When multiple pathogens cocirculate this can lead to competitive or cooperative forms of pathogen–pathogen interactions. It is believed that such interactions occur among cold and flu viruses, perhaps through broad-acting immunity, resulting in interlinked epidemiological patterns of infection. However, to date, quantitative evidence has been limited. We analyzed a large collection of diagnostic reports collected over multiple years for 11 respiratory viruses. Our analyses provide strong statistical support for the existence of interactions among respiratory viruses. Using computer simulations, we found that very short-lived interferences may explain why common cold infections are less frequent during flu seasons. Improved understanding of how the epidemiology of viral infections is interlinked can help improve disease forecasting and evaluation of disease control interventions. The human respiratory tract hosts a diverse community of cocirculating viruses that are responsible for acute respiratory infections. This shared niche provides the opportunity for virus–virus interactions which have the potential to affect individual infection risks and in turn influence dynamics of infection at population scales. However, quantitative evidence for interactions has lacked suitable data and appropriate analytical tools. Here, we expose and quantify interactions among respiratory viruses using bespoke analyses of infection time series at the population scale and coinfections at the individual host scale. We analyzed diagnostic data from 44,230 cases of respiratory illness that were tested for 11 taxonomically broad groups of respiratory viruses over 9 y. Key to our analyses was accounting for alternative drivers of correlated infection frequency, such as age and seasonal dependencies in infection risk, allowing us to obtain strong support for the existence of negative interactions between influenza and noninfluenza viruses and positive interactions among noninfluenza viruses. In mathematical simulations that mimic 2-pathogen dynamics, we show that transient immune-mediated interference can cause a relatively ubiquitous common cold-like virus to diminish during peak activity of a seasonal virus, supporting the potential role of innate immunity in driving the asynchronous circulation of influenza A and rhinovirus. These findings have important implications for understanding the linked epidemiological dynamics of viral respiratory infections, an important step towards improved accuracy of disease forecasting models and evaluation of disease control interventions. The human respiratory tract hosts a community of viruses that cocirculate in time and space, and as such it forms an ecological niche. Shared niches are expected to facilitate interspecific interactions which may lead to linked population dynamics among distinct pathogen species , 2. In t5Early interest in the potential for interference among taxonomically distinct groups of respiratory viruses stemmed from epidemiological observations of the temporal patterns of infection in respiratory virus outbreaks –11. MoreThe role of adaptive immunity in driving virus interferences that alter the population dynamics of antigenically similar virus strains is well known , 19. For22However, despite epidemiological, clinical, and experimental indications of interactions among respiratory viruses, quantitatively robust evidence is lacking. Such evidence has been hard to acquire, due to a paucity of consistently derived epidemiological time-series data over a sufficient time frame to provide well-powered analyses, and the lack of analytical approaches to differentiate true virus–virus interactions from other drivers of coseasonality, such as age-dependent host mixing patterns , 28 and 29Here, we apply a series of statistical approaches and provide robust statistical evidence for the existence of interactions among respiratory viruses. We examined virological diagnostic data from 44,230 episodes of respiratory illness accrued over a 9-y time frame in a study made possible by the implementation of multiplex-PCR methods in routine diagnostics that allow the simultaneous detection of multiple viruses from a single respiratory specimen. Each patient was tested for 11 virus groups , 29, proWe first evaluated the total monthly infection prevalences across all viral respiratory infections from 2005 to 2013. As typically observed in temperate regions, the proportion of patients with respiratory illness testing positive to at least one respiratory virus peaked during winter, with the exception of the influenza A H1N1 pandemic in the summer of 2009 A. NevertSI Appendix, Tables S1 and S2 and We evaluated correlations in the monthly prevalence time series for each pair of respiratory viruses. We first employed a simple bivariate nonparametric cross-correlation analysis by estimating Spearman’s rank correlation coefficients and observed 26 significant virus–virus correlations, 13 negative and 13 positive . These interactions can be seen empirically as asynchronous . See This bespoke approach revealed many fewer statistically supported epidemiological interactions, with negative interactions between IAV and RV and between influenza B virus (IBV) and adenovirus (AdV)  when inferring disease–disease associations from hospital-based case-control data . To accoWe infer signatures of virus–virus interactions from the nonrandom patterns of virus mixing captured by coinfection information by assessing whether the propensity of a given virus X to coinfect with another virus Y was higher, lower, or equal to the overall propensity of any (remaining) virus group to coinfect with virus Y. We adjusted for the effects of age, gender, patient origin , and the time period (with respect to the 3 major waves of the 2009 IAV pandemic). To distinguish interactions between explanatory and response viruses from unrelated seasonal changes in infection risk, we also adjusted for the monthly background prevalence of response virus infections.P < 0.05 before correction for multiple testing: 4 negative interactions (OR < 1) among 3 virus pairs involving influenza and 13 positive interactions (OR > 1) among 8 pairs of noninfluenza viruses (P < 0.001) and a positive interaction between AdV and PIVB . See SI Appendix, Tables S15–S17 and As our data did not allow us to infer the directionality of virus–virus interactions, and nor did we have an a priori basis to inform this, we first performed 72 statistical tests to evaluate all 36 virus-pair hypotheses in 9 virus models  were expected when applying the permutation test of the global null hypothesis to all 72 tests. See SI Appendix, Figs. S2 and S3 and We also used a permutation method to test the global null hypothesis that there were no interactions among any of the remaining 5 virus groups . The distribution of Our statistical analyses provide strong support for a negative interaction between seasonal IAV and the relatively ubiquitous RV, at both population and individual host scales. This negative interaction may be driven by virus competition for susceptible cells, for example as a consequence of influenza-induced destruction of cell-surface receptors  and/or cSI Appendix, Fig. S4 and Table S18 and To address this question, we performed epidemiological simulations of the cocirculatory transmission dynamics of a seasonal influenza-like virus, such as IAV, and a nonseasonal common cold-like virus, such as RV, using ordinary differential equation (ODE) mathematical modeling  seasonal pattern, typically peaking in winter months, AdV typically peaks around May. However, because our Bayesian hierarchical model adjusts for virus seasonality on a month-by-month basis, it is not seasonal differences that explain the negative relationship between this virus pair. In the absence of a seasonal driver or a host-scale mechanism, it is possible that the lack of cooccurrence of IBV and AdV is explained by other ecological drivers. For example, convalescence or hospitalization induced by one virus may reduce the susceptible pool at risk of exposure to other viruses, as previously discussed by others in the context of childhood diseases , 38.SI Appendix, Fig. S1) and thus their cooccurrence with other respiratory viruses is expected to vary. Based on these differences between IAV and IBV, it is feasible that their ecological relationships with other viruses have evolved differently.Both IAV and IBV viruses exhibited only negative interactions at both host and population levels, although the specifics differed. That they differ in their exact pairwise interactions is unsurprising when considering that these viruses are antigenically distinct, constitute different taxonomical genera, and exhibit different viral evolutionary rates , 42, as 45Of further note is the lack of interaction detected between IAV and IBV, since there is some suggestion from global data of a short lag between their outbreak peaks. However, epidemiological data are inconsistent in that they report both asynchrony and codominance , 47. We Age patterns of infection may provide important insights into the opportunity for, or alternatively the consequences of, virus–virus interactions. A lag in epidemic peaks across children and adults has been observed in the case of RSV , 51. SucOur study describes positive interactions among respiratory viruses at the population scale. These positive epidemiological interactions were not mirrored at the host scale, which suggests they are independent of host-scale factors and may instead be explained by variables that were not captured by our study. For example, some respiratory viruses, such as RSV and MPV, are known to enhance the incidence of pneumococcal pneumonia , 52. It Although not mirrored at the population level, our 72 pairwise tests did detect the existence of positive interactions among 8 pairs of viruses, including strong statistical support for AdV/PIVB following Holm’s correction for multiple comparisons. This finding is consistent with a recent, smaller-scale clinical study of children diagnosed with pneumonia, which detected 2 pairs of positively associated noninfluenza viruses . PlausibIt is important to consider that virus–virus interactions are likely shaped by evolutionary processes and thus their existence, magnitude, and dominant form (whether competitive or facilitative) may not be static in nature, and instead depend on particular virus strains, as well as host and environmental factors . The virOur study provides strong statistical support for the existence of interactions among genetically broad groups of respiratory viruses at both population and individual host scales. Our findings imply that the incidence of influenza infections is interlinked with the incidence of noninfluenza viral infections with implications for the improved design of disease forecasting models and the evaluation of disease control interventions. Future experimental studies are required to decipher the biological mechanisms that underpin virus–virus interactions and their effects on the within-host dynamics of infection.Our study was based on routine diagnostic test data used to inform the laboratory-based surveillance of acute respiratory infections in NHS Greater Glasgow and Clyde , spanning primary, secondary, and tertiary healthcare settings. Clinical specimens were submitted to the West of Scotland Specialist Virology Centre for virological testing by multiplex real-time RT-PCR , 59. PatData were available to our study for the period January 2005 to December 2013 comprising 61,427 clinical samples, most of which were nasal and/or throat swabs (98%). The test results of individual samples were aggregated to the patient level using a window of 30 d to define a single episode of illness, giving an overall infection status per episode of respiratory illness. This yielded a total of 44,230 episodes of respiratory illness from 36,157 individual patients. In this study population, 35%  of patient episodes were virus-positive, 8% (1247) of which were coinfected with more than one virus group.These data provide a coherent source of routine laboratory-based data for inferring epidemiological patterns of respiratory illness, reflecting typical community-acquired respiratory virus infections in a large urban population . Of the These diagnostic data included test-negative results providing the necessary denominator data to account for fluctuations in testing frequencies across patient groups and over time. Such changes in testing patterns may be expected to arise in healthcare data accrued over extended time frames, for example because of changes in healthcare-seeking behavior and/or the clinical management of patients. We refer readers to ref. SI Appendix, Tables S1 and S2 for the estimated correlation coefficients, distributions under the null hypothesis, and P values.Spearman’s rank correlation coefficients were computed and tested between all pairs of virus infection prevalences (the proportion positive among those tested) in each month using cor.test in R  version Although the simple bivariate cross-correlation analysis revealed several significantly correlated virus pairs, inferring genuine virus–virus interactions using diagnostic patient data requires several features to be accounted for: Absolute infection counts must be adjusted for fluctuations in testing frequency, temporal autocorrelation, and alternative confounding explanations of temporal dependency between virus groups.SI Appendix, Tables S3 and S4 for the pairwise correlation estimates summarized in To address these methodological limitations, we developed and applied a statistical approach that extends a multivariate Bayesian hierarchical modeling method to times-series data . The metSI Appendix, Table S19 for details).We analyzed the patient-level coinfection data with a series of binary logistic regression models and evaluate whether virus–virus interactions may be explained by interactions operating at the scale of individual hosts. Of the 44,230 total episodes of respiratory illness, 73% represent patients experiencing a single episode during the 9-y study period, while the remaining 27% represent patients experiencing 2 or more episodes . Retaining the first observed episodes generated 36,157 patient records and 20,703 with complete testing across all virus groups [note that pressures on laboratory resources led to high partial testing during the major waves of A(H1N1)pdm09 virus circulation]. Of these 20,703 patients, 6,884 were virus-positive, 11% of which were coinfected with 2 or more viruses. We note that the coinfection prevalence and detected virus–virus interactions were robust to the choice of window used to define an episode of illness when aggregating individual samples , patient sex (SEX), hospital vs. GP patient origin (ORIGIN), and time period of sample collection with respect to the influenza A(H1N1)pdm09 virus pandemic (PANDEMIC). In addition, we ensured that virus–virus associations were not simply explained by independent monthly fluctuations in the response virus infection risk. To do so, we adjusted the total number of infections with the response virus (VCOUNT) and the total number tested (TCOUNT) within a 15-d window either side of each (earliest) sample collection date for each individual observation. Model variables were either binary or categorized; see SI Appendix, Tables S6–S14 for details). Due to low infection frequencies, we regrouped PIVs into 2 groups: PIVA  and PIVB .Specifically, the relative odds of coinfection with virus Y (versus any other virus group) was estimated for each of the 8 explanatory viruses, for each response virus Y. Thus, each virus model  used a specific subset of the virus-positive population (because each virus model excluded virus-negative patients and single infections involving only the response virus Y) with each data subset ranging from 4,629 to 6,743  out of the 72 tests . Not all virus-pair interactions were bidirectional; however, we note that these analyses do not allow the differentiation of cause from effect.Analyses were conducted using the glm function in R version 3.4.4 . The quaP < 0.001 following correction) of the existence of both negative (IAV/RV) and positive (AdV/PIVB) interactions . A permutation test of the global null hypothesis was then applied to the 5 remaining virus groups  to test the hypothesis that the 20 remaining null hypotheses tested were true. Using Fisher’s method of combining P values , although we expect nonindependence between these tests. We therefore accounted for nonindependence among the pairwise tests by using permutations to simulate the null distribution of combined P values. Each generalized linear model was fitted to 10,000 datasets where the null hypothesis was simulated by permuting the response variable (virus Y). The significant tendency of the distribution of P values toward zero (and away from the uniform distribution expected under the null hypothesis) was illustrated using a quantile–quantile (QQ) plot to compare the 20 observed P values with the 10,000 distributions simulated under the global null hypothesis —only 3 (95% CI: 0 to 9) interactions were expected by chance assuming the null hypothesis of no interactions, in comparison to the 17 interactions we detected.Applying Holm’s method to control the probability of one or more false discoveries arising among the family of 72 tests [using the p.adjust function in R version 3.4.4 ] yieldedP values , we founpothesis C. The siSI Appendix, Fig. S4. The temporal dynamics of the viruses were distinguished in 2 key ways. First, seasonal forcing was applied to the influenza-like virus (virus 1) via a sinusoidally varying transmission rate. Second, the rate of waning immunity of the common cold-like virus (virus 2) was assumed to be 10 times faster than for the influenza-like virus. This more rapid replenishment of susceptible individuals was designed to reflect the high year-round prevalence and diversity of circulating subtypes that are characteristic of RV infections , assuming frequency-dependent transmission, and subsequently transition from susceptible (S) to an infectious refractory phase (I). Infected individuals were assumed not to be susceptible to further infections with the primary infecting virus. Our assumption is that multiple exposures to similar virus strains are unlikely to alter the within-host dynamics during this short period. At the end of the infectious period, individuals entered a noninfectious refractory phase (J) at rate 1/γ (days−1). This second refractory phase was designed to reflect immune effects that may persist for a period beyond viral clearance , 0 < φ < 1 induced a partial reduction in susceptibility, and φ = 1 induced a complete loss of susceptibility.Individuals were assumed to acquire primary infections at rate β  (R). During this phase, individuals were not susceptible to the primary infection but could acquire secondary infections if previously unexposed. Following a period of long-term immunity individuals reverted to susceptible (S) at a rate σ (days−1) representing the waning of antibodies against the primary infection.At the end of the second refractory phase, infected individuals developed long-lasting immunity to infection due to antibodies generated at rate The full model ODEs are given as follows:1(t) denotes the time-dependent rate at which new infections with virus 1 are generated and comprises a fixed transmission parameter B together with a seasonal amplitude parameter b; and φ represents a background, nonacute-respiratory-infection–induced rate of mortality given by 1/the average life expectancy (days−1). The peak proportion of individuals coinfected with both viruses was 0.39% during each simulated season of influenza-like virus circulation. The R0s of these 2 viruses assuming a completely susceptible homogeneous population are 1.6 (virus 1) and 2 (virus 2). Full parameter values and ranges are provided in SI Appendix, Table S18.where {1,2} denotes the infection status of individuals with respect to virus 1  and virus 2  for each of 12 combinations of infection classes, whereby S = susceptible, I = infectious refractory phase, J = noninfectious refractory phase, R = immunity phase, and subscripts 1 and 2 denote the corresponding virus-specific parameters. For example, {SI} indicates the state of being susceptible to virus 1 and infectious with virus 2. This framework was implemented in MATLAB software v.R2013b using the ode45 differential equation solver. Each simulation was run for a period of 20 y; a “burn-in” period of 10 y was excluded from the analyses. Using this framework, we quantified the effect of transient immune-mediated viral interactions on the percentage decrease in daily nonseasonal common cold-like virus prevalence during peak seasonal influenza-like virus activity.https://www.informationgovernance.scot.nhs.uk/pbpphsc/home/for-applicants/). Aggregated forms of summary data and computer code may be made available upon request to the corresponding author.The patient-level data used in this study are available upon request to NHS Scotland (Supplementary File


Haliaeetus leucocephalus) once experienced near-extinction but has since rebounded. For decades, bald eagles near the Wisconsin River, USA, have experienced a lethal syndrome with characteristic clinical and pathological features but unknown etiology. Here, we describe a novel hepacivirus-like virus (Flaviviridae: Hepacivirus) identified during an investigation of Wisconsin River eagle syndrome (WRES). Bald eagle hepacivirus (BeHV) belongs to a divergent clade of avian viruses that share features with members of the genera Hepacivirus and Pegivirus. BeHV infected 31.9% of eagles spanning 4,254 km of the coterminous USA, with negative strand viral RNA demonstrating active replication in liver tissues. Eagles from Wisconsin were approximately 10-fold more likely to be infected than eagles from elsewhere. Eagle mitochondrial DNA sequences were homogeneous and geographically unstructured, likely reflecting a recent population bottleneck, whereas BeHV envelope gene sequences showed strong population genetic substructure and isolation by distance, suggesting localized transmission. Cophylogenetic analyses showed no congruity between eagles and their viruses, supporting horizontal rather than vertical transmission. These results expand our knowledge of the Flaviviridae, reveal a striking pattern of decoupled host/virus coevolution on a continental scale, and highlight knowledge gaps about health and conservation in even the most iconic of wildlife species.The bald eagle ( Haliaeetus leucocephalus) was selected as the emblem of United States of America by the Continental Congress of 17821. For millennia prior, the bird was recognized as sacred by diverse Native American peoples2. Despite its importance to North American histories and cultures, the bald eagle nearly went extinct in the 1960s due to eggshell thinning caused by dichlorodiphenyldichloroethylene , a biodegradation product of dichlorodiphenyltrichloroethane (DDT)4. The publication of Rachel Carson’s Silent Spring in 19625, subsequent banning of agricultural DDT use in the USA and Canada in the early 1970s6, and improved protections under the Bald and Golden Eagle Protection Act of 1940 and the Endangered Species Act of 19738 helped the species recover from a nadir of 487 breeding pairs in the coterminous United States in 1963 to over 16,000 breeding pairs in 200911. The bald eagle was removed from the US federal list of threatened and endangered species in 20077. Currently, major causes of bald eagle mortality, especially for birds living near people, include poisoning, trauma, electrocution, and illegal hunting13.For its majestic splendor, the bald eagle (15) have generally been considered sporadic and incidental17. Among 2,980 bald eagle carcasses submitted to the U.S. Geological Survey National Wildlife Health Center (NWHC) in Madison, Wisconsin, USA, between 1982 and 2013, only 5.2% of deaths were attributed to infectious diseases12. Documented infectious diseases of bald eagles include: ectoparasitoses19, helminthoses24, aspergillosis25, coccidiosis26, toxoplasmosis29, sarcocystosis32, leucocytozoonosis33, avian malaria36, avian cholera37, mycobacteriosis39, trichomoniasis40, other bacterioses43, avian pox44, herpes45, avian influenza47, Newcastle disease48, eastern equine encephalitis49, and West Nile encephalitis15. However, many of these infections are known from only single cases or case clusters and affected birds often present with comorbidities, such that the importance of infection for bald eagle population health remains unclear.Infections of bald eagles have been documented since the early 1900s but . The condition is refractory to treatment, and death or euthanasia follow shortly. WRES is characterized by hepatocellular cytoplasmic vacuolation, with vasculitis and cerebral microhemorrhages sometimes observed. The neurologic signs and hepatic pathology characteristic of WRES initially led to investigations of potential toxic causes, but no suspect chemical compounds have been identified. Similarly, testing for known pathogens, including neurotropic viruses such as West Nile virus, has failed to identify an infectious etiology.Since approximately 1994, bald eagles near the lower Wisconsin River in Wisconsin, USA, have experienced a lethal and enigmatic clinical syndrome, Wisconsin River eagle syndrome (WRES)15) and the observation of neurologic signs and hepatic pathology in affected eagles, we investigated potential viral causes. Our analyses led to the identification of a novel hepacivirus-like virus (Flaviviridae: Hepacivirus), the type-virus of which is the globally important human pathogen hepatitis C virus (HCV)51. Herein we characterize the virus and describe our subsequent investigations into its distribution across the coterminous United States, its potential association with clinical disease, the population genetic structure of host and virus, and patterns of host-virus coevolution.In an effort to identify the cause of WRES, we examined bald eagle tissues archived at the Wisconsin Department of Natural Resources and the NWHC. Submissions of carcasses to these agencies included cases from eagles in Wisconsin that had died of WRES as well as eagles from Wisconsin and across the coterminous USA that had putatively died from other causes. Given the known susceptibility of raptors to viral encephalitides  and generally more similar to HCV than to human pegivirus (HPgV), including immediately downstream of the E1/E2 junction, where homology between BeHV and other viruses is undiscernible . The virus was identified in serum from a bird collected on December 29, 2002, from Sauk County, Wisconsin, and the bird displayed hepatocellular cytoplasmic vacuolation characteristic of WRES Fig. . The 11,dae Fig. , includible Fig. . Similarses Fig. . Phyloge56, is low between BeHV and homologous regions of HCV (50.5%) and the human pegivirus HPgV (41.8%). Minimum free energy prediction of RNA secondary structure shows this portion of the BeHV 5′UTR to exhibit three large and well-defined stem-loop (SL) structures  more closely resembles those of certain pegiviruses, as has been noted for DuHV52.Nucleotide-level similarity within the 5′UTR, which has proven useful for inferring hepacivirus taxonomy and functionres Fig. . This prrus Fig. , and it Testing of 47 eagle liver tissues (which we chose to perform based on the known hepatic tropism of the hepaciviruses) from Wisconsin and elsewhere in the United States Table  using meReal time quantitative reverse transcription PCR (RT-qPCR) of RNA from liver tissues of the 15 BeHV-positive eagles yielded Ct values ranging from 26.1 to 36.4, with an overall mean of 30.26 Fig. . Ct valuP = 0.0086). Similarly, the prevalence of BeHV in eagles from counties in Wisconsin where WRES had been diagnosed was 83.3% , whereas the prevalence of BeHV in eagles from elsewhere was 24.4% , making eagles from counties in Wisconsin where WRES had been diagnosed 14.5 times more likely to test positive for BeHV than eagles from elsewhere .Between 1995 and 2018, the NWHC received 1188 bald eagle submissions, of which 260 (21.9%) were from Wisconsin. Of the 260 eagle submissions from Wisconsin, 28 (10.8%) were diagnosed clinically and histopathologically with WRES. No cases of WRES were diagnosed outside of Wisconsin. Of the 28 eagles diagnosed with WRES in Wisconsin, 14 (50.0%) came from counties bordering the lower Wisconsin River. Based on molecular testing of tissues from 47 eagles (described above), the prevalence of BeHV in Wisconsin was 75.0% , whereas the prevalence of BeHV elsewhere was 23.1% , making eagles from Wisconsin 9.4 times more likely to test positive for BeHV than eagles from elsewhere . Nucleotide level genetic diversity among virus envelope gene sequences from those same eagles (GenBank accession numbers MN062563-MN062576) was higher (π = 4.61 × 10−2 ± 3.43 × 10−3). Analysis of isolation-by-distance revealed different patterns for hosts and viruses  but a strong and statistically significant positive correlation between geographic and genetic distances for envelope gene sequences of viruses infecting those same eagles . Virus pairs tended to sort into clusters of genetic similarity at discrete classes of geographic separation, revealing a spatial signal of viral population genetic substructuring across the coterminous USA that was not evident in eagle hosts  among eagle mitochondrial DNA sequences (GenBank accession numbers MN062428-MN062562) was low  and only one statistically significant individual association .Phylogenetic analyses of eagle mitochondrial DNA sequences and virus envelope gene sequences yielded different topologies Fig. . Maximum4. Banning of DDT in the 1970s6 and improved Federal protections8 allowed the species to rebound, but eagles still experience significant mortality from anthropogenic factors such as poisoning, illegal hunting, trauma, habitat loss, and antagonistic interactions with people12. Our results highlight two additional factors that have received comparatively little attention with respect to bald eagle conservation: infectious disease and lack of genetic diversity. We describe a novel hepacivirus-like virus, bald eagle hepacivirus (BeHV), discovered during an investigation into Wisconsin River eagle syndrome, a lethal affliction of bald eagles. We also document remarkably low genetic diversity in bald eagles across the coterminous United States, consistent with the species’ recent recovery from a population bottleneck59.Bald eagles are an iconic North American wildlife species that has faced myriad challenges. In the 1960s, bald eagles were nearly extirpated from the coterminous USA due to the toxic effects of DDT50, but the role of the virus in clinical disease remains uncertain. We identified the virus in the serum of a single affected eagle, but we did not find BeHV in this bird’s brain tissue or in serum or brain tissues from eight other eagles that had died of WRES. We also found BeHV in 31.9% of eagle liver samples from across the USA. BeHV viremia is therefore likely transient or intermittent, and the site of persistence is probably the liver, as is characteristic of hepaciviruses51. Our initial analyses focused on brain and serum were therefore based on erroneous assumptions that a neurtropic virus was involved in WRES, because of the neurologic signs observed in many affected birds50.We discovered BeHV during an attempt to determine the cause of WRES, which has defied etiologic diagnosis for decades62. Outwardly, the liver pathology characteristic of many WRES-affected eagles  may indicate locally favorable conditions for transmission or, alternatively, reporting or diagnostic bias.67. BeHV could predispose eagles to such factors. If so, region-specific co-factors could interact with BeHV to precipitate particular disease manifestations. For example, parts of the lower Wisconsin River remain ice-free in Winter, providing eagles with rare fishing opportunities and creating dense aggregations of birds that attract tourists. Eagles could transmit infections under such circumstances14, and indeed WRES tends to strike in the Winter months50. The same favorable conditions could also allow eagles to survive more readily to the end stages of infection, perhaps explaining the good body condition characteristic of WRES-affected eagles50. Nevertheless, positive eagles from Wisconsin had viral loads no higher than those of birds from elsewhere. Unfortunately, our sample included only moribund or dead eagles submitted to state and federal agencies, which is not a representative sample of the population of eagles at large, such that additional effort will be required to ascertain whether BeHV is causally related to WRES in the wild.Infections can weaken wildlife, making animals more vulnerable to other causes of mortality, such as predation, trauma, and starvation59 and their ability to fly long distances. By contrast, BeHV showed higher genetic diversity, strong evidence of isolation by distance, and distinct sorting of pairwise genetic distances into discrete geographic clusters , where they interact aggressively with conspecifics and with other predatory birds during competition over carrion or prey items (“piracy”)71, an aspect of their natural history that famously inspired Benjamin Franklin to impugn the species as “a bird of bad moral character”1. Such features of eagle social behavior could predispose them to cross-species and horizontal viral transmission. The reservoir of BeHV could be bald eagles, their prey/carrion, or other species with which they interact. Although no hepaciviruses are known to be vector-borne, this possibility should also not be discounted.Eagle mitochondrial DNA and virus envelope gene sequence phylogenies were uncorrelated Fig. . BecauseFlaviviridae currently contains four genera: Flavivirus, Pestivirus, Pegivirus, and Hepacivirus72. Members of the genus Flavivirus commonly infect birds (e.g. West Nile virus) and can cause lethal disease73. The genus Pestivirus contains viruses that have so far only been found in mammals75. The genus Pegivirus, also so far found only in mammals, contains an equine virus associated with Theiler’s disease of horses77 but no other known pathogens. HPgV is nevertheless of clinical interest because it may slow disease progression in patients with AIDS and Ebola virus disease through mechanisms that remain incompletely understood79. Members of the genus Hepacivirus infect diverse mammals76, and hepacivirus-like viruses have been found in reptiles, amphibians, fishes and domestic ducks82. The hepaciviruses and pegiviruses are gaining recognition as examples of rapidly expanding viral genera that may hold important clues about the origins and pathogenesis of human and animal diseases alike81.The family Hepacivirus may be necessary, especially if additional discoveries support the association of clades of hepacivirus-like viruses with particular host taxonomic groups 75.Our results support the notion that the diversity and host range of the hepaciviruses will likely continue to expand. BeHV and DuHV form an avian lineage of hepacivirus-like viruses that also share genomic features with the pegiviruses, suggesting an undiscovered diversity of related avian viruses that may possess similar intriguing attributes. Across the genome, BeHV is more similar to the hepacivirus type virus (HCV) than to the pegivirus type virus (HPgV), suggesting a greater affinity of BeHV for the hepaciviruses than the pegiviruses. More broadly, our results show that the mammalian hepaciviruses form a sub-clade within a diverse clade of hepacivirus-like viruses from non-mammalian hosts, and the mammalian hepaciviruses and non-mammalian hepacivirus-like viruses form a clade distinct from the pegiviruses Fig. . TaxonomOverall, our results show that bald eagles across the coterminous United States are infected with a novel hepacivirus-like virus that displays intriguing genomic features, epidemiologic patterns, and evolutionary history. BeHV evolution and population genetics are strikingly decoupled from those of its host, perhaps reflecting a “lag effect” due to the recent recovery of eagles from a severe population bottleneck. The extent to which BeHV represents a threat to bald eagle health and conservation will require further investigation, as will determining its origin, reservoir(s) and mode(s) of transmission. Additional studies using expanded sample sets and associated clinical data from bald eagles and other birds should add to our understanding of BeHV and its relatives. Our findings already reveal how much remains to be discovered about health and conservation in North American wildlife, even in species central to the history and cultures of the continent.We obtained bald eagle samples from tissue archives maintained by the NWHC and the WDNR from carcasses submitted post-mortem. We used the records system of the NWHC to obtain data on numbers of eagles submitted per year, their geographic origins, causes of death, and pathologic findings. Tissues were submitted, archived, and analyzed in accordance with all local, state, tribal, and federal laws and policies, and with appropriate permits. No live animals were used in this research.84. Briefly, we isolated viral nucleic acids using the QIAamp MinElute virus kit , converted RNA to double-stranded cDNA using random hexamers, and prepared libraries for sequencing on an Illumina MiSeq intrument  using the Nextera XT DNA sample preparation kit . We analyzed sequence data using CLC Genomics Workbench version 11.0 , first trimming low-quality bases  and discarding short reads (<75 bp). We then analyzed unassembled reads and contiguous sequences derived from de novo assembly of raw reads for nucleotide-level (blastn) and protein-level (blastx) similarity to viruses in GenBank as previously described84.We processed serum and brain tissues from nine eagles that had died of WRES for deep sequencing and virus discovery as described previouslyg for 5 minutes at 4 °C and supplemented 100 µL  of supernatant with 900 µL of Trizol. We performed phase separation in 2 mL Phase Lock Gel Heavy tubes  and purified nucleic acids from the aqueous phase using the RNA Clean & Concentrator-5 kit . We then conducted deep sequencing as described above.Based on the known hepatic tropism of the hepaciviruses, we next examined archived liver tissues of 47 birds (fresh frozen at −80 °C), including the aforementioned juvenile eagle, of which eight were from Wisconsin (three of these from counties bordering the lower Wisconsin River) and 39 were from elsewhere Table . From eaTo test eagle liver samples for BeHV, we designed a nested RT-PCR using primers annealing to the highly conserved NTPase region of the viral NS3 gene Table . We perfTo assess viral genetic variation, we amplified and sequenced the viral envelope gene in all positive tissues using nested RT-PCR assay Table  and the To assess eagle genetic variation and to provide host relatedness data for cophylogenetic analyses, we assembled sequences of 9 mitochondrial genes  from deep sequence data generated from liver tissues of all BeHV-positive eagles liver samples sequenced and analyzed as described above. We then concatenated the resulting sequences for population genetic and phylogenetic analyses.et al.85. We used the same primers, reagents and cycling conditions for nested RT-PCR described above, except that we appended the primer annealing to negative-strand RNA during reverse transcription (BeHV-NS3-EX-F3869) with a 5′ blocking sequence  RNA following the methods of Lin ce Table . During ce Table  as the fTo assess relative viral loads, we conducted RT-qPCR using primers Table  designedin situ, we attempted to apply RNAscope technology65 to histologic sections of eagle livers using RNAscope double “Z” oligonucleotide probes designed to hybridize to the BeHV polymerase gene; however, all attempts failed, likely because of the degraded nature of RNA in tissues from carcasses collected under field conditions.To visualize BeHV RNA 86, we used SignalP87 to identify signal peptide sequences, and we used the IRESPred web server88 to characterize the BeHV IRES. We also conducted analyses of core and NS5A protein sequences to identify intrinsically disordered regions (IDRs) and sites within IDRs with capacity to undergo disorder-to-order transitions for binding interactions using ANCHOR90. Because of previous work showing that the 5′ untranslated region (UTR) of the hepaciviruses is informative for viral taxonomy and prediction of function55, we analyzed homologous 3′ ends of this feature in BeHV, HCV and HPgV to predict RNA secondary structure using the mfold Web Server91 and the RNAfold algorithm executed on the Vienna RNA Websuite server93.We determined the putative translation initiation codon for the BeHV open reading frame using the ATGpr prediction server94 with the Gblocks algorithm96 applied to remove poorly aligned regions using TranslatorX97. We inferred phylogenies from the resulting 3,579-position nucleotide sequence alignment using PhyML98, with the model of molecular evolution (GTR + Γ + I) estimated from the data99 and 1,000 bootstrap replicates to assess statistical confidence in clades, and we displayed trees using FigTree100. To compare BeHV and select hepacivirus-like viruses, hepaciviruses, and pegiviruses, we aligned full viral genomes as described above and examined variation in amino acid-level similarity across the length of the alignment using the sliding-window method implemented in SimPlot101.To infer the phylogenetic position of BeHV, we compiled nucleotide sequences of complete viral polyprotein genes available in Genbank representing known lineages within the hepaciviruses and pegiviruses. We then generated codon-based sequence alignments using the PRANK algorighmπ102) from aligned eagle and virus gene sequences using the computer program MEGA7103. To assess isolation by distance, we computed pairwise geographic distances between centroids of the US county of origin from which each eagle was collected using Geographic Distance Matrix Generator104. We then computed uncorrected pairwise genetic distances between eagles  and viruses (envelope gene sequences) and compared each to pairwise geographic distances using Mantel tests of matrix correlation105 with 10,000 permutations of the data to assess statistical significance, implemented in the APE package in R106.To assess host and viral genetic diversity, we calculated nucleotide diversity (107 and tested for overall phylogenetic congruence and the significance of individual host/virus associations using the AxParafit algorighm108 implemented in CopyCat, version 2.04109.To assess host-virus coevolution, we conducted cophylogenetic analyses. We generated codon-based sequence alignments of eagle concatenated mitochondrial DNA sequences and virus envelope gene sequences and inferred maximum likelihood phylogenies as described above. We then constructed tanglegrams using DendroscopeSupplementary Figure S1Supplementary Figure S2Supplementary informationSupplementary informationSupplementary information


Twenty-two states have turned to managed care organizations to finance and deliver long-term services and supports (LTSS) as a way to control costs, improve quality and shift the locus of care away from institutional settings. These programs, referred to as Managed Long-Term Services and Supports (MLTSS) have been undertaken under several CMS authorities, and vary in terms of the program design and populations covered. Some programs such as the financial alignment demonstration, have integrated Medicaid and Medicare financing, while others have used waiver authority and three-party agreements to achieve coordination. However, all efforts share the same goal of improving the linkage between LTSS and both physical and behavioral health care. This presentation will provide conceptual framework for understanding and assessing program impact using the example of the multi-method evaluation of the $4 billion Pennsylvania Community HealthChoices program, one of the largest transitions in Medicaid policy to date.


The temperature responsive behavior investigated by transmittance and dynamic light scattering showed that some of the obtained mPEG-PAAHPs exhibited obvious temperature responsiveness and could be used to prepare nanoparticles by quickly heating. Drug paclitaxel can be encapsulated into mPEG-PAAHP based nanoparticles with a high encapsulation efficiency up to 99% (corresponding to a drug loading content of around 9.9%). Dynamic light scattering results showed that the PTX-loaded nanoparticles had a mean size around 80 nm (PDI<0.2) and good stability in PBS with 150 mM ionic strength. In vitro cytotoxicity results showed that mPEG-PAAHP did not show any toxicity to HeLa cells, but the PTX-loaded nanoparticles based on mPEG-PAAHP exhibited obvious anti-cancer activity. Thus, the temperature responsive PEGylated polyaspartamide derivative mPEG-PAAHP may be a promising drug delivery system.The temperature responsive PEGylated polyaspartamide derivative, denoted as mPEG-PAAHP, was synthesized by the click reaction. FTIR and  Polymeric drug carriers with various stimuli responsiveness have attracted great attention in the past two decades ,2. TheseL-aspartic acid (NK105  were synthesized via the click reaction successfully. The structures of obtained mPEG-PAAHPs were confirmed by FTIR and


Development of effective and environmentally friendly disinfectants, or virucidal agents, should help prevent the spread of infectious diseases through human contact with contaminated surfaces. These agents may also be used, if non-toxic to cells and tissues, as chemotherapeutic agents against infectious diseases. We have shown that arginine has a synergistic effect with a variety of virucidal conditions, namely acidic pH and high temperature, on virus inactivation. All of these treatments are effective, however, at the expense of toxicity. The ability of arginine to lower the effective threshold of these parameters may reduce the occurrence of potential toxic side effects. While it is clear that arginine can be safely used, the mechanism of its virus inactivation has not yet been elucidated. Here we examine the damages that viruses suffer from various physical and chemical stresses and their relations to virus inactivation and aggregation. Based on the relationship between the stress-induced structural damages and the infectivity of a virus, we will propose several plausible mechanisms describing the effects of arginine on virus inactivation using the current knowledge of aqueous arginine solution properties. Acquiring specific immunity through vaccination is the most effective way to prevent viral infections ,2,3. Daii.e., arginine is highly effective in suppressing protein aggregation [in vivo for topical infectious diseases. Here we review both the in vitro and in vivo virus inactivation effects of arginine, the mechanism of various stresses that cause virus inactivation, and propose a plausible mechanism by which arginine inactivates viruses. Arginine is one of the twenty natural amino acids and, as a normal cell metabolite, is considered to be safe for human use. This amino acid has been found to have very unique properties; regation ,15,16, iregation ,18, interegation ,20, and regation ,25,26,27regation ,29. We hregation ,11,12,13In vitro virus inactivation effects of aqueous arginine solution were evaluated by incubating the virus preparations with various solvents as a function of pH or temperature. After incubation, the surviving infectious viruses were determined by infecting appropriate host cells with viruses and measuring the plaque formation of the infected cells. With enveloped viruses, arginine has consistently shown a synergistic effect with solution pH and temperature on virus inactivation. Typical inactivation data with an enveloped virus, HSV-2 (herpes simplex virus type-2), are shown in 10 reduction was observed at pH 3.8. Neither the increased citrate concentration at 0.7 M (open square) nor the addition of 0.6 M NaCl (closed triangle) resulted in a shift on the pH titration curve. Although marginal, higher concentration of citrate and NaCl appeared to suppress pH-induced virus inactivation above pH 4.2. i.e., 0.6 M) have a synergistic effect with pH  on virus inactivation. Conversely, arginine demonstrated synergy with pH in inactivating viruses. 10 reduction, the solution pH can be maintained at ~4.0–4.1 in the presence of 0.7M arginine, while the same reduction in infectivity can only be achieved at a pH of ~3.8–3.9 in its absence. Although this difference in effective pH range appears to be marginal, the difference could have a significant impact on patient safety if these acidic solutions are utilized in circumstances that require application for human body surface, or perhaps accidental contact. Similar synergistic effects of arginine with a variety of environmental conditions  have been observed with several other enveloped viruses [The above results indicate that neither 0.6 M NaCl nor excess citrate  showed severe lesion. In contrast, the eyes treated with a commercially available acycloguanosine ointment demonstrated potent efficacy with no lesion. The application of an acidic arginine solution onto the infected eyes resulted in lesion on the cornea, however to a lesser extent than those treated with saline alone. Citrate of the same acidity did not exhibit such a clear suppression. Although the concentration and pH of the acidic arginine solution were not at physiological conditions, it should be noted that no apparent injuries to the cornea of the treated rabbit’s eyes were observed . The decWe then examined the effects of arginine treatment on the establishment of genital infection with HSV-2 . HSV-2 iIt is evident that virus inactivation by arginine can only be observed in the presence of other stresses, which may include pH, temperature, freezing, drying, and the presence of antibodies/proteins: so far only pH and temperature were tested. In other words, arginine may synergize with these stresses; in the previous section, the stresses described were heat and acidification. It is important to understand the impact of these stresses on viruses in order to evaluate the synergistic effects of arginine. In general, when both temperature and pH were varied, each virus demonstrated a range of solution pH values in which it was stable and a temperature threshold below which it exhibited structural stability. The most important parameter that is affected by the stresses may be the presence of a viral envelope. Enveloped viruses, such as measles virus and RSV , demonstrate increased stability under alkaline conditions ,33, whilThe physical properties of the virus may also have a profound impact on determining its stability, with respect to changes in solution pH and temperature. One critical physical parameter is virus aggregation, as also depicted in Mechanism-2 is related to aggregation of infectious viruses . SpecifiCertain stresses can affect membrane lipids or protein-lipid interface. These stresses may cause conformational changes in proteins, which in turn lead to alteration in membrane properties. Alternatively, they can directly damage membrane lipids, as was the case for defensins, which were shown to induce pore formation in the viral lipids, leading to exposure of viral core genome ,39. ThesLow pH alone, or in combination with elevated temperature, has been implicated as the major factor influencing the structure of viral proteins and virus infectivity. Adenovirus 2 (Ad2) underwent a structural transition at elevated temperatures, accompanied by the loss of penton bases and their fibres at the icosahederal vertices, which are required for the maintenance of viral infectivity . While tSimilarly, Ad5 (Adenovirus 5) demonstrated structural change of capsid with increasing temperature, which was enhanced further at higher pH . Using ai.e., pH-induced structure change is reversible. However, response to high temperature stress is different. Both the local structural changes of the proteins and the capsid structure upon heating were irreversible. It appears that this irreversible heat-induced change is due to aggregation of viral proteins. Norwalk virus like particles (VLP), a non-enveloped virus, demonstrates a unique dependence on pH and temperature stresses. When the virus preparation was brought to pH 8.0, intact VLP virus disappeared, most likely due to changes in the local conformational changes of the viral proteins . When thin vitro and in vivo. Measles virus is unstable in the dried state at elevated temperatures [i.e., dehydration) to cause virus inactivation.Another stress that causes virus inactivation is dehydration, which can occur both eratures . Althougeratures . Since deratures . In thiswww.ap-lab.com).Aggregation of virus is a common observation , althougi.e., bivalent binding and/or consequent conformational changes. This polymerization resulted in loss of infectivity. As the size of oligomers increased, infectivity was reduced (e.g. the dimeric virus has 50% reduced infectivity), suggesting that the loss of infectivity was due to steric hindrance of virus accessibility to the host cells and not due to irreversible damages that the virus has suffered from the bound antibody. This was confirmed by the release of infectious virus from the polymerized structure through papain treatment. Polymerization of poliovirus was at least in part due to bivalent antibody binding, as excess antibody resulted in monomeric virion particles: this is expected from a large excess of antibody per binding site on viral particle [Aggregation of infectious poliovirus has been extensively studied . A monocparticle . Aggregaparticle . In thiset al. have used DLS to study the aggregation behavior of MS2 bacteriophage, which ranged in size up to 500 nm, and have correlated aggregation to its reduced efficacy [Structural damages on viruses are often accompanied by virus aggregation. In such cases, virus inactivation and aggregation are associated with each other and hence it is difficult to distinguish between damage-induced inactivation and aggregation-induced inactivation, as illustrated in the following examples. Pinto efficacy . DLS wasefficacy . Furtherefficacy . The sizefficacy . With ind) at which the RSV membrane proteins lost secondary structural content varied depending on the solution pH; significant loss was observed below 40 ºC at pH 3–5 and in between 40–60 ºC for solution pH between 6 and 8. FTIR (Fourier transform infrared spectroscopy) analysis indicated that the cause of this shift in Td was primarily due to the formation of intermolecular β-sheets, which may be due to aggregation. Thus, there is an abundance of data implicating the structural instability of viral proteins to be the main cause for virus aggregation, which consequently leads to its inactivation. It is still unclear, however, why the viral membrane is unstable under acidic condition and not under alkaline condition. In another example of low pH-mediated aggregation, respiratory syncytial virus (RSV) underwent extensive aggregation under acidic conditions; aggregation of RSV at pH 3–5 during dialysis was confirmed by elevated optical density values . FurtherFreezing causes loss of infectivity of enveloped viruses , suggest10 reduction in infectivity of HSV-1 in the presence of 25 μg/mL HNP-1 at pH 7.4, while increasing the pH from 6 to 8 in the presence of HNP-1, reduced the infectivity by >1 Log10 at 37 ºC. Defensins are not effective against all viruses, however. HNP-1 was not effective against echovirus and reovirus, which are both non-enveloped viruses. Similarly, arginine was shown to be ineffective against poliovirus, a non-enveloped virus [p-xylene-bis-pyridinium bromide) system encapsulated within a POPG  vesicle, was conducted to observe the effect of increasing HNP-2 concentration on the integrity of the liposome. The addition of 1.3 μM HNP-2 to POPG vesicles resulted in enhanced fluorescence, which further increased with incubation time. Decreasing the HNP-2 concentration from 1.3 to 0.3 μM resulted in a decrease in both the fluorescence signal and the release kinetics, suggesting that the interaction is concentration dependent. The similarity observed between arginine and defensins may not be unexpected, as mature neutrophil defensins are reported to comprise of high arginine content [Defensins, which comprise a family of broad-spectrum antimicrobial peptides, are among the key mediators of the innate immune system. One of the defensin family member, human neutrophil peptide (HNP-1), was shown to be effective in inactivating several viruses, including HSV types 1 and 2, cytomegalovirus, vesicular stomatitis virus, and influenza virus A/WSN . Similared virus . A fluor content . in vitro and in vivo experiments are usually heterogeneous, containing cell debris, aggregated particles, and chemically or physically modified viruses. Thus, the observed virus inactivation effects of arginine can be complicated by the heterogenous nature of virus preparation.As the surface of enveloped viruses is composed of spike proteins and membrane lipids, these are the most likely targets of arginine. However, any preparations of viruses used for It appears that pH and temperature are the major cause of alteration in the structure of spike proteins, leading to virus inactivation often accompanied by aggregation. Does arginine further enhance structural changes in viral proteins See . ArgininArginine suppresses protein-protein interactions and aggregation ,16,17. IAlthough unrelated to virus aggregation, arginine can also inhibit agglutination processes. Certain viruses induce agglutination of erythrocytes, and the effects of solvents on agglutination may potentially mimic virus inactivation processes. Namely, the solvents that alter viral protein structure, and consequently reduce agglutination, may cause virus inactivation. Measles, EMC and NDV viruses associate with erythrocyte cell membrane and induce hamagglutination . These iAggregated viruses may respond to pH and temperature differently. They are persistent to virus inactivation ,44. As si.e., binding to phosphate and insertion into lipid membrane, may be responsible for the virus inactivation observed upon the addition of arginine, as depicted in Arginine has also been shown to suppress the interactions of proteins with non-polar surfaces ,53,54. Si.e., electrostatic ionic binding and weak salting-out effects [i.e., influenza virus) and stabilize  a virus against the same stress, heat, in this case. Similarly, sucrose stabilized RSV [The effects of arginine are due to weak interactions with virion components, independent of the inactivation mechanisms. This is evident from the requirement of high concentrations of arginine. Such weak, but extensive interactions of arginine and many other compounds have been shown to occur with a variety of proteins and macromolecules ,64,65,66ized RSV . When 20Weak interactions have two important implications. Binding of arginine is normally reversible. When arginine concentration is reduced, it dissociate from the complexes. The consequence of binding can be, however, irreversible as in the case of virus inactivation. This implies that arginine could be less toxic than those disinfectants that strongly (and irreversibly) bind. Another important implication is that multiple arginine binding at different sites on a virus will unlikely cause drug resistance. It would be a daunting task for the virus to mutate all of the potential targets of arginine see . On the We have shown three possible modes that cause virus inactivation: structural changes of viral spike proteins, virus aggregation and pore formation in virus envelope. Arginine can inactivate viruses synergistically with other stresses, e.g., elevated temperature or acidic pH through any one or combinations of these modes. The mechanism by which arginine exerts its virus inactivation effect is most likely due to the suppression of protein interactions with other molecules or surfaces. More importantly, such effect of arginine is due to its weak interactions, which are unlikely to lead to the development of virus resistance against arginine.


Paramyxoviridae, was isolated from a Sri Lankan Giant squirrel (Ratufa macroura), which originated in Sri Lanka and deceased with severe pneumonia in a German zoo. The full-genome characterization of this novel virus, tentatively named Giant squirrel respirovirus (GSqRV), revealed similarities to murine (71%), as well as human respiroviruses (68%) with unique features, for example, a different genome length and a putative additional accessory protein. Congruently, phylogenetic analyses showed a solitary position of GSqRV between known murine and human respiroviruses, implicating a putative zoonotic potential. A tailored real-time reverse transcription-polymerase chain reaction (RT-qPCR) for specific detection of GSqRV confirmed a very high viral load in the lung, and, to a lesser extent, in the brain of the deceased animal. A pilot study on indigenous and exotic squirrels did not reveal additional cases in Germany. Therefore, further research is essential to assess the geographic distribution, host range, and zoonotic potential of this novel viral pathogen.In a globalized world, the threat of emerging pathogens plays an increasing role, especially if their zoonotic potential is unknown. In this study, a novel respirovirus, family  Paramyxoviridae in terms of genomic structure, genome length, and encoded proteins [In recent decades, several novel paramyxoviruses have been discovered, which diversified the characteristics of the family proteins . Many ofproteins ,3,4,5,6.proteins .Paramyxoviridae, order Mononegavirales, is a large and rapidly growing group of viruses [Paramyxoviridae contains seven genera, among them the genus Respirovirus, which consists of five species: Human Respirovirus 1 , Human Respirovirus 3 (former Human Parainfluenza Virus 3), Porcine Respirovirus 1 (former Porcine Parainfluenza Virus 1), Bovine Respirovirus 3 (former Bovine Parainfluenza Virus 3), and Murine Respirovirus  [The family  viruses . They ari virus) .In 1952, an outbreak of pneumonia in human newborns occurred in Sendai, Japan. A novel virus, initially designated Sendai virus (now MRV), was isolated in laboratory mice that were intranasally inoculated with patient-derived suspensions of the necropsied lungs. The infected mice died after a week with pathological findings, similar to those observed in the deceased human newborns  and feraMRV is able to cause severe respiratory disease with high mortality in immunocompetent adult mice, being easily transmitted between mice through direct contact or airborne transmission . Other lRatufa macroura). Sri Lankan Giant squirrels are distributed throughout Sri Lanka, where it also represents the national animal, and southern India. The International Union for Conservation of Nature (IUCN) lists this species as near threatened as a result of habitat loss and hunting [In the present study, we were able to isolate a novel respirovirus, tentatively designated “Giant squirrel respirovirus” (GSqRV), exhibiting sequence similarities to both MRV and HRV1. The virus was identified in a Sri Lankan Giant squirrel ( hunting . In addiSciurus variegatoides), 27 Prevost’s Squirrels , 7 Eurasian Red Squirrels (Sciurus vulgaris), and 2 Sri Lankan Giant squirrels were previously collected during investigations on Variegated squirrel bornavirus 1 [The deceased squirrel was dissected and sample materials of different organs were provided for diagnostic purposes. A cold-chain was maintained at all times to ensure the good quality of sample materials. For the routine post-mortem examination, the tissue samples were fixed in 10% neutral buffered formalin  for at least 24 h, embedded in paraffin , sectioned at 3–4 µm, and stained with hemalum and eosin (H.-E.) . The hisavirus 1 ,20.Salmonella spp.Bacteriological examination of tissue samples was performed by standard protocols  on diffew/v) of lung and intestine, and an organ pool consisting of liver, spleen, and kidney samples were used for virus isolation. These samples were prepared by first mincing small pieces of tissue into 1 mm cubes in a sterile Petri dish with dissecting scissors, followed by macerating the tissue cubes further in serum-free culture medium  containing antibiotics  using a mechanical tissue grinder. After incubation for 0.5–1 h at room temperature and centrifugation at 1000× g for 15 min, 0.3 mL of the supernatant was inoculated into cell monolayers in duplicates. Virus isolation was performed with freshly prepared confluent monolayers of baby hamster kidney , Insel Riems, Germany), rabbit kidney , and primary porcine thyroid cells. After inoculation, the cells were incubated at 37 °C for 24 h. Subsequently, the medium containing antibiotics was changed completely and the inoculated cells further incubated at 37 °C for up to one week. Monolayers were monitored microscopically for occurrence of a cytopathic effect (CPE), that is, formation of syncytia. Cultures exhibiting no viral CPE after one week of incubation were additionally passaged twice into freshly prepared cell monolayers before being discarded as negative. The presence of virus was assessed by electron microscopy, hemadsorption, and hemagglutination.Tissue homogenates  for Newcastle disease virus.Cell culture supernatants and homogenized tissue material were negatively stained with 2% phosphotungstic acid  and examined with a Philips EM 208S transmission electron microscope .For sequencing purposes, RNA was extracted using Trizol LS Reagent  in combination with RNeasy Mini Kit  including DNase digestion (Qiagen) on the spin column.Formalin-fixed paraffin-embedded (FFPE) materials of the squirrel including samples of lung, brain, heart, liver, kidney, spleen, and intestine were extracted using the RNeasy FFPE Kit (Qiagen) as described by the manufacturer.For screening purposes, nucleic acids of squirrel samples were extracted using a combination of Trizol Reagent (Thermo Fisher) and automated extraction with the NucleoMagVet kit . Sample materials were lung, brain, swabs, or organ pools.p-value of 10−6 and filter for strand bias. MAFFT v7.308 [Extracted RNA of the cell culture supernatant (fourth passage) was used for reverse transcription, library preparation, and sequencing of the resulting library lib01567 on a MiSeq instrument  as previously described . For speT v7.308  was usedT v7.308 , executiT v7.308   long sequence in the L gene. The oligonucleotide sequences for the specific RT-qPCR were as follows: GSqRV-fw 5′-TCACAGGCTCAAGGATACCG-3′, GSqRV-rv 5′-TCCTTGAGGGCCATGTTGTC-3′, Resp-probe 5′-6-FAM-ACYCAGATGAAGTTCTCHAGTGCMA-CACT-BHQ-1-3′. For a pan rodent respirovirus RT-qPCR (MGSqRV RT-qPCR) simultaneously detecting MRV and GSqRV, primers binding to MRV  were added. Cycling conditions followed the QuantiTect Probe RT-PCR Kit (Qiagen) manufacturer protocol. For validation, RNA covering the amplification site of MRV and HRV1 was transcribed in vitro using T7-Polymerase (Thermo Fisher) as described by the manufacturer, followed by DNase digestion , and spiked into background squirrel RNA. As an internal control, a primer-probe-system detecting β-actin was applied .In 2009, a male adult Sri Lankan Giant squirrel died during quarantine in a German zoo. The 960-g animal originated from Sri Lanka. Pathologic–anatomic and histologic examinations revealed a moderate to severe acute hemorrhagic-necrotizing pneumonia with intranuclear eosinophilic inclusion bodies, severe diffuse alveolar damage, and multifocal moderate intraalveolar hemorrhages in the lung . AdditioRoutine diagnostics in virology and bacteriology were performed for identification of the responsible pathogen. Inoculation of homogenized lung material on primary porcine thyroid cells resulted in a CPE including the formation of syncytia on day 3 post inoculation . No CPE Electron microscopy of cell culture supernatant showed typical paramyxovirus-like particles . HemadsoEscherichia coli in the lung, small intestine, and colon, while investigations for Salmonella spp. were negative.Bacteriological examination revealed a high concentration of non-serotypable 6 reads marked approximately each 1% as low quality or unclassified, respectively. Most importantly, with a proportion of 63.8% of the reads being classified as sequences representing the family Paramyxoviridae, this analysis confirmed the previous findings but provided no hint at other potentially present pathogens. In addition, 33.8% were eukaryotic sequences, that is, sequences representing the used cell line.To confirm the electron microscopic results and specify the viral species, the obtained raw sequencing data was first analyzed using RIEMS . This inq 16), and FFPE material (Cq 27). The untreated brain material was also positive (Cq 27), whereas in the FFPE material, no viral RNA could be detected. Equally, in the FFPE material of heart, liver, kidney, spleen, and intestine, no viral RNA could be detected. The strong presence of viral RNA in the lung points to GSqRV being accountable for the hemorrhagic-necrotizing pneumonia. The detection of GSqRV RNA in the brain material, although at a much lesser extent, indicates a severe systemic infection with GSqRV, potentially causing the observed multifocal scattered glial nodes. Acute encephalopathy associated with respiroviruses has also been described for some human cases upon infection with HRV1, although extra-pulmonary symptoms are rare [On the basis of the full-genome sequence , a specific RT-qPCR was developed for GSqRV detection using specific primers amplifying a fragment of the L-gene. The viral load in the lung tissue of the squirrel was high, as observed in both the untreated . A unique feature of GSqRV is the genome length of 15,378 nucleotides (nt), complying to the rule of six for members of the lication . In contThe genome comprises the six genes 3′-N-P/C-M-F-HN-L-5′. The localization and length of the coding sequences (CDS) for the nucleoprotein (N), phosphoprotein (P), the accessory proteins , and the matrix protein (M) are analogous to the genome organization of MRV. In contrast, the CDS for the fusion protein (F), hemagglutinin–neuraminidase (HN), and polymerase (L) differ in their positions compared with MRV and HRV1 because of six missing nucleotides in the intergenic region between M and F . The CDSAs a unique feature of GSqRV, an additional open reading frame (ORF) is located in the P gene, leading to the hypothetical expression of an additional accessory protein (Y0), not present in MRV and HRV1. The putative fifth translation site is located in the P gene, upstream of the initiation sites for Y1 and Y2 . The expWithin MRV, the P gene serves also as a template for the proteins V and W, being produced by a leaky scanning mechanism of the polymerase, and thereby insertion of non-templated G nucleotides into the mRNA, resulting in a shift of the reading frame . The resIn the genome sequence of the GSqRV, 13 SNVs were detected (cutoff = 10%), suggesting the existence of at least one variant. Four SNVs are located in the HN-CDS, and an additional four in the L-CDS see . Only 2 The overall pairwise nucleotide sequence identity of the GSqRV is only slightly higher to MRV  than to HRV1 , while the sequence identities within a sliding window of 200 nt differ from 11 to 94% .Paramyxovirinae, the sequence similarity between corresponding proteins is greater for N, M, and L, with F and HN being slightly less conserved, and C and P being poorly conserved [The amino acid sequence identities vary between 55% and 89% to MRV, strain Nagoya, and between 49% and 87% to HRV1, strain Washington/1964, respectively . The M gTo the best of our knowledge, no other squirrel-specific respiroviruses have been reported in the literature or the sequence databases to date. There has been serological evidence of MRV in wild grey squirrels in North Wales, however, in this study, no virus was characterized . In anotRespirovirus, most closely related to MRV and HRV1 (Respirovirus in the 9th ICTV report [Sequence alignment with subsequent phylogenetic analysis shows that GSqRV can be classified within the genus and HRV1 . The outV report , each viV report . BecauseV report , the que, 27 Prevost’s Squirrels, and two Sri Lankan Giant squirrels from holdings in Germany, as well as seven wildlife Eurasian Red Squirrels, neither GSqRV nor MRV were detected. Therefore, GSqRV infections in squirrels might be very rare in Germany, however, the number of animals in this study was quite limited. Additionally, the natural host of GSqRV is not known. The deceased squirrel could embody the reservoir host or an accidental host. Because of acute stress caused by transportation and adaptation to a new environment, it may have succumbed to the disease, while being unaffected in its natural habitat. The examination of more samples of different rodents is necessary, starting in Sri Lanka and south India, to determine the reservoir, host range, and geographic distribution of GSqRV. Future studies should also include serological analyses, as the viremic phase is probably short and direct detection of viral RNA is thus restricted.For identification of potential further cases in Germany, squirrels living wild or in captivity were screened via RT-qPCR. For simultaneous detection of MRV and GSqRV, a pan rodent respirovirus RT-qPCR assay was developed and applied. Sample materials consisted of lung, brain, oral swabs, or organ pools. In 88 squirrels tested, comprising 52 Variegated SquirrelsThe genome similarity of GSqRV and MRV/HRV1 raises concerns of the potential for zoonotic and interspecies transmission of GSqRV. Both MRV and HRV1 are known to be pathogenic for their respective hosts. However, while HRV1 replicates poorly and is nonpathogenic in mice, MRV has been shown to replicate as efficiently as HRV1 in the upper and lower respiratory tract of African green monkeys, and may therefore lack host range restriction . SimilarHere, we were able to detect and characterize a novel virus isolated from a deceased Sri Lankan Giant squirrel with severe pneumonia that was imported to Germany. The unique position of the virus in phylogenetic analysis as an “intermediate” between MRV and HRV1 is remarkable and demonstrates the need for further research regarding its zoonotic potential. An initial pilot screening of indigenous and exotic squirrels living in Germany by RT-qPCR did not identify further cases, nevertheless, more squirrels in different areas of the world should be examined to provide more information on host specificity, transmission, and geographic distribution of this novel virus. These investigations will expand our knowledge about wildlife associated viruses that might represent a threat to human and animal health.


Begomovirus have been documented as the most genetically diverse in the family Geminiviridae and present a serious threat for global horticultural production, especially considering climate change. It is important to characterize naturally existing begomoviruses, since viral genetic diversity in non-cultivated plants could lead to future disease epidemics in crops. In this study, high-throughput sequencing (HTS) was employed to determine viral diversity of samples collected in a survey performed during 2012–2016 in seven states of Northern-Pacific Mexico, areas of diverse climatic conditions where different vegetable crops are subject to intensive farming. In total, 132 plant species, belonging to 34 families, were identified and sampled in the natural ecosystems surrounding cultivated areas . HTS analysis and subsequent de novo assembly revealed a number of geminivirus-related DNA signatures with 80 to 100% DNA similarity with begomoviral sequences present in the genome databank. The analysis revealed DNA signatures corresponding to 52 crop-infecting and 35 non-cultivated-infecting geminiviruses that, interestingly, were present in different plant species. Such an analysis deepens our knowledge of geminiviral diversity and could help detecting emerging viruses affecting crops in different agro-climatic regions.Plant DNA viruses of the genus  Agroecosystems are used for the production of food, feed, fuel, fiber, and other harvestable goods providing human support and health . Mexico Geminiviridae) are characterized by their geminate particles that encapsidate a circular single-stranded (ss) DNA genome (monopartite or bipartite) of about 2.8 kb in size. They are transmitted via whitefly (Bemisia tabaci), infecting a large number of plant species worldwide and causing serious crop losses, and constitute a major global threat [Chino del tomato virus (CdTV) [Pepper huasteco yellow vein virus (PHYVV) and Pepper golden mosaic virus (PepGMV) [Tomato yellow leaf curl virus (TYLCV) in Sinaloa has dealt a devastating impact on tomato production. In different agro-climatic regions of Mexico, begomovirus-associated diseases are commonly caused by mixed infections with diverse begomoviruses, and affect tomato and pepper crops [Tomato yellow leaf curl Malaga virus (TYLMaV) is a recombinant of Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus mild strain (TYLCV) which, unlike its “parental” genomes, has the ability to infect common bean and wild Solanum nigrum [Plant diseases caused by begomoviruses and RNA viruses have been the main concern in Mexican horticulture throughout the years, having a significant negative impact on the crop production of tomato, pepper, bean, pumpkin, melon, soybean, tomatillo, tobacco, watermelon, and cotton ,4,5,6,7.l threat , cassaval threat , cotton l threat , and toml threat . In Mexis (CdTV) . However(PepGMV) . Recentler crops ,16. Coiner crops ,18,19. Ger crops ,21,22,23m nigrum . More imm nigrum .It has been proposed that global warming will influence the epidemiology of virus-related plant diseases, mainly due to alterations in the distribution of insect vectors and in their host range ,27. EmerBemisia tabaci, while aphid vectoring was recently confirmed for the Capulovirus Alfalfa leaf curl virus (ALCV) [New mutant or recombinant viral genomes that have arisen in non-cultivated plants could achieve successful infection of plant hosts to which they have newly adapted and cause significant crop damage, in a process subject to effective transmission between plant species. Vector-transmission is an evolutionary barrier for plant viruses to expand their host range. Alterations in vector-dependent transmission, most likely through mutations in the viral coat protein sequences, could increase the risk of emergence of a plant pathogen in a given crop. Begomoviruses are transmitted by the whitefly s (ALCV) ,33. Vects (ALCV)  due to aHigh-throughput sequencing (HTS) has provided the means to detect known viruses as well as to identify novel viruses in plants ,37,38,39de novo via short read assembly, a process that may create chimeras deriving from the different genomes present in a sample, and secondly, the accurate differentiation of sequences; thus, confirmation is required by cloning followed by Sanger sequencing. The hope is that the latest single-molecule NGS technologies (3rd generation), where long reads are obtained, could address both of the above-mentioned issues, especially when their sequence reading error rates drop significantly compared to the levels of the previous NGS technologies. The resolution of the metagenome of a given sample could identify genetic variations in a viral population, providing the necessary input to study viral genome evolution, and determine which environmental factors might influence the generation of novel plant pathogens from previously benign viruses.The current next generation sequence (NGS) technologies have a couple of drawbacks: firstly, the contigs/singletons need to be annotated It is a well accepted notion that non-cultivated (wild) host plants play a key role in the generation of viral genetic variation, maintaining a certain degree of sequence heterogeneity convenient for viral adaptation in nature, without altering essential consensus sequences. Only recently, metagenomics studies on non-cultivated plant species have attracted the attention of plant researchers ,41. MexiA total of 422 non-cultivated plants (both symptomatic and asymptomatic) located in the area between crops and wild vegetation zones , in seven states in the northern-pacific region of Mexico during the period 2012–2016, were collected, GPS-documented, photographed, and identified to the species level. Thus, 132 species of plants belonging to 34 families were identified. The sampling regions were grouped as follows: (1) Baja California (BC), (2) Sonora (SO), (3) Sinaloa (SI), (4) Colima-Nayarit (CN), and (5) Coahuila-Durango (CD) states of Mexico. Samples were placed on ice and brought to the laboratory and stored at −80 °C until processed. Plants were collected in non-protected areas; additionally, an herbarium was established with most of the plant specimens.Begomovirus detection using degenerate universal primers  using the illustra TempliPhi DNA Amplification Kit , following the manufacturer´s instructions. Then, all RCA products obtained per sampling region were pooled in equimolar concentrations, and cleaned using phenol:chloroform:isoamyl alcohol (25:24:1)/potassium acetate (5 M) and 100 ethanol % precipitation . DNA integrity was analyzed by agarose gel electrophoresis, and the cleaned RCA mixtures were used for NGS library construction that was sequenced by a commercial facility  using Illumina Nextera XT paired end 2 × 150 bp protocol on a MiSeq 500. The same procedure was followed for each sampling region to obtain one library per region .Total DNA was extracted from individual plants using the CTAB method , and the primers . For eachttps://www.bioinformatics.babraham.ac.uk). Each library was filtered for human, bacteria, plant, and eukaryotic viruses reads using the ViromeScan pipeline [https://www.dropbox.com/sh/ha6pkzls9217dhf/AAADNUa0TfYj3EZ8bb315cSga?dl=0.Reads obtained from each library were trimmed employing the trimmomatic tool   and SnapGene  software. All pairwise comparisons were performed using the MUSCLE algorithm implemented in Mega 7 [Full-length geminiviral genomes were obtained following a previously described protocol . In brien Mega 7  and maxi2-emission scenario (A1FI) put forward by the Intergovernmental Panel on Climate Change [2, heavy rains, increased humidity, drought, and warmer winter temperatures [It is an accepted notion that global warming will have an impact on global food security. In particular, crop yields are predicted to significantly decrease considering the “worst” COe Change . Plant pe Change , imposineratures . StudiesAstaraceae, Solanaceae, Malvaceae, and Fabaceae. It is worth mentioning that those families were found to be the most predominant in natural ecosystems considering 200 plant families described in Mexico [Begomovirus, we were able to amplify the expected DNA fragment of 950–1100 bp in 252 out of the 422 (60%) tested individual plant specimens, indicating that begomoviruses were present in 29 plant families collected from all five regions sampled  present in four regions, Pepper golden mosaic virus (PepGMV-signature) present in four regions, Pepper leafroll virus (PepLRV-signature) present in one region, Tomato chino la Paz virus (ToChLPV-signature) present in two regions, Tomato severe leaf curl virus (ToSLCV-signature) present in two regions, Tomato yellow spot virus (ToYSV) present in three regions, Potato yellow mosaic virus (PYMV) present in one region, Okra yellow mosaic mottle virus (OYMMV-signature) present in four regions, Cabbage leaf curl virus (CabLCV-signature) present in two regions, Bean calico mosaic virus (BCaMV-signature) present in four regions, Bean yellow mosaic Mexico virus (BYMMV-signature) present in one region, Vigna yellow mosaic virus (ViYMV-signature) present in two regions, Water melon chlorotic stunt virus (WmCSV-signature) present in one region and Squash leaf curl virus (SLCV-signature) present in three regions; and five with monopartite genomes, three belonging to the genus Begomovirus, namely Chilli leaf curl virus (ChiLCV-signature) present in one region, and Tomato yellow leaf curl virus (TYLCV-signature) present in four regions, Sweet potato leaf curl virus (SPLCV-signature) present in one region; additionally, one belonging to the genus Curtovirus, namely Beet curly top virus (BCTV-signature) and another belonging to genus Topocovirus, namely Tomato pseudo-curly top virus (TPCTV), both present in one region (Solanum mosaic Bolivia virus (SoMBoV-signature), present in two regions, Sida mosaic Sinaloa virus (SiMSiV-signature) present in five regions, Sida golden yellow spot virus (SiGYSV-singnature) present in one region, Malvastrum bright yellow mosaic virus (MaBYMV-signature) present in three region, Rhyncosia golden mosaic virus (RhGMV-signature) present in five regions, and Rhyncosia golden mosaic Sinaloa virus (RhGMSV-signature) present in two regions, Euphorbia mosaic virus (EuMV-signature) present in three regions, Euphorbia yellow mosaic virus (EuYMV) present in one region and Blechum leaf curl virus (BlelCV-signature) present in one region, (Tomato pseudo-curly top virus (TPCTV) in samples from Colima-Nayarit is the first report of the genus Topocovirus in Mexico. The list of geminiviruses are grouped as a potential of new viruses or strain of the best match virus, with molecular and biological validation being necessary.The analysis of the highest geminivirus-related signature sequences revealed a list of both bipartite and monopartite begomoviruses, including crop-adapted viruses in different plant families; 14 with bipartite genomes such as e region . Further region, . InteresBegomovirus comprises the most common DNA viruses responsible for several virus-associated plant diseases in Mexico. Among them PHYVV, an endemic virus, and PepGMV have been documented as the most widespread and predominant in pepper crops [Sida rombifolia [Sida rombifolia and Rhyncosia minima, respectively), with different wild species (acting as reservoirs) being suspected as the origin of the inoculum. Moreover, EuMV and EuYMV were reported previously in Euphorbia heterophylla in Mexico and Brazil [The genus er crops ,14,57,63er crops . Intereser crops . Howeverer crops . The other crops . The detmbifolia  without mbifolia . It is wd Brazil ,69; wherd Brazil . To the d Brazil ,72,73.The ecology of begomoviruses identified by HTS studies in non-cultivated plants requires more effort in order to understand the contribution of these plants for disease development ,75,76. NFabaceae, Malvaceae, and Solanaceae families are the most widely distributed in all sampled areas. The HTS analysis revealed that signatures corresponding to TYLCV, SiMSiV, and RhGMV were present in all five NGS libraries, whereas the RhGMSV-signature was detected only in two out of five NGS libraries, suggesting that these species of begomovirus are predominant. Initially, the presence of these four viruses was confirmed by using sequence-specific primers for each viral species ; of Sida acuta from Colima (SiMSiV PCR-positive), and two Rhynchosia minima both from Sinaloa (PCR-positive for RhGMV/RhGMSV), were used for RCA amplification and cloning. Sequence analysis of obtained clones is summarized in According to our survey, plants belonging to the  species , testingN. glauca was 2781 nt in length and showed high nucleotide homology (99.9%) to TYLCV (Accession number: EF523478.1). Clones LV15-Sa-03 and LV15-Sa-02 , from S. acuta, were 2611 nt and 2583 nt in length and showed nucleotide homology of 95.1 and 91.3% with DNA-A and DNA-B of SiMSiV . Clones LV17-Rm-02 LV17-Rm-06 , from R. minima, were 2605 nt and 2568 nt in length and showed nucleotide homology of 98.6 and 91% with DNA-A and DNA-B of RhGMV . Finally, clones LV15-Rm-02 LV15-Rm-08 , from R. minima, were 2578 nt and 2525 nt in length and showed nucleotide homology of 91.9 and 85.9% with DNA-A and DNA-B of RhGMSV . For all viral genomes obtained, the predicted stem-loop region containing the sequence TAATATTAC, found in the common region of family Geminiviridae, was identified. For bipartite begomoviruses, high nucleotide homology of the common region (CR), of 98, 91, and 87% (DNA-A versus DNA-B) was observed for SiMSiV, RhGMV, and RhGMSV, respectively. Furthermore, the array of regulatory elements (iterons and TATA boxes) was conserved in all cases, suggesting that corresponding DNA-A and DNB-B are cognates. According to the present taxonomic classification of ICTV [Geminiviridae, the clones of TYLCV, SiMSiV, and RhGMV obtained in this study are classified as strains (≥94% DNA-A nucleotide homology), whereas the RhGMSV clones are classified as different isolates (≥91% DNA-A nucleotide homology).Clone LV15-Ng-04 (Accession number: MK643155) from  of ICTV , for famN. glauca cluster together with different TYLCV isolates from different regions of the world, and segregate more closely to Mexican and Asian isolates (Israel and China)  isolated from S. acute is closely related to an isolate of SiMSiV from Sinaloa state  A. These oa state B,C. Simioa state B,C. AltoThe metagenomic approach carried out in the present work, allowed us to identify the geminiviral diversity present in different plant families and geographical regions; however, it is crucial from an ecological point of view to determine the occurrence and dynamics of the viral community in non-cultivated plants.Fabaceae, Malvaceae, and Solanaceae) were examined for the dynamics of the individual plant-virus infections in the five sampling regions , the Malvaceae family , and the Solanaceae family . PHYVV is an endemic virus of Mexico, described as a major concern for pepper production [Fabaceae family , the Malvaceae family , and the Solanaceae family . On the other hand, SiMSiV was reported infecting Sida rombifolia in Mexico; interestingly, this virus was detected not only in several other malvaceous plants  but also in the Fabaceae family  and the Solanaceae family . Furthermore, RhGMV was first reported infecting Rhynchosia minima in Honduras [Rhynchosia minima but also other fabaceous plants , and other hosts belonging to the Malvaceae , and the Solanaceae  families. This part of the study was oriented to individual plant samples, providing evidence about the ecology of the virus. The existence of several plant species harboring different viruses in multiple infections represents an important melting pot for the geminivirus to evolve in different directions triggered by vector and environmental factors [TYLCV is the major viral concern for tomato production worldwide . It was lanaceae ,97,98,99oduction ,14,63,65oduction ,100. HerHonduras  and afteHonduras . The dat factors . Previou factors ,69,102.  factors ,103. The factors ,76, therDatura leaf curl virus (DaLCV) in Sudan [The evolution of plant viruses is a complex process involving multiple ecological and genetic factors resulting in host-pathogen co-evolution. Studies on viral diversity have been documented in a wide number of non-cultivated plants, with new entries described either as different strains of existing viruses or as new viruses ,105,106.in Sudan . In the