/ml trypsin treated with t-test. When more than two groups were compared, one-way ANOVA was applied in combination with the post hoc Bonferroni test.All of the statistical analyses were performed using Prism 7 software . Data were expressed as mean ± SD, except for analysis of mouse body weight change, where mean ± SEM was used. For comparison between two groups, statistical significance was assessed using a two-tailed Student’s 6 EID50. All of the infected mice survived up to 14 days after virus challenge. Both viruses caused a slight weight decrease in the infected mice in the early period that lasted for 5 and 8 days for CK/F98-4664HA and CK/F98 virus, respectively. After that, the weight of the mice in both groups start to increase. CK/F98-4664HA infection had weaker weight effects than the parental virus CK/F98, including a higher nadir and faster rebound. Accordingly, compared to the weight before infection, the former group attained an average weight of approximately 5% higher at 14 dpi, whereas the CK/F98 group showed essentially no gain  virus on the background of H9N2 virus. To evaluate the pathogenecity of that virus, mice were intranasally infected, as described above. All of the CK/F98-4664HA/NA-infected mice survived at 14 dpi. As compared to the CF/F98 group, the CK/F98-4664HA/NA group displayed more rapid weight loss in the early stage, reaching the nadir at 4 dpi . In addi6 EID50/g, which was significantly higher than that of CK/F98-infected mice  by reverse genetics. This reassortant virus, designated as CK/F98-4664Int, was successfully generated and subsequently examined in a mouse infection model. Unlike the surface gene replacement viruses and parental virus CK/F98, CK/F98-4664Int caused drastic weight loss, reaching a nadir of 28% weight loss within 9 dpi . There wP < 0.001; 10 TCID50 in the supernatant at 24 hours post inoculation (h.p.i.), whereas there was no virus detectable in the supernatant of cells infected with the latter. The virus titer of CK/F98-4664Int-infected cells was 100–1000 times higher than that of CK/F98 infected cells at 48 and 72 h.p.i. (P < 0.001; We further analyzed the kinetics of replication of CK/F98-4664int versus parental CK/F98 on MDCK cells. MDCK cells were infected with each of the two viruses at an MOI of 0.001, and the virus titers in the supernatant were tested on MDCK cells at different numbers of hours post-infection. CK/F98-4664Int proliferated significantly faster than CK/F98, and the titer reached 3.5 logP < 0.001; To test the activities of vRNPs, minigenomes derived from H9N2 and H7N9 viruses were analyzed on 293T cells. The relative activity of vRNPs from H7N9 virus was about 7-fold higher than that of vRNPs from H9N2 virus (As one of most widely circulating influenza viruses in chickens , H9N2 isPrevious studies have suggested that H9N2 AIVs can act as either donors or recipients of genes when co-circulated with other virus strains . Here, win vivo pathogenicity was highly correlated with virus titers. Thus, the CK/F98-4664Int virus with internal genes entirely from the H7N9 strain combined with a couple of H9N2 surface genes yielded significantly higher viral titer in lung and nasal turbinate as compared to H9N2 CK/F98, in line with substantially increased mortality. On the contrary, either the CK/F98-4664HA or CK/F98-4664HA/NA virus, with HA or both HA and NA from H7N9 virus and retaining the CK/F98 internal gene constellation, showed a similar low level of viral replication to CK/F98 in infected animal and consequently remained low in pathogenicity. That CK/F98-4664Int virus grew better than CK/F98 virus in cultured MDCK cells further indicated that the replication difference could be attributed at least in part to direct functioning of the internal gene constellation. This notion was further evidenced by the higher activity of H7N9-derived vRNP when compared to that of H9N2-derived vRNP in minigenome assay. Thus, the subunits of viral polymerase, PA, PB1, and PB2, along with NP, are strong candidates for virulence determinants distinguishing H7N9 from H9N2. However, we cannot exclude the candidacy of the other two internal gene-encoding segments, the NS and M segments, which respectively produced two viral proteins, namely NS1 and NS2 and M1 and M2, by alternative splicing. Indeed, previous studies have suggested the involvement of NS and M genes in the emergence of reassortants between H9N2 and H5N1 viruses. NS1 protein, which is only expressed in infected cells, is particularly of interest, given its critical role in evading host innate immunity. Thus, it will be important for future studies to pinpoint which component or combination of internal genes from H7N9 is responsible for the conversion of H9N2 to a mammalian-adapted virus on the advent of H7N9-H9N2 reassortment.Our animal studies revealed that the In summary, our study demonstrated the genetic compatibility between human-origin H7N9 and avian-origin H9N2 virus and revealed gain of the internal gene cassette from human H7N9 virus by reassortment as a potential path by which for avian H9N2 to evolve to become a causative agent in mammals. This is in agreement with the concept that the zoonotic potential of an influenza virus is determined by two critical factors: the entry and spread capability associated with HA and cellular permissiveness dictated by internal gene products. A future comparison between CK/F98-4664Int and parental 4664 virus will be needed to validate that the internal gene cassette is fully responsible for the difference between the replication capabilities of H9N2 CK/F98 and H7N9 SH/4664 strains in mammalian cells. It will also be of great interest to determine the detailed mechanism by which gene acquirement shapes the virulence of H9N2 virus. We propose that it is important to closely monitor for the emergence of H9N2 reassortant virus containing a human-derived H7N9 internal gene cassette, which might pose a serious threat to human health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Shanghai Public Health Clinical Center.MZ, JX, XZ, and ZL designed the experiments, analyzed the data, and wrote the manuscript. MZ, HC, QT, CZ, SY, LJ, XL, and DF performed the experiments. All authors reviewed and revised the first and final drafts of this 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.


Echocardiography is the most frequently used non -invasive diagnostic tool to evaluate cardiac anatomy and function in domestic species but increasingly also in non -domestic species, especially since cardiac disease is being recognized as an important cause of death in captive primates. The purpose of this cross -sectional study was to investigate the feasibility of transthoracic echocardiography in healthy squirrel monkeys as well as to provide species specific normal values for standard echocardiographic measurements. A secondary aim was to determine plasma and serum levels of the cardiac biomarkers, N -terminal pro -brain natriuretic peptide (NT -proBNP) and cardiac troponin T (cTnT). Furthermore, a commercial, non -invasive, smartphone -based ECG (AliveCor Vet TM) monitoring device was used to evaluate the heart rate and rhythm and to diagnose possible arrhythmias.In this study, transthoracic echocardiography of 14 squirrel monkeys was performed in right and left lateral recumbency. Similar standard right parasternal and left apical images were obtained as in dogs and cats and normal values for routine two -dimensional, time motion mode and Doppler mode measurements were generated. Thirteen animals were considered healthy and one squirrel monkey was identified with significant aortic dilation and regurgitation and consequently values obtained from this animal were not used when species specific normal values were calculated. NT -ProBNP and cTnT concentrations were available for 7 of the 13 healthy monkeys with NT -proBNP concentrations below detection limit in all animals and a mean cTnT concentration of 0.049 ng/mL. Electrocardiography was performed in all squirrel monkeys. The mean heart rate was 172 bpm. Frequent supraventricular premature beats were diagnosed in the squirrel monkey suffering from significant aortic dilation and regurgitation.This study presents echocardiographic normal values and additional cardiovascular data in anaesthetised Saimiri monkeys, fundamental from both the perspective of zoo animal health care as well as scientific research, since the squirrel monkey is often used as an animal model for human disease. Squirrel monkeys  are among the most commonly studied animal models in biomedical research, both in veterinary and human medicine, the latter due to their phylogenetic similarities . These aMacaca mulatta), owl monkey (Aotus sp.) and squirrel monkey [Several studies in both human and nonhuman primates have identified cardiovascular disease as a major cause of death with mortality rates ranging from 41% in gorillas to approximately 81% in chimpanzees . Eal monkey , 15–24.Given the significant mortality secondary to cardiovascular disease, previous research has concentrated on thoracic radiographic, electrocardiographic and echocardiographic characteristics of squirrel monkeys , 26. HowEchocardiography is the most commonly used diagnostic tool for non -invasive assessment of cardiac structures and function. Elevated blood concentrations of cardiac biomarkers such as N -terminal pro -brain natriuretic peptide (NT -proBNP), cardiac troponin I (cTnI) and T (cTnT) are associated with several types of cardiac disease in human and veterinary medicine –30. CardAlthough an echocardiographic protocol in great apes has been described and is currently being applied in a large international study in chimpanzees, gorillas, bonobos and orangutans, no such protocol has been defined in the squirrel monkey –38. The The primary aims of this study were to test the feasibility of transthoracic echocardiography (TTE), following a protocol similar to canine and feline echocardiography, in healthy squirrel monkeys as well as to provide species specific normal values for standard echocardiographic measurements. A secondary aim was to determine blood concentrations of two cardiac biomarkers, NT -proBNP and cTnT, and to evaluate heart rate and rhythm using a non -invasive, smartphone based ECG (AliveCor Vet TM) monitoring device.Saimiri boliviensis peruviensis presented with a significant abnormality (severe dilation of the ascending aorta with aortic regurgitation) on echocardiography and arrhythmia (frequent supraventricular beats) on electrocardiography and was excluded from the study. The age of the 13 remaining animals ranged from 2.25 to 21.3 years (mean age = 9.12 years ± SD of 6.95 years). The minimum and maximum weights of the animals were 467 g and 756 g respectively (mean weight = 615 g ± SD of 92 g). There were 11 females and 2 males.One Saimiri spp.) showed signs of trace valvular insufficiency without clinical relevance, namely of the mitral valve (n = 2), tricuspid valve (n = 6), aortic valve (n = 2) and pulmonic valve (n = 7). Furthermore, one mild mitral valve insufficiency was diagnosed without signs of cardiac remodelling.Good quality echocardiographic examinations were obtained in all 13 healthy animals and 25 variables were measured  during the echocardiographic examination was 172 ± 40 bpm (121 -236 bpm). No cardiac arrhythmias were detected , there is little data available regarding echocardiographic evaluation of the heart in this particular species [Despite the fact that cardiomyopathy is among the most common causes of death in aging squirrel monkeys . The relevance of this statement was proven by the fact that one apparently healthy squirrel monkey was diagnosed with significant aortic dilation and insufficiency and frequent supraventricular premature beats .This study shows that TTE represents a useful, non -invasive imaging technique for antemortem evaluation of cardiac structure and function. Furthermore, TTE could also be a valuable tool for performing cardiovascular physiological studies and/or non -invasive routine cardiac screening in squirrel monkeys . In healthy humans and animals, both circulating cTnT and NT -proBNP values are low. As myocardial cells are damaged secondary to inflammation or infarction, cTnT, which is tightly bound to sarcomeric actin filaments under physiological circumstances, leaks into the cytosol and interstitial space. Both in human and veterinary medicine, cTnT has proven its diagnostic value in evaluating the presence and severity of myocardial damage. Given its high conservation across species, we expected it will be of value in squirrel monkeys as well [Additionally, cardiac biomarkers cTnT and NT -proBNP were determined. To the best of the authors’ knowledge, this is the first study evaluating the cTnT or NT -proBNP values in squirrel monkeys  have low NT -proBNP values or the human NT -proBNP test used in this study is not applicable in this species. Recent research in Cynomologous monkeys has shown that caution should be used with the interpretation of NT -proBNP when applying human assay kits as good cross reactivity was not observed despite 90% sequence homology of NT -PROBNP between humans and monkeys [NT -proBNP is the prohormone of B -type natriuretic peptide . It will become elevated secondary due to myocardial stress, which can be caused by multiple cardiomyopathies. Given its longer circulating half -life, higher concentration and more stable nature in stored (frozen) plasma or serum compared to BNP, NT -proBNP is preferred as a cardiac biomarker over the latter –57. Both monkeys .As is often the case in zoological studies and because this study was limited to the zoo colony of Pairi Daiza, the low number of squirrel monkeys included is a limitation of the study. The sample size does not allow an assessment of statistical differences in echocardiographic variables between male and female monkeys or young and older animals. Furthermore, given the low number of squirrel monkeys and the fact that all NT -proBNP concentrations were below detection limit, this study was not able to establish reference values for cTnT nor NT -proBNP. Despite these limitations, we were able to demonstrate that a standard veterinary echocardiographic scanning protocol can be used to generate an extensive set of echocardiographic normal values and described it in detail. Nonetheless, additional studies with a larger number of animals allowing differentiation between male versus female monkeys and also young versus older animals are necessary in the future.Saimiri spp.), whereas concentrations of NT -proBNP were below detection limit. Results of this study are valuable in zoological medicine as well as in future research involving non -human primate models of cardiovascular disorders and morphologic studies on squirrel monkeys (Saimiri spp.).This study showed that qualitative echocardiographic images can be obtained in squirrel monkeys, following an echocardiographic scanning protocol similar as has been described in canine and feline cardiology. Secondly, normal values for 2D, M -Mode and spectral Doppler measurements were established. Lastly, to the best of our knowledge, this is the first study to report normal values of cTnT in squirrel monkeys . A description of the thoracic radiographic findings of this cohort of monkeys has been previously published [Fourteen captive squirrel monkeys (Saimiri spp.), 9 ublished . Monkeysublished .The general health examination was the sole reason of the general anesthesia in all included animals. General anesthesia, including both induction and maintenance, was by mask. To minimize resistance and dead space, an Ayre’s T -piece was used. The volume of oxygen was fixed at 2 L/min at all times and the percentage of isoflurane  adjusted to the anaesthetic monitoring of the patient by checking eyelid  and cornea reflexes. The percentage was on the maximum value (5%) for induction and was steadily reduced for maintenance until 1,5 ± 2%. Twenty milliliter of saline solution (Ringer Lactate1 solution) was administered subcutaneously to all animals at the beginning of the procedure. Throughout the course of the complete anaesthesia, respiratory rate was counted visually, heart rate and oxygen saturation were monitored using pulse -oxymetry placed on the hand and rectal temperature was continuously monitored with a digital probe. In order to maintain the latter, gloves were filled with hot water (37 °C), a heating carpet (38 °C) and a pre -heated sonographic gel were used. After completion of the general health examination, animals were recovered in a small dark transport cage in a heated room and transported back to their enclosure thereafter. Animals were monitored after anaesthesia for 3 consecutive days. No adverse effects were recorded .A complete echocardiographic examination was performed by a board -certified cardiologist and an electrocardiogram (ECG) with the Alivecor system (AliveCor Vet TM®) after the radiographic thoracic examination. The mean time of anesthesia was 31 min, ranging from 22 min to 35 min .Blood samples were taken in seven squirrel monkeys by femoral venipuncture and collected in 4 -mL blood tubes, EDTA (ethylenediaminetetraacetic acid) and serum separator tubes. The remaining 6 squirrels monkeys could either not be sampled or blood sample volume was insufficient. Samples were centrifuged within 30 min, plasma and serum samples were stored at 4 °C until testing. cTnT serum and NT -proBNP concentrations were determined using the chemiluminescent sandwich ELISA human high sensitive (hs) Troponin T (Cobas®) and human proBNP II (Cobas®) tests, respectively.Echocardiographic examinations were performed by a single operator (PS) using a GE Vivid -I portable ultrasound machine and a 3.5 -7.5 MHz phased array transducer ) and analysed offline with GE EchoPAC software®. A detailed step -by -step operation procedure of the echocardiographic examination has been added as ‘The right parasternal long -axis four -chamber image was obtained by placing the transducer within the 3rd to 6th intercostal space, close to the sternum, with the reference mark toward the neck and a 45° angle between the transducer and the squirrel monkey Figs. . FinallyThe right apical four -chamber long -axis image was generated with the sound plane cranially along the heart, the transducer placed near the apex of the heart, starting close to the xyphoid, moving cranially along the sternum and the crystals directed toward the shoulder and base of the heart. Whilst remaining in the same intercostal space, the left ventricular outflow tract could be visualised by slightly rotating the transducer. The transvalvular flow of the atrioventricular valves and aortic velocity were evaluated by applying spectral Doppler mode Fig. . Mitral Immediately before echocardiography an electrocardiogram (ECG) was recorded via a commercial, non -invasive, smartphone -based monitoring device (AliveCor Vet TM®). Similar to the use of the AliveCor Vet TM® in cats, the hands of the monkeys were placed at the contact pads. Electrocardiograms were recorded in right lateral recumbency for at least 30 s by using a chart speed of 25 mm/s whilst the gain was adjusted for 10 mm of deflection per millivolt. Electrical contact was enhanced by the application of alcohol. During the echocardiographic examination, ECG was also performed, using live monitoring on the GE Vivid -I portable ultrasound machine. Figure All variables were evaluated visually for normality using Q -Q plots. As all variables were normally distributed, the mean and standard deviation (SD) are given, together with the range. Only animals considered healthy were included when the species -specific normal values are reported. The statistical analysis was conducted in R version 3.4.4.Additional file 1. Supplement I: Operating procedure of the echocardiographic examination in healthy captive male and female squirrel monkeys.


Severe acute respiratory illness caused by 2019 novel coronavirus (2019-nCoV), officially named severe acute respiratory syndrome coronavirus (SARS-CoV-2) in late December 2019 is an extremely communicable disease. World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) as a pandemic as it has spread to at least 200 countries in a short span of time. Being a new disease there is lack of information about pathogenesis and proliferation pathways of this new coronavirus. Currently there is no effective treatment for coronavirus infection; major effort is to develop vaccine against the virus and development of therapeutic drugs for the disease. The development of genome-based vaccine and therapeutic antibodies require thorough testing for safety and will be available after some time. In the meanwhile, the available practical approach is to repurpose existing therapeutic agents, with proven safety record as a rapid response measure for the current pandemic. Here we discuss the presently used repurposed drugs for COVID-19 and the potential for ivermectin (IVM) to be used as a therapeutic option in COVID-19. The severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2) has been detected as the pathogenic virus in the current outbreak which started in the city of Wuhan, Hubei Province of China in early December 2019. The Centre of Disease Control and Prevention in China, detected a new type of corona virus on 7th January 2020 causing coronavirus disease of 2019 (COVID-19), provisionally named as 2019 novel coronavirus (2019-nCoV) by World Health Organization (WHO) and later renamed as SARS-CoV-2 by International Committee on Taxonomy of Virus  that makes this agent a broad antiviral agent . Though n = 99) has concluded that the combination was not more effective than standard care treatment and had similar recovery process in severe COVID-19  and EC90  values of 0.77 μM and 1.76 μM, respectively , including SARS-CoV-2 with ECectively .50 of favipiravir against SARS-CoV-2 was 61.88 μM/L in Vero E6 cells  polymerase, inhibiting viral replication. In vitro, the ECE6 cells .Streptomyces avermitilis and purified by Campbell, has greater potency and lower toxicity . IVM, an anthelminthic drug is used to treat various parasitic infestations. This includes river blindness (onchocerchiasis), head lice, scabies, lymphatic filariasis, ascariasis, entrobiasis, strongyloidiasis and trichuriasis . IVM wastoxicity . They actoxicity . The peatoxicity .Independent of its antiparasitic action, IVM has demonstrated broad spectrum antiviral property in vitro. IVM is shown to be effective in vitro against RNA and deoxyribonucleic acid (DNA) viruses, including human immunodeficiency virus-1 (HIV-1), dengue virus (DENV), influenza, Venezuelan equine encephalitis virus (VEEV) and Zika virus . NuclearPseudorabies virus (PRV) is the causative agent for pseudorabies (PR), which is an important swine disease. Virus can cause lifelong infection in pigs by residing in the trigeminal ganglia. Vaccines are used widely but are unable to provide absolute protection. Use of IVM, blocked the nuclear/nucleolar translocation of the accessory subunit of the DNA polymerase UL42 and reduced the lesions in mice caused by PRV infection in vitro and in vivo . This im50 for inhibition of YFV replication was between 3.1 nM and 6.3 nM. IVM proved less active against DENV in the CPE reduction assay (EC50 0.1 mM), although inhibition in virus yield reduction assays was observed (EC50 0.7 mM). IVM has proved to be most potent against YFV and inhibit the in vitro replication of DENV, JEV and WNV, though less efficiently  and DENV nonstructural (NS1) protein 5. In DENV, a single daily oral dose of IVM has resulted in a significant reduction in serum level of viral NS1 protein, but no change in viral load has been observed . Infectiiciently . Liposomiciently . Recentliciently .IVM is used in a dose of 0.15 mg/kg–0.2 mg/kg body weight for most of the parasitic infestations as oral tablet and is well tolerated. It will be reasonable to use similar dose empirically in COVID-19 patients with mild to moderate symptoms, PCR positive for SARS-CoV-2 virus, and without comorbidities on first encounter, till there is conclusive evidence from randomised controlled trials. Rarely adverse effects such as seizure, hypotension and worsening of asthma are observed with IVM and should be avoided in patients with history of allergy, liver disease and asthma. A combination of IVM (200 mcg/day once stat) with doxycycline (200 mg/day for 5 days) can also be attempted empirically as it has been administered previously for parasitic infections too.COVID-19 pandemic has affected over 200 countries in a span of less than 4 months burdening the entire health care system with no viable drug which can inhibit the viral transmission or infectivity. Most of the drugs which are being used to treat this novel disease are not so effective and the development of vaccines is distant in near future, thus there is enormous requirement to repurpose the available drugs using concrete evidences. Present circumstances demand a therapeutically effective option against SARS-CoV-2 without delay. IVM which is a widely used as antiparasitic drug has shown to have antiviral activity in in vitro studies against HIV, dengue, influenza, VEEV and Zika virus. The broad-spectrum antiviral activity of IVM against both RNA and DNA viruses offers an added advantage to its potential use over other antiviral drugs. Studies are available for its use against RNA virus and have also been tested for its effectiveness against SARS-CoV-2 in vitro. Based on evidences it seems prudent to use IVM empirically and conduct well designed controlled randomised trials to prove its efficacy in vivo.


Viral encephalitis, an inflammation of the brain parenchyma caused by virus infection, poses a serious threat to global public health. The major causal agents worldwide are herpes viruses and arboviruses. HSV-1 is the leading cause of adult encephalitis in developed countries. While Japanese encephalitis is the most common encephalitis worldwide, West Nile virus is the most widespread virus. Recently, Zika virus, chikungunya virus, and Dengue virus have co-circulated in many regions of the Americas Silva, .Despite the advance in studying the complex interplay between viruses and infected cells, the pathogenesis of viral encephalitis is largely unknown. For example, the regulation of blood brain barrier (BBB) permeability and the regulation of immune responses to virus infection in the CNS remain to be explored.This Research Topic combines 10 publications from 64 authors, including 3 review articles and 7 research articles, covering many aspects of RNA and DNA virus-associated encephalitis.Chen B-S et al. and Majer et al. provide readers a convenient way to understand the most up-to-date knowledge of non-polio, enterovirus-associated encephalitis. These two articles cover all aspects of recent advances in enterovirus-induced encephalitis including the routes of CNS infection, tropism, virulence, immune response, and molecular pathogenesis. These two reviews provide insightful information for the development of effective antiviral strategies and vaccines.The two in-depth reviews by Chen J. et al. described the epidemiological and phylogenetic analysis of the very first large-scale Echovirus 30  outbreak in northwest China in 2015. The study shows children aged 6–15 years are more susceptible to E-30 infection. The highly similar E-30 genomes in this outbreak suggested an aggregate outbreak of E-30. The dominant lineage has a complex genetic transmission that indicates the infection initiated from coastal provinces of China and then spread to other parts of the country. The study provides valuable information for future surveillance.Giannitti et al. characterized the first case of bovine astrovirus-associated encephalitis in Uruguay (South America). Phylogeographic analysis suggests the virus was introduced to South America from Europe and later spread to North American and Japan. The study advances the understanding of the geographic distribution and genetic diversity of astroviruses.Globalization promotes virus transmission and disease spreading among continents. The ongoing COVID-19 pandemic is a devastating example  restricting virus replication in West Nile virus (WNV)-induced encephalitis. WNV is the leading cause of viral encephalitis in the United States. ZBP1 plays an essential role in triggering robust immune responses. WNV infection dramatically up-regulates ZBP1 expression in mouse brain and primary mouse cells. Deletion of ZBP1 results in higher morbidity and mortality after WNV infection in mice. Infection of ZBP1–/– mice with the virus is lethal, indicating that ZBP1 is required for survival after WNV infection.The vast majority of papers in this Research Topic are related to Flavivirus-associated encephalitis with distant emphasis on different viruses. Tomar et al. reported the development of a polymerase spiral reaction assay for real time detection of WNV from clinical samples. As there is no FDA-approved vaccine or antiviral against WNV, early diagnosis of WNV infection is critical for clinical management and disease control. The reverse transcription polymerase spiral reaction (RT-PSR) assay rapidly and accurately detects the envelope gene of WNV using real-time turbidimeter or visual detection by the addition of SYBR Green I dye. The assay is validated and is able to detect as low as one copy of RNA, which is 100-fold higher than conventional RT-PCR. The assay has a potential to be used for rapid testing of a large number of clinical samples.Chiu et al. used human placenta trophoblasts cells (JEG-3) and human brain-derived endothelial cells (hCMEC/D3) as in vitro models to study the mechanisms ZIKV uses to cross the physiological barriers. The study shows ZIKV infection changes the permeability of JEG-3 cells by disrupting the tight junction and by transcytosis. ZIKV crosses the BBB by transcytosis.Zika virus (ZIKV) is another important member in the Flavivirus family that causes severe neurological complications such as meningoencephalitis in adults and microcephaly in fetus  mice and in RIPK3-knockdown neuro2a cells with a significantly increased level of interferon (IFN)-induced protein 44-like gene (IFI44L). Over-expression of IFI44L decreases JEV replication in neuro2a cells and double knockdown of RIPK3 and IFI44L promotes virus replication. On the other hand, RIPK3 inhibits IFI44L expression to promote JEV propagation in neurons.Japanese encephalitis virus (JEV) is the major cause of viral encephalitis worldwide. JEV infection targets neurons and receptor interacting serine/threonine-protein kinase 3 (RIPK3), which contributes to neuron inflammation and death. Oladunni et al. reviewed the discovery of the virus, the latest developments of treatment and disease control, and summarizes recent advances in the research of EHV-1 pathogenesis. The information presented is useful for development of strategies to limit the spread of EHV-1 in equine populations.DNA virus infection can induce encephalitis as well. Equine herpesvirus-1 (EHV-1) is one of the most important pathogens of horse and causes a constant threat to the equine industry worldwide. There are increasing numbers of devastating equine herpesviral myeloencephalopathy outbreaks. Qi et al. used a fosmid library as a platform and Red/ET recombination technology to generate recombinant PRV fused with EGFP. The rescued recombinant virus is used to monitor retrograde and anterograde moving in the axon, making it a powerful tool for neuronal circuit analysis. The study is helpful for development of vaccines against PRV and other herpesviruses.Pseudorabies virus (PRV) is a member of the Herpevirus family. We hope that this Research Topic provides readers a better understanding of virus-associated encephalitis. A deeper understanding of viral encephalitis will facilitate the development of innovative antiviral approaches.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Aedes aegypti and Culex pipiens complex), from different geographic locations and environments  for highly sensitive next-generation sequencing-based virome characterization. We found a rich virus community associated with a great diversity of host species. Among those, we detected a large diversity of novel virus sequences that we could predominately assign to circular Rep-encoding single-stranded (CRESS) DNA viruses, including the full-length genome of a yet undescribed Gemykrogvirus species. Moreover, we report for the first time the detection of a potentially zoonotic CRESS-DNA virus (Cyclovirus VN) in mosquito vectors. This study expands the knowledge on virus diversity in medically important mosquito vectors, especially for CRESS-DNA viruses that have previously been shown to easily recombine and jump the species barrier.Mosquitoes are the most important vectors for arthropod-borne viral diseases. Mixed viral infections of mosquitoes allow genetic recombination or reassortment of diverse viruses, turning mosquitoes into potential virologic mixing bowls. In this study, we field-collected mosquitoes of different species ( Aedes and Culex genera are well adapted to human environments, making them the most important transmission vectors for arthropod-borne viruses (arboviruses) such as Dengue virus, Zika virus, or West Nile virus [Mosquitoes represent the most medically important group of arthropod vectors. In particular, mosquitoes of the le virus . Neverthle virus ,3,4,5.Flaviviridae, Togaviridae, the order of Bunyavirales, and, recently, Mesoniviridae [Aedes cell lines in vitro, including West Nile virus, Japanese encephalitis virus, and Zika virus [Mosquitoes may be infected by a range of insect-specific viruses (ISVs) that replicate only in arthropod cells, unlike arboviruses that also replicate in vertebrate cells. Arboviruses are closely related to ISVs in terms of biological properties and phylogenetic distance and may have arisen from ISVs that occasionally gained the capacity to infect secondary hosts ,7,8. Mosiviridae . Despiteiviridae ,10,11,12ka virus ,14. In vka virus .After identification of the first ISV (cell-fusing agent virus) in 1975, it took almost 25 years for the second ISV to be discovered (Kamiti river virus) and seven more years for the third (Culex flavivirus) . While aIn addition to arboviruses and ISVs, mosquitoes may temporarily host additional virus species that do not replicate in the arthropod hosts but become transiently associated with the mosquito vector through dynamic interaction with virus sources of the prevailing environment. For example, depending on species and sex, mosquitoes can be nourished on the blood of vertebrate hosts  or on the nectar of pollinating plants . The diversity of the mosquito virome is, therefore, in constant flux, depending on the surrounding environment . VirusesThe presence of a range of viruses from different hosts may drive interspecies recombination or reassortment within mosquitoes and promote the emergence of novel virus species . The likAedes aegypti (Ae.ae.) and Culex pipiens complex (C.pip.cl.) mosquitoes from the Caribbean  and central Europe  to conduct comparative virome analysis of the two ecologically divergent areas. We characterized phylogenetic virome patterns and the spectrum of virus-associated hosts. Viral hits were categorized by concordance with database entries, hence revealing any novel viruses. Among those, we describe the genome organization of a novel virus of the genus of Gemykrogvirus and, for the first time, identify a potentially zoonotic Cyclovirus (CyCV-VN) in mosquito vectors.The replication of arboviruses and ISVs within the same biological compartment and the transfer of viruses between ecological niches provide the prerequisite for the emergence of novel mutant viruses with pathogenic potential. Metagenomic analysis of the mosquito virome can promote unbiased detection of viruses that might not have been associated with mosquito vectors before, including zoonotic viruses. In this study, we aim to analyze the metagenomic virome diversity of medically important mosquito vectors. We performed highly sensitive virus metagenome sequencing of field-collected Mosquito collection was performed as previously described, using carbon dioxide-equipped BG sentinel traps  for 24-h time periods . To comp® Inc, Mountain View, CA, USA) and land cover maps provided by the Austrian Environment Agency  were used for this study. Trapped mosquitoes were shock-frozen at −80°C within a maximum time of 60 min after trap disassembly and transferred to the research laboratory of the University of Veterinary Medicine, Vienna. Female mosquitoes were specified  using the key of Becker and (single legs) genetically verified with molecular barcoding, as previously reported [In Austria, traps were set up at two trapping sites at the municipal area of Vienna . Locatioreported ,24. Noneg and 15 min at 4800× g, filtration through a 0.45 μM syringe filter, ultrafiltration using 50 kDa molecular weight cut-off filtration units , DNase I digestion (Qiagen), DNA and RNA preparation using a QIAamp UCP Micro Kit (Qiagen), blocking of ribosomal RNA sequences using a set of 5 specific oligonucleotides, cDNA synthesis using nonribosomal hexanucleotides together with the Super Script IV enzyme (Thermo Fisher Scientific), and final amplification of the total nucleic acids using a Repli-g kit (Qiagen).Mosquito individuals of each spot were pooled in numbers of 50. Mechanical homogenization was performed using a TissueLyser II  with five 2.8 mm ceramic beads  and 1 mL of Dulbecco’s phosphate-buffered saline (DPBS) buffer at a frequency of 30 strokes per second for a duration of 4 × 30 s interspersed by a 30 s pause. Virus purification and enrichment protocol (VIPEP) was performed on mosquito homogenates in combination with an additional SpeedVac  concentration step, as previously described . In shorMDA-amplified double-stranded cDNA and genomic DNA were quantified with a Qubit dsDNA High Sensitivity Kit on a Qubit 4 fluorometer (Thermo Fisher Scientific) according to the manufacturer’s instructions. Shotgun library preparation was done with a NEBNext Ultra II DNA Library Prep Kit for Illumina in combination with the Index Primers Set 1 and 2  according to the manufacturer’s instructions. Briefly, up to 30 ng dsDNA were fragmented by ultrasonication in a Bioruptor Pico sonication system  in a total volume of 55 μL 1× TE for 5 cycles of 15 s on and 30 s off. Then, 50 μL of fragmented DNA was used for end repair and adapter ligation reactions, according to the manufacturer’s instructions, for a DNA input of less than 100 ng. Size selection and purification were performed according to instructions for 500 to 700 bp insert size. Subsequent PCR amplification was performed with 12 cycles, and libraries eluted after amplification and purification in 33 μL 1× TE buffer (pH 8.0). For quality control, libraries were analyzed with a DNA High Sensitivity Kit on a 2100 Bioanalyzer system  and quantified on a Quantus™ fluorometer . Following library preparation, equimolar pools were sequenced on an Illumina MiSeq desktop sequencer . Libraries were diluted to 8 pM and run without PhiX control for 600 cycles with version three chemistry according to the manufacturer’s instructions. FASTQ Files were used for data analysis.https://adapterremoval.readthedocs.io), keeping only sequences longer than 30 bp, with a maximum error rate of 3 and trimming ambiguous bases in 3′ and 5′ ends. Trimmed datasets were de novo assembled using metaSPAdes v3.12.0 (https://cab.spbu.ru/spades) with default parameters. Contigs larger than 500 bp were used for downstream analyses. Open reading frames (ORF) were predicted on the contig sequences using MetaGeneMark v3.38. BLASTN analysis (Blast+ v2.5.0) (http://exon.gatech.edu/meta_gmhmmp.cgi) against the NCBI nt database was used as a first approach to determine contig identity, employing a minimum e-value threshold of 1 × 10−5 and keeping the first 25 hits. A BLASTP analysis of the predicted ORF sequences (in amino acids) against the NCBI nr database  was used as a second approach. Depending on the case (see the Results section), taxonomic annotation of contigs was resolved by either importing BLAST results to MEGAN v6.10.13, which uses the lowest common ancestor algorithm for classification, or by considering the best BLAST hit. The relative abundance of each contig in each sample was calculated using the RPKM metric (reads per kilobase per million mapped reads), which normalizes by contig lengths and sequencing depth of samples. For this, reads were remapped on contigs using bwa v0.7.10-r789 with the “aln” method, while coverage and depth were calculated on the resulting bam files using custom scripts and bedtools v2.26.0 (https://bedtools.readthedocs.io). In addition, the abundance of each individual taxa (based on NCBI taxID) was also estimated using the RPKM method, utilizing the average of RPKM of all contigs assigned to the same taxID.Raw sequencing data were quality trimmed and adaptors removed using AdapterRemoval v2.2.0  and mean coverage of query sequence (% subject coverage) were used for the analysis. Alternatively, when contigs were found to hit different sequences but with the same NCBI taxonomy ID, mean % ID and mean % query coverage were calculated for each subject sequence.Genomoviridae, according to Zhao et al., and (ii) ten other Genomoviridae species identified by BlastN were retrieved from the NCBI database [Gemycircularvirus type species was included as the outlier. Sequence alignments were created in T-Coffee using the PSI-Coffee protein alignment algorithm, including protein structure information [Predicted Rep and Cap protein sequences of the novel virus were used for phylogenetic analysis. Amino acid sequences of Rep and Cap proteins of (i) proposed type species of the nine phylogenetic genera of the family of database . Additioormation . AlignmeThe phylogenetic tree was generated using the maximum likelihood algorithm based on the LG model including discrete Gamma distribution (+G) and by assuming that a certain fraction of sites are evolutionarily invariable (+I). This model was identified as the best fitting model with the lowest BIC (Bayesian information criterion) in MEGA10 ,29. The http://primer3.ut.ee) software, and primer sequences were tested for specificity by Primer Blast analysis (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Amplicon length was chosen to be less than 200 nt (132 bp for BatCV and 160 bp for CyCV-VN), as required for testing shared sequencing libraries. The following primer pairs were chosen: BatCV_fw (5′- ATCCAGCCGTAGAAGTCGTC-3′) and BatCV_rv (5′-CGGAAAATCAAAGCGTGCAC-3′), CyCV_fw (5′- TGAAGGAGGAGAGACATGCC-3′) and CyCV_rv (5′- TGTTCCAGTCGATCCCCAAA-3′). The PCR mixture contained 1× iTaq PCR buffer , 200 mM of each deoxynucleotide triphosphate (dNTP) mix, 2 mM MgCl2, 5 mM of each forward and reverse primer, and 0.4 U iTaq DNA polymerase (Bio-Rad) in a 20 μL reaction volume. Additionally, 2 μL of VIPEP enriched Ae.ae.BRB or C.pip.cl.BRB was used as a template and 2 μL of nucleic acid-free water as a negative control. PCR reactions included initial denaturation in a thermal cycler at 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 59 °C (BatCV) or 54 °C (CyCV-VN) for 60 s and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. Amplicons were visualized by electrophoresis on 2% agarose gel.PCR reactions were performed for verification of metagenomic detection of Bat circovirus POA/2012/II (BatCv) and Cyclovirus VN isolate hcf1 (CyCV-VN). Primers were designed in Primer3 (Verification of human Torquet Teno virus (TTV) was done by real-time PCR (RT-PCR), as previously described by Maggi et al. . Briefly6 to 1.41 × 107 raw sequencing read pairs per sample; read pairs were consequently trimmed and assembled to contigs. Taxonomic annotation matched 66% to 83% of contigs longer than 500 bp to NCBI database entries  shared eight viral hits while mosquitoes from Barbados (Ae.ae.BRB and C.pip.cl.BRB) shared a set of four mutual viral hits  than compared to the pools of Cx. pipiens complex mosquitoes from Austria . However, these findings are limited by the small number of pools tested.Mosquito pools from similar environments were found to share mutual virus hits, referred to as ecosystem signature viruses. ral hits . We did Cx. pipiens complex mosquitoes from the collection spots. Governmental land coverage maps were used to characterize the immediate surroundings  and introduced variable numbers of random point mutations between 10% to 50% of total length . AssignmWolbachia-infecting phage species. Moreover, we found that ecosystem signature viruses were predominantly assigned with a high degree of confidence. We used this method of analysis to identify those hits of the mosquito virome that closely resembled assigned virus taxa . Among tWolbachia-infecting bacteriophages. Daesongdong virus 2 (DaedV2) was previously identified in C.pip.cl. mosquitoes from South Korea, while different strains of Biggievirus Mos11 (BigV) have been identified in C.pip.cl. mosquitoes from the US, Italy, and India . Imjin River virus 1 (IRV1), an ssRNA virus taxonomically related to Wuhan mosquito virus 8 (WMV8), has been previously identified in virus metagenomes of Cx. bitaeniorhynchus from South Korea [Torquet Teno virus (TTV) and Bat circovirus POA/2012/II). Notably, the fraction of CRESS-DNA viruses in the group of high-confidence hits, across all samples, was less than in the overall metagenomes , we designed specific PCR assays on the respective metagenomic sequences. All four PCR assays yielded uniformly positive results, and sequencing of PCR amplicons verified NGS-derived metagenomic sequences .Cyclovirus VN (CyCV-VN) virus species. Sequence analysis of the contig sequence annotated as CycV-ZM38 revealed a pairwise sequence identity to CyCV-VN of 78%, including a 141 nt sequence that is identical to the last 141 nt (1855–1995) of the contig, indicative of the circular genomic structure. The contig sequence displayed a conserved genome organization, with two ORFs resembling a capsid protein (cap) coding gene and a replicase (Rep) coding gene in BlastP analysis. According to the species demarcation criteria for circoviruses, being <75% of genome nucleotide identity, the metagenomic sequence resembles a closely related, novel variant of CyCV-VN.CycV-ZM38 resembles a strain of p = 0.001).Hits with 0–60% query coverage matched database sequences only at short stretches, thereby resembling widely novel sequences. Of these, 64.9% were assigned as CRESS-DNA viruses . CompareHigh-fidelity virus assignments were associated with hosts that fit into the biological context . Hits of lower goodness-of-assignment fit corresponded to hosts that were inappropriate to the ecosystem of mosquito collection . Moreover, environmental viruses, plant viruses, and the majority of bacteriophage sequences were clustered in this field of low goodness-of-assignment fit. Genomoviridae. The full-length contig sequence k141_1014 (2222 nucleotide) resembled the circular genome of a novel CRESS-DNA virus of the family of Genomoviridae, tentatively named mosquito-associated virus Barbados  and a capsid protein (Cap) and presented the structural features of two palindromic sequences flanking a putative origin of replication. In the phylogenetic tree constructed using Rep amino acid sequences from viruses of the family of Genomoviridae, MaVBRB clusters were within the genus of Gemykrogvirus  trapped at distinct ecosystems (Austria and Barbados). We assembled a large diversity of viral sequences matching database entries of viruses that were previously associated with a range of hosts. Separating hits by their goodness-of-assignment fit, we provide evidence that mosquito vectors host a “core virome” that is closely related to previously identified virus taxa. Strikingly, a large fraction of this set of viruses was shared among mosquito pools from the same ecosystem, even though collection points were separated by several kilometers in distance. Ecosystem signature viruses of the Austrian pools predominantly resembled close taxonomic relatives of mosquito-specific viruses that have been identified previously in Culex mosquitoes from different parts of the world [C.pip.cl. and emphasize the global abundance of these mosquito-specific viruses. However, despite the limiting number of samples, we observed more similarity in viromes from closely related environments than in the same mosquito species from different environments. We, therefore, hypothesize that the local ecosystem may play an important role  in shaping the viral communities in mosquitoes as individuals of different species are exposed to common sources of virus infection.In this study, we analyzed and compared virome patterns of two medically relevant mosquito species  and Wolbachia phage WOVitA2 (Tax ID 949125) in field-collected mosquitoes from central Europe. However, we did not recover full genomic sequences as corresponding contigs covered just parts of the bacteriophage genomes that might represent conserved sections of this taxonomic group. Moreover, we found evidence that different pod host . Therefopod host . This isAnalyzing the viral metagenome on a taxonomic level, we found that CRESS-DNA viruses account for a disproportionally prominent fraction of the taxonomic richness and form the most abundant taxonomic group in the mosquito virome. The taxonomic clade of Rep-encoding ssDNA viruses (CRESS-DNA viruses) is highly diverse and abundant. CRESS-DNA viruses share a similar genome organization, all encoding a highly conserved replication-associated protein (Rep) and at least one further, less conserved capsid (Cap) protein. In many virus metagenomic studies, multiple displacement amplification (MDA) is used as a standard method for unspecific sequence amplification, whereby small circular ssDNA sequences such as CRESS-DNA viral genomes are preferentially amplified. Hence, it has to be taken into account that relative virus representation might thereby be biased . HoweverAe. ae. virome. Bat circovirus POA/2012/II (BatCv POA/II) is a CRESS-DNA virus of the family of Circoviridae that was initially detected in bat feces collected in Southern Brazil [Cycloviruses may only infect arthropods while they are currently being associated with diverse vertebrate hosts [Ae. ae. virome were exclusively identified by metagenomic sequencing of bat feces. Final host assignment can only be done by further virus isolation experiments. However, the current findings suggest that a spillover of CRESS-DNA viruses does not only occur from vertebrate hosts downwards onto mosquitoes but also from mosquitoes upwards. Thereby, mosquitoes act as mixing vessels for CRESS-DNA viruses from different environmental sources, which may promote recombination events leading to novel virus variants.Among CRESS-DNA viruses of vertebrate hosts, bat viruses accounted for a disproportionally high fraction of the n Brazil . Besidesn Brazil . Hence, te hosts . All fouGemykrogvirus, tentatively named MaVBRB. MaVBRB forms a phylogenetic cluster with a later identified AmGV-2, infecting honeybees. Most of the other members of the genus of Gemykrogviruses have previously been detected in the feces of various animal species [Gemykrogviruses. It is still a matter of speculation what the true hosts of Gemykrogviruses are. One possibility is that Gemykrogviruses actually infect plants, which would explain their detection in the feces of herbivorous animals and in mosquitoes that feed on pollen. CRESS-DNA viruses evolve rapidly, with comparable evolution rates to RNA viruses . High mu species . Since vAnelloviridae family, accounts for the most abundant virus infection of humans, with estimated worldwide infection rates of 70–100% [The host range of eukaryotic CRESS-DNA viruses is highly diverse and includes plants and various invertebrate and vertebrate species, including humans ,42. Inde 70–100% ,44,45. IAedes aegypti. However, further studies are needed to confirm these findings in larger cohorts and to investigate mosquito vector competence for CyCV-VN transmission. Moreover, single CRESS-DNA viruses have been identified in serum and brain biopsies of multiple sclerosis patients, in the pericardial fluid of pericarditis patients, and the cerebrospinal fluid of encephalitis patients ,47,48. CC.pip.cl. and Ae. ae. mosquitoes from two distinctive geographic locations. Among all phylogenetic virus clades, CRESS-DNA viruses constituted a diverse and highly abundant portion of the mosquito virome. We present evidence that mosquito vectors are important hubs for CRESS-DNA virus transmission between different environmental sources. Thus, mosquitoes contain a large pool of novel CRESS-DNA viruses from which we identified a novel variant of a human-infecting CRESS-DNA virus, currently not linked to vector-related transmission.In conclusion, this study comprehensively characterized the viral metagenome of field-collected


Odontonema tubaeforme) plants exhibiting leaf curling and chlorosis. The molecular features of the viral genomic RNA and proteins resemble those of ampeloviruses. Based on sequence comparisons and phylogenetic analysis, we propose a new species in the genus Ampelovirus, which we have tentatively named Firespike leafroll-associated virus (FLRaV). Bioassays showed that the virus is mechanically transmissible to Nicotiana benthamiana. In addition, a full-length cDNA clone of FLRaV could successfully infect N. benthamiana via agroinfiltration.A novel RNA virus was identified in firespike ( Ampelovirus (family Closteroviridae) are plant RNA viruses that infect mainly fruit crops in orchards and stock nurseries, causing serious reductions in fruit yield and quality worldwide . A . A Ampeloviruses b 12]. O. OAmpelooviruses c 13]. O. OAmpeloc region . Putativc region d 15]. O. OAmpeloectively ,23.Ampelovirus species showed a range of 22.5–50.6% sequence identity  of inoculated plants  The virus contains a large, single-stranded, positive-sense RNA genome  ThA genome ; (iii) Pelovirus . In addielovirus , suggestAmpelovirus through phylogenetic analysis, the FLRaV virus genome possesses some features similar to members of subgroup I. For example, unlike members of subgroup II, the FLRaV virus genome has a small ORF positioned between the RGB and the QGB, and two additional ORFs located at the 3′ end of the genome. To the best of our knowledge, it is the first identified ampelovirus that possesses features of both subgroups. By analogy to similarly located ORFs of other members of the family Closteroviridae , FLRaV P25 and P37 might be involved in the suppression of host RNA interference [Although clustered in the subgroup II of rference ,31,32. TClosterovirus is mechanically transmissible to the model plant N. benthamiana [Ampelovirus was successfully transmitted to N. benthamiana, although the transmission rates were very low and the best transmission rates were obtained using transgenic N. benthamiana plants expressing HC-Pro from the turnip mosaic virus [N. benthamiana. It should be noted that reads with similarity to cucumber mosaic virus (CMV) and columnea latent viroid were identified in the BLASTx analysis as well, and firespike is a common host of CMV [N. benthamiana without inducing visible symptoms, similar to the full-length cDNA clone of GLRaV-3 [N. benthamiana.It has been shown that GLRaV-2 in the genus thamiana ,34, but ic virus . Here, wt of CMV . Further GLRaV-3 . Future N. benthamiana. This experimental system can be used for further analysis of ampelovirus-host interactions.By using unbiased high-throughput sequencing and conventional molecular biology tools, a new ampelovirus, tentatively named FLRaV, was identified. Full-length cDNA of FLRaV was infectious to a convenient laboratory plant host


ObjectiveIn this study, we aimed to determine the correlation between costs/charges related to admissions for pediatric tonsillectomy in New York State (NYS) and variables including discharge year, All Patient Refined (APR) severity of illness, length of hospital stay, payment typology, location, race, and institutional factors during 2009-2017.MethodsData were extracted from the Statewide Planning and Research Cooperative System (SPARCS) Hospital Inpatient Discharges database developed by the NYS Department of Health. Statistical analysis was employed to determine multiple linear regression coefficients with the costs and charges set as the dependent variable.ResultsCosts increased by an estimated $230.73 (p<.001) each year, and charges increased by an estimated $1,231.41 (p<.001) annually. For each categorical increase in severity of illness, costs increased by $1,019.21 (p<.001), and charges increased by $3,088.41 (p<.001). For each day spent in the hospital, costs increased by $3,539.23 (p<.001), and charges increased by $8,908.01 (p<.001). Unspecified managed care had the highest mean costs and charges (p<.001). Bronx County had the highest costs, and Queens County had the highest charges. Queens County demonstrated the largest gap between costs and charges.ConclusionThis study revealed that the costs and charges related to admissions for elective tonsillectomy had risen from 2009 to 2017, and these changes were not accounted for by inflation alone. We found that the costs and charges for inpatient pediatric tonsillectomy were significantly correlated with discharge year, APR severity of illness, length of hospital stay, location of the hospital, and primary payer. One of the most pronounced trends in healthcare has been the rise in the costs and charges to deliver common services. In order to make healthcare a more accessible market, it is crucial to investigate the trends and sources of these costs and charges, particularly for common procedures such as pediatric tonsillectomies. In this study, our goal was to evaluate the differences in costs and charges for inpatient elective tonsillectomy performed in New York State (NYS) in order to characterize the factors contributing to these differences.Data were extracted from the Statewide Planning and Research Cooperative System (SPARCS) Hospital Inpatient Discharges database developed by the NYS Department of Health. This database is composed of public-use data and contains de-identified information comprising basic admission-level detail from all NYS hospitals. Data from 2009 to 2017 were filtered for ages 0-17 years and Clinical Classification Software (CCS) procedure code 30, “Tonsil-/adenoidectomy”. All were elective admissions. Variables available and analyzed include gender, race/ethnicity, length of stay (LOS), the All Patient Refined (APR) severity of illness score, primary payer, total costs, and total charges.The APR severity of illness scale was used to categorize patients’ clinical severity into four subclasses: minor, moderate, major, and extreme. Severity was classified based on seven dimensions intended to reflect the burden of disease: stage of the principal diagnosis, complications of the principal condition, concurrent interacting conditions that affected the hospital course, dependency on hospital staff, extent of nonoperating room life support procedures, rate of response to therapy or rate of recovery, and impairment remaining after therapy for the acute aspect of the hospitalization. Each of these dimensions was given a score of 1-4 with 1 being minor and 4 being extreme. These values were then integrated into one single score from 1 to 4 [The cost was defined as the expense incurred to deliver the service. It was unclear as to what specific parameters were included in the cost. The charge was defined as the sum of total accommodation charges plus total ancillary charges for the patient's stay . All monetary values were adjusted for inflation and reported in US Dollars. A linear regression coefficient analysis was employed to analyze the impact of discharge year, severity of illness, and length of hospital stay on costs and charges.Data from 6,020 inpatients were analyzed. Approximately 60% and 40% of these patients were male and female, respectively Table . Most paDischarge yearAnalysis of overall costs and charges revealed a steady increase in both the costs and charges annually Figure . AdditioSeverity of illnessThe APR severity of illness for each patient was categorized on a scale of minor, moderate, major, and extreme. It was estimated that for each categorical increase in the severity of illness, costs increased by $1,019.21 (p<.001), and charges increased by $3,088.41 (p<.001) . For each day spent in the hospital, costs increased by $3,539.23 (p<.001), and charges increased by $8,908.01 (p<.001) . The order of insurance payers pertaining to increase in costs and charges (p<.001) was as follows: government insurance < private insurance < self-pay.LocationThe six counties with the highest case volumes  were chosen for analysis: Bronx, Erie, Kings, Manhattan, Queens, and Richmond. In each county, charges increased faster than costs Figure . Bronx CRaceAnalysis performed on race as a variable affecting costs and charges revealed that multiracial patients incurred the highest costs and charges.InstitutionalPediatric tonsillectomies are performed in one of three hospital settings: non-children’s teaching hospitals, children’s teaching hospitals, and non-teaching hospitals. An analysis of the facility types revealed that pediatric tonsillectomies were more commonly performed in children’s teaching hospitals throughout the study period Figure .This study showed that the costs and charges of pediatric tonsillectomies in NYS increased from 2009 to 2017. Charges were observed to increase over the study period despite adjusting for inflation and relatively constant costs. This discrepancy may be explained by changes in practice guidelines, surgical/technological advancements, operative/postoperative practices, healthcare market trends, and some other factors.Discharge yearThe yearly increase in healthcare dollars spent on pediatric tonsillectomies was expected as healthcare prices continue to rise annually on a national level. Studies show that in the past 20 years, the Consumer Price Index (CPI) - defined as the average change in prices paid by urban consumers for various goods and services - has grown annually at an average of 2.1%, while the CPI for medical care as a whole has grown at an average rate of 3.5% annually [Although there are no specific recommendations set forth by the American Academy of Otolaryngologists (AAO) for obtaining histopathologic samples, current practice patterns in some places may include histopathology of tissues during tonsillectomies. Studies show that unusual histopathology in children is rare, suggesting that routine pediatric specimen examination may be unnecessary in cases when suspicion of malignancy is absent . It is uTechnological advancement over time in pediatric tonsillectomies may contribute to increased healthcare costs. Previously, the most common operative method was dissection and snare, which employed reusable instruments . Over tiAnother factor to consider is the change in healthcare trends over time. Between 2009 and 2017, the consolidation of hospital systems may have translated into more market power with fewer choices for patients. Less competition in the healthcare market may allow administrators to control healthcare prices. In 2018, Definitive HealthCare tracked 803 mergers and acquisitions and 858 affiliation and partnership announcements. This organization also conducted a survey of over 1,000 healthcare professionals, who reported that healthcare consolidation has become the most critical healthcare trend . There aInstitutional factors may also play a role in increasing tonsillectomy expenditures. Pediatric tonsillectomies performed in children’s teaching hospitals or non-children’s teaching hospitals incur higher costs and charges compared to other hospital types . The incStudies show that public hospitals may be more efficient than private hospitals in terms of technological productivity, the number of inactive beds, transfer of care, minimizing the number of healthcare personnel, and time spent per patient . While tSeverity of illnessPatients who are more severely ill are at higher risk of an unfavorable clinical course and more postoperative complications, contributing to increasing costs and charges. Patients in this study who were considered more severely ill had more significant healthcare expenditures due to more preoperative interventions , higher intraoperative costs , and postoperative surveillance. Hospital administrators may consider these variables when factoring in numbers for reimbursement.Length of hospital stayThe length of hospital stay plays a significant role in determining costs and charges. The increase in expenditures associated with longer hospital stays is mainly a reflection of more services received , more medical supplies, and more medications used.LocationPatients often prioritize geographic convenience when making healthcare decisions. This study revealed that location plays a significant role in pediatric tonsillectomy costs and charges. The etiology behind geographical discrepancies is likely multifactorial. The large gap between costs and charges in Queens County warrants further consideration. Queens County is one of the most expensive places to live and work in the US . It is aInsurance typeAnalysis of payment types revealed a significant discrepancy between costs and charges when using different payment methods for tonsillectomies. Self-pay patients incurred the highest costs and charges. From 2010 to 2015, healthcare providers saw at least a 10% increase in self-pay accounts . The numThe discrepancies between different payer systems may stem from private negotiations with healthcare providers. It is possible that some insurers are not paying the listed charges determined by hospitals, and there are various price tags for the same procedure at the same location . For eacRaceThe results of this study demonstrated disproportionately elevated costs and charges for the multiracial population. It is challenging to classify race-based trends because of the ambiguity of “unknown” or “other” categories. Also, multiracial patients comprised a small percentage of subjects. It is worth considering that racial minorities may experience more difficulty in obtaining healthcare access and coverage . Racial LimitationsIt is unclear as to what parameters were included in the cost and if they were distributed the same way in all hospitals over the study period. Access to this information may have improved the study by allowing for control of bundling of services, administrative costs, and strategic pricing. Also, there were some outliers in the data. Erie County data showed two outlier years in 2012 and 2015. In 2012, there were three cases with a hospital LOS of greater than 15 days, whereas the mean LOS in 2012 was 1.51 days. These three cases incurred charges of approximately $48,000, $70,000, and $100,000, respectively. The mean charges in 2012 in all of NYS were approximately $15,000. In 2015, there was a case with a hospital LOS of greater than 120 days, which incurred charges greater than $500,000. These outlier cases may alter mean values, linear regression, and coefficient analysis. Our study was not able to describe these outlier cases. This raises the bigger question as to whether significant socioeconomic factors influence the APR severity of illness among different counties. Our study could not fully demonstrate a statistically significant discrepancy between different racial factors, though significant research supports the association between healthcare disparities and race in the US .Overall, this study was not able to demonstrate causative factors that affect costs and charges. We have revealed associations between multiple variables and cost/charge, warranting further research to understand the root causes of these expenditures.This study revealed that costs and charges incurred to perform pediatric tonsillectomy had risen during 2009-2017, and these changes were not accounted for by inflation alone. We found that inpatient pediatric tonsillectomy costs and charges were significantly related to several factors such as discharge year, APR severity of illness, length of hospital stay, location of the hospital, and primary payer. A more detailed analysis would highlight some of the daily services that hospitals offer to surgical patients. By gaining greater insight into daily hospital logistics and services, hospitals can employ cost-saving techniques to shrink the financial burden of each extra day spent in the hospital. It is very difficult to universalize healthcare prices across different hospital and payer systems because of competition and negotiation between entities.There is a highly complex system involved in generating medical costs and charges. By laying out the foundation of these costs/charges and understanding where they come from, this study opens up opportunities for researchers to conduct further analyses of these driving factors.


All species evolve to adapt to the environment in which they exist. As we sit in the middle of a global viral pandemic with our own environment changing every day at an uncomfortably rapid pace, one must wonder how much we are microevolving ourselves. Some changes are obvious, of course, the increased wearing of masks and the maintenance of physical distancing, but it will be years before we truly see how much this situation has affected us, and more importantly, our children. It is also clear and obvious that we are evolving, or at least adapting, on a professional level as scientists who are studying viruses and the immune system. A colleague recently mentioned to me that it must be an exciting time to be in this field, and it is. All fields of research evolve as new ideas and technologies emerge, but in the viral immunology world, a completely new disease can appear with the emergence of almost every new virus.Anyone studying viruses knows that they can fall in and out of fashion quite quickly. The virus that is cool today might not be so cool tomorrow, and the hot new virus of this year might be gone by next year. Some virus researchers study a single virus for their whole career, whereas others study numerous viruses, sometimes unrelated, but I think all virology researchers at some point wonder whether they should switch to a different virus, or jump on the bandwagon and follow the money available to study the newest virus. What is clear is that our field is constantly changing, and most of us have to change with it because funding will always follow what is publically relevant. The resilience of immunology and virology researchers has always been apparent because the landscapes of our research fields have changed over the years. This notion is clear if we consider a few viruses such as HIV, HCV, and now SARS-CoV-2.As with all viruses, when HIV was first discovered, understanding the virus and the immune response against it was the priority, but as we began to understand how the viral proteins functioned, and multiple classes of antiviral agents were discovered, we turned our attention to virus–host interactions and the identification of cellular factors that were critical for virus replication. Thirty years later, HIV infection is a chronic manageable disease in countries where therapy is available, but rightfully, the affected community said that a lifetime of treatment was not enough, and they asked for a cure. In response, funding priorities shifted, and many HIV researchers adapted and evolved, and we now have exciting efforts focused on finding new ways to target the latent pool of virus hiding in our own DNA. In this way, the status of HIV research forced immunology and virology researchers to rapidly adapt and evolve along with their field of research.The evolution of HCV research took a similar path as that of HIV. We studied the virus, then the cellular factors, and the immune response for many years, but not everyone expected 99% cure rates to come along when they did, and that left many HCV researchers wondering whether studying this virus was still in their future. At that point, there was adaptation in many directions. Some stayed the course because there are indeed many important questions that still need to be answered regarding HCV, but some branched out conservatively, and others completely moved to different viruses. And as with HIV, the pursuit of an HCV vaccine remains to be a major priority, especially since we are still seeing more new infections annually than we are seeing individuals cured.So here we are again in 2020. Our personal and professional environments have changed more in the past 6 months than they have in a 100 years, and all over the world immunologists and virologists, as well as scientists from numerous other fields, are joining the fight. Perhaps for some the decision is motivated by a need for research funding, but the result is still the same, we are adapting and evolving along with the field we work in. Some research programs are shifting focus completely, at least temporarily, while others are “growing new limbs” to incorporate coronavirus research. Hopefully we will not see another viral pandemic for at least another 100 years, but if we do, immunologists and virologists all over the world will be ready to adapt, evolve, and join the fight just as we have this time.