Chrosomus eos) and finescale dace (C. neogaeus) display a wide sympatric distribution in North America. The asexual reproduction of their hybrids allows determining where and when hybridization occurred. The aim of this study was twofold: first, to assess whether temperature affected reproductive isolation, and second, whether the effects of climate warming resulted in a latitudinal progression of hybridization. We performed a 500 km latitudinal survey (51 sites) in southeastern Quebec (Canada) and determined the distribution of clonal hybrid lineages. Results revealed a total of 78 hybrid lineages, including 70 which originated locally. We detected a significant difference between the southern and northern range of the survey in terms of the proportion of sites harboring local hybrids  and hybrid diversity . This confirmed that there was more frequent interspecific mating in the warmest sites. In the southern range, diversity of lineages and simulations suggest that hybridization first took place  in sites characterized by a longer growing season, followed by northerly adjacent sites . Moreover, evidence of hybridization occurring in present‐day time was detected. This suggests that the current warming episode is going beyond the limits of the previous warmest period of the Holocene.Climate warming may affect reproductive isolation between sympatric sister species by modifying reproductive phenology or mate choice. This is expected to result in a latitudinal progression of hybridization in response to the shifting of environmental conditions. The fish species northern redbelly dace  revealed a significant difference in terms of the proportion of sites harboring local hybrids and hybrid diversity, between the southern and northern halves of the survey. This more frequent interspecific mating in the warmest sites and evidence of hybridization occurring in present‐day time suggest that the current warming episode is going beyond the limits of the previous warmest period of the Holocene.The asexual reproduction of  The modifications of environmental conditions brought by the rise of the temperature have major impacts on premating barriers, making hybridization a serious consequence of global warming Chunco, . FirstlyThe effect of warming is not uniform across the latitudinal range of species because temperature varies with latitude . If temperature must reach a threshold to trigger a given change, warmer regions will be first affected, and with time (and continuous warming), the geographic limit reaching this threshold will move latitudinally. The modification of premating barriers between sympatric related species is therefore expected to result in a wave of hybridization through the sympatric area in response to climate warming.Moreover, the current climatic warming is not an isolated event. The transition from Pleistocene to Holocene ca. 12,000 years BP was marked by a strong increase in temperature, while the following Kyears were punctuated by cooling and warming episodes , and second, whether effects of climate warming  resulted in a latitudinal progression of hybridization through time . To address these objectives, we performed a 500 km latitudinal survey in the southeastern Quebec region to investigate the large‐scale geographic variation in distributions of the C. eos–neogaeus hybrids, as a consequence of local hybridization between C. eos and C. neogaeus.Since local hybridization events appear to be rare in most sperm‐dependent unisexual hybrids, this raises questions about the causes of such high rates of spontaneous hybridization in that region. The aim of this study was twofold: first, to assess whether temperature  affected reproductive isolation between Chrosomus neogaeus first reproduces in early spring, while C. eos reproduces later . In addition, the asexual reproduction of all‐female clonal hybrids is expected to result in a rapid demographic expansion of hybrid population . However, through generations, random processes related to lineage sorting, as well as natural selection, progressively reduce the number of lineages. Therefore, the diversity of lineages at a given site depends on the time elapsed since the hybridization events . Relative time since local hybridization events can be correlated to lineage diversity.In this contextual framework, we can formulate specific predictions for each objective. First, we expect a higher occurrence of hybridization in southern sites than in northern sites due to the higher temperature at lower latitude (closer to the threshold expected to weaken premating barriers). In northern sites, where premating barriers are not yet disturbed, the presence of locally produced hybrids is expected to be infrequent, even in the presence of sympatric parental species. Second, if climate warming (previous episodes during Holocene as well as the current episode) modifies premating barriers, hybridization is expected to progress northward through time. We therefore predict that the hybrid diversity resulting from local hybridization will vary according to a latitudinal gradient. Since the number of lineages decreases with time since hybridization, diversity is expected to increase going northward, as hybridization events are more and more recent.22.1We analyzed a total of 51 sites along a 500‐km‐long transect, from 45°02′ to 48°29′ north Figure . Nine ofIn addition, individuals from 32 new sites were sampled in six additional hydrographic networks: Chaudière R. (CH) northerly adjacent to SF, then moving northward Saint John R. (SJ), Ouelle R. (OU), Du Loup R. (LO), Rimouski R. (RM), and Mitis R. (MI).This research was performed under institutional animal care guidelines  and conforms to the mandatory guidelines of the Canadian Council on Animal Care. Sampling permits were provided by the Quebec Ministry of Natural Resources and Wildlife (MRNF).This transect encompassed a broad latitudinal range and temperature gradient. For instance, there is a difference of 3.0°C in the mean annual temperature between the southernmost and the northernmost sites. The length of the growing season (LGS) was used to represent the latitudinal variation of temperature and other associated environmental conditions for which it can be used as a proxy. Indeed, the LGS is limited by different factors, such as air temperature, frost days, rainfall, and daylight hours; it provides an integrative index of climate differences among regions .For all individuals identified as hybrids, a multilocus genotype was determined according to the size variation of microsatellite loci Seat412, Ca‐12, Pho‐60, Pho‐1, Pho‐2, and Rhca‐20  across LGS categories was tested by Fisher's exact test, while the abundance of species was compared by a chi‐square test.2.5C. eos and C. neogaeus, the difference in the number of sites harboring private hybrid lineages  and diversity of private lineages were tested according to the LGS using Fisher's exact test and nonparametric Mann–Whitney U tests, respectively. The diversity of private lineages across a latitudinal gradient was also represented using a LOESS/LOWESS  regression  from populations generated by EASYPOP 1.7  of one site (CH‐4) has been underestimated by the sampling due to the presence of several lineages with low frequency. To assess the effective number of lineages in this population, we estimated the number of lineages from 100 random samplings . This species displayed an even distribution; the proportion of sites occupied by C. eos did not differ among LGS categories (Fisher's exact test p = .3745).Both s Figure , Table 1C. neogaeus was only detected in 18 sites (35.3%) and was less abundant than C. eos . The distribution of this species is uneven (Fisher's exact test p = .0429), being less abundant in the LGS of 150–160 days . However, their distribution across LGS categories is also uneven (Fisher's exact test p = .0124). At the opposite of C. neogaeus, hybrids were less abundant in the northern sites  and were as abundant as 3.2R2 = 0.08; p = .1).The genetic survey of hybrid individuals revealed a total of 78 distinct lineages Table ; 41 lineHybrids could be discriminated into two categories according to their geographic distribution. On the one hand, eight lineages displayed a large geographic distribution, being shared between at least two distant sites  due to the low proportion of sites with private lineages in both 120–130 and 130–140 days LGS categories . Their distribution across LGS categories is also uneven (Mann–Whitney 2 = 19.33 df = 3 p = .0002). A total of 57 private hybrid lineages (81.4%) were detected in the southern half of the survey (LGS of 140–150 and 150–160 days), contrasting with the northern half (LGS of 120–130 and 130–140 days) with only 13 hybrid lineages  even when excluding the site CH‐4 . For instance, four sites from LGS of 140–150 days displayed five private lineages or more (two sites in SF and two sites in CH network), while all sites from LGS of 150–160 days harbored three lineages or less.We assessed the temporal change, more specifically whether hybridization first occurred in the southernmost sites (LGS of 150–160 days) before progressing northwardly (LGS of 140–150 days). Indeed, a higher diversity was observed in sites from LGS of 140–150 days  than in southernmost sites from LGS of 150–160 days  with a diversity of three lineages or less is detected when hybridization occurred >7,000 years BP. Finally, hybridization that occurred 10,000 years ago  still displays diversity and some systems could maintain up to three lineages.4C. eos and C. neogaeus  and second whether climate warming (previous or current episodes) resulted in a latitudinal progression of hybridization through time . The clonal hybrids that keep track of hybridization between those species are therefore a useful biological indicator through which to infer the effects of climate warming.The aim of this study was first to assess whether temperature  affected reproductive isolation between C. eos–neogaeus hybrid lineages. This striking result contrasts with diversity from the other regions of North America surveyed so far. Previous studies reported a single or a very low number of widespread lineages per region  were specific to a single site or were detected in a few geographically close sites that shared the same lineage assemblage. The very high diversity of lineages and their restricted distribution are consistent with the hypothesis that hybridization events occurred locally. Genetic analyses provided another support to this hypothesis as the multilocus genotype of hybrids from RI, YA, and SF networks matched that of The latitudinal pattern of diversity is an additional argument to reject the hypothesis that hybrids have colonized this region. The increase in hybrid lineage diversity with latitude in the southern half of the survey  sympatric in most of the hydrographic networks analyzed, the diversity of private hybrid lineages strongly varied latitudinally. We observed a higher proportion of sites where hybridization occurred locally and a higher number of private lineages in the southern half of the survey than in the northern sites.The spatial distribution of private hybrid lineages is consistent with a temperature or environmental condition effect on hybridization. While C. eos and C. neogaeus species. For instance, both species were found in sympatry in eight sites  in the northern half of the survey, even in the presence of both species in sympatry. The low number of private lineages indicated that premating barriers have not been disturbed yet in regions characterized by a LGS shorter than 140 days.This suggests that sympatry alone is not sufficient to explain hybridization between 4.2C. neogaeus from the networks of southeastern Quebec  performed in the LGS of 150–160 days  revealed an exceptionally high number of lineages, one order of magnitude higher than in other sites. The unusually high diversity of CH‐4 contrasts with that of the other sites of the survey. The low diversity of those sites is expected to be representative of the population. For example, the same lineages were detected when sampling geographically close sites  and the finescale dace (C. neogaeus).Results of the simulations revealed that 100 lineages produced by a local hybridization can rapidly decrease to 50 lineages per population (the number expected in site CH‐4) in less than 20 generations. This result suggests that a LGS of 140–150 days could serve as a reference for conditions triggering hybridization between the northern redbelly dace (In conclusion, the present study provides an empirical framework to assess how historical climate warming influenced hybridization between sympatric sister species. However, detection of contemporary hybridization close to the northern limit of historical hybridization suggests that the current warming episode is going beyond the limits of the Holocene Optimum, a conclusion recently reached by Porter et al.  on warmiNone declared.


Reliable serologic tests are needed for diagnosis and surveillance of Zika virus infection. We evaluated the Euroimmun and Dia.Pro serologic tests for detection of Zika virus IgM and IgG by using a panel of 199 samples from a region endemic for flaviviruses. Kinetics of Zika virus antibodies were monitored from 300 sequential specimens sampled over a period of 10 months after infection. We observed suboptimal performance; sensitivity for Zika virus IgM was low, especially in the Euroimmun assay (49%), whereas IgM could be detected for months with the Dia.pro assay. The specificity of the Zika virus IgG assays was also low, especially that of Dia.Pro (62%); findings were strongly influenced by the epidemiologic context. These results highlight the complexity of serologic diagnosis of Zika virus infection in regions endemic for flaviviruses. Accurate analysis of the performance of assays is required to adapt and interpret algorithms. Flavivirus, and is an arbovirus transmitted mainly by mosquitoes of the genus Aedes (Stegomyia). Initially isolated in 1947 from a sentinel monkey during yellow fever surveillance in Uganda, Zika virus was reported as causing only sporadic human infections, associated with asymptomatic or mild, self-limiting illness, until 2007  and Dia.Pro Zika virus IgM and IgG ELISAs . The second objective was to determine the kinetics of Zika virus IgM and IgG induced after infection, as defined by these kits.The first objective of this study was to evaluate the performance of two commercial serologic kits for the detection of Zika virus–specific IgM and IgG in serum samples from patients with an arboviral-like syndrome in a region where other arboviruses are known to circulate, including dengue and chikungunya viruses. The 2 commercial kits (4 assays) studied were the Euroimmun Zika virus IgM and IgG ELISAs  was used for Zika virus detection and NRC in-house real-time RT-PCR for dengue and chikungunya viruses.The clinical samples used in this study were selected according to the standards for reporting diagnostic accuracy requirements. Two thirds came from the serum collection of the National Reference Centre (NRC) for Arboviruses in French Guiana and one third from samples collected for a descriptive prospective study of Zika virus disease in the French military community in French Guiana (ZIFAG)  . We evalTo determine the kinetics of Zika virus antibodies, we used a panel of 300 serum samples collected from 124 patients with confirmed Zika virus infection from day 0 through day 300 after the onset of symptoms . We coll>0.8 to <1.1 as equivocal, and >1.1 as positive. We tested all serum specimens with the respective Dia.Pro ELISAs for qualitative determination of IgM and IgG, according to the manufacturer’s instructions; we interpreted results as positive if the S/CO ratio was >1.1, negative if <0.9, and equivocal if 0.9–1.1. Recombinant Zika virus nonstructural protein 1 was the antigen in both the Euroimmun and Dia.Pro assays.We tested all samples with the Euroimmun ELISAs according to the manufacturer’s recommendations and calculated signal-to-cutoff (S/CO) ratios; values <0.8 were regarded as negative, t test. We performed all statistical analyses using R 3.4 statistical software (https://www.r-project.org).Continuous variables were expressed as mean (± SD) or median with interquartile range (IQR) and discrete variables as percentages and 95% CIs. We calculated the sensitivity and specificity of the assays with 95% CIs. The differences in the S/CO ratios of the IgM and IgG assays at each time were compared with the Student + SD age of 36 + 16 years . This panel was composed of the Zika virus–positive subgroup (n = 90), 51 (57%) female and 39 (43%) male, with a mean age + SD of 39 + 12 years; and the Zika virus–negative subgroup (n = 109), 62 (57%) female and 45 (43%) male, with a mean + SD age of 34 + 18 years .The 300 samples used to determine the kinetics of Zika virus IgM and IgG came from 124 patients, 82 (66%) female and 42 (34%) male. The age range of these patients was 8–74 years ; sensitivity of the Dia.Pro test was 69% . Only 1 of the 109 non-Zika subgroup serum samples was positive in the Euroimmun test, indicating 99% specificity (95% CI 97%–100%). Four non-Zika subgroup samples were detected as positive by the Dia.Pro IgM test, including 3 samples from patients with acute dengue virus infection, indicating a specificity of 96% .We used the same panel to evaluate the performance of Euroimmun and Dia.Pro Zika virus IgG kits . With thThe false positivity rate of the 2 Zika virus IgG assays varied according to the subpanel used. These rates were 40%–58.6% for positive dengue virus or chikungunya virus sample subgroups and 11.9%–21.4% for the negative dengue virus, negative chikungunya virus, and negative Zika virus sample subgroup .The sensitivity of the combined Euroimmun Zika virus IgM and IgG assays was 82%  and the specificity was 69% . The sensitivity of the combined Dia.Pro Zika virus IgM and IgG assays was 87%  and specificity was 62% .We used the panel of sequential samples from patients with confirmed Zika virus infection to determine the kinetics of Zika virus IgM and IgG over 10 months after clinical onset . For the>5 sequential samples are  than the Euroimmun IgM test (49%), the performance of both IgM assays was suboptimal. The sensitivity of a screening test is a major factor in determining its usefulness, because poor sensitivity of the first test used in a diagnostic algorithm could lead to false-negative results that would not be further evaluated.–. The differences might be caused by differences in study design, selection criteria, and the few positive samples in the previous studies  was false positive. Maximum cross-reactivity of IgG tests was seen in the subpanel collected in the post–dengue epidemic period in 2014–2015. The false-positivity rate was 40% (14/35) in the acute dengue subpanel for both commercial tests; the rate grew to 45.8% (11/24) for the Euroimmun Zika virus IgG test and 58.6% (17/29) for the Dia.Pro Zika virus IgG test in the subpanel of acute chikungunya virus samples collected right after the dengue epidemic period in 2014–2015. The IgG cross-reactions tended to decrease with time: 2 years after the end of the latest dengue epidemic in French Guiana, 5/42 (11.9%) samples were falsely positive by the Euroimmun IgG test and 9/42 (21.4%) samples were falsely positive with the Dia.Pro test. All samples except 2 with a false-positive result for Zika virus IgG were positive for dengue virus IgG by our in-house IgG antibody capture ELISA technique. The predictive positive value of both Zika virus IgG assays therefore largely depends on the epidemiologic situation of other flaviviruses, like dengue virus.. At the time of the study, only sporadic dengue cases were reported in Martinique, as the previous epidemic occurred in 2013–2014, >2 years earlier. In our assessment, the IgG S/C ratio value of false Zika virus IgG–positive samples varied from 1.1 to 6.7 (mean 3.9) for acute dengue samples to 1.2 to 3.7 (mean 2.0) for other non–Zika virus samples. These results underline the importance and potential efficacy of using selected panels to evaluate performance and, especially, the specificity of serologic assays. These results also indicate that when there is major cocirculation of dengue virus and Zika virus, the interpretation of serologic assays could be increasingly complex.A study performed in Martinique during March–June 2016 during the Zika virus epidemic showed a good correlation between a high Euroimmun Zika virus IgG ratio and a positive Zika virus seroneutralization result. Ratios >4 were associated with positive seroneutralization in >95% of cases, whereas ratios >5 were associated with seroneutralization in 100% of cases  of samples positive by the Euroimmun assays and 21% (16/75) for the Dia.Pro assays correspond to IgM negative/IgG positive samples, for which distinction between recent and past infections is not possible. Thus, a combined interpretation is not suitable for Zika infection diagnosis in the context of endemic–epidemic circulation.As reported previously by others, when IgM and IgG results were combined, sensitivity increased to 82% for Euroimmun and 87% for Dia.Pro assays, whereas specificity decreased to 69% for Euroimmun and 62% for Dia.Pro , high IgM ratios during the acute phase were associated with delayed and moderate increases in Zika virus IgG ratios, possibly reflecting a primary flavivirus or Zika virus infection; for other patients , an early increase in the IgG ratio was combined with a low or even negative Zika virus IgM ratio throughout follow-up, indicating secondary flavivirus infections. These 2 types of kinetics showed contrasted signal intensities, which are not observed with our in-house ELISA assays, in which whole Zika virus is used as an antigen (data not shown).We not only assessed the performance of Euroimmun and Dia.Pro Zika virus IgM and IgG tests but also evaluated the kinetics of the antibodies through 300 days after the onset of symptoms. A major concern in serologic diagnosis of Zika virus infection is determining the date of infection, due to the high proportion of asymptomatic forms. Data on the duration of IgM persistence after Zika virus infection are still limited, but our results indicate that this antibody could persist for at least several months, as described for other arboviruses (,A limitation of our study is that the Zika virus–positive samples were confirmed by RT-PCR and thus were all from symptomatic cases. If antibody levels are different in symptomatic and asymptomatic infections, as described for dengue, the performance and antibody kinetics observed in this study might be considerably different in samples from asymptomatic infections (This study highlights the complexity of interpreting serologic assays in areas where various arboviruses cocirculate and demonstrates the importance of evaluating serologic assays with serum specimens from persons living in endemic–epidemic areas and use of parallel testing antibodies to maximize the reliability of diagnosis. Further studies are also needed to identify specific biomarkers of each flavivirus infection for diagnosis after the acute phase of disease.


Gorham’s disease (GSD) is a rare osteolytic disease with unclear etiology, and no known prevention or effective treatment. Here we report a new surgical treatment for a case of GSD in September 2017.We report GSD in a 52-year-old woman. She had disappearance of her humeral head and a defect of the glenoid bone in her left shoulder joint, which were serious obstacles to joint function. We used an autologous iliac bone graft to repair the glenoid bone defect and a reverse total shoulder arthroplasty. After surgery, humeral osteolysis did not continue, and her shoulder function recovered well.This case suggests that autologous bone grafting can still be used to treat GSD despite it being an osteolytic disease. The successful treatment suggests that this method could be used for GSD in other bones. Gorham’s disease (GSD) was first reported by Jackson in 1838 then later in 1872 –7. It prWe report a case of GSD with extremely rapid progression of idiopathic osteolysis in the proximal humerus and glenoid in only 2 months . It was A 52-year-old woman presented with pain and active function loss in her left shoulder, and was admitted to our hospital in June 2017. Her shoulder problem had started 3 months earlier, and there was no history of trauma or fracture. At first the pain was intermittent and bearable, but then gradually increased. On examination, there were no positive signs except for localized pain. A radiologic examination on 3 April 2017 found no destruction of the shoulder (Fig. The patient is a healthy, active individual with no history of weight loss, anorexia, or fever during this period. General and systemic examination findings were within normal limits. Routine laboratory investigations were also normal, including levels of serum calcium, phosphate, alkaline phosphatase, high-sensitivity C-reactive protein, and erythrocyte sedimentation rate. An open biopsy of the lesion revealed that the bony tissue had been replaced by fibrous connective tissue, and small areas of bony trabeculae with occasional osteoclasts were visible Fig. . There wWe performed surgery involving glenoid cavity amplification with an autologous iliac bone graft and a reverse total shoulder arthroplasty. The grafts were from autologous iliac bone Fig. , Fig. 7.GSD is an unusual nonmalignant condition characterized by spontaneous and massive osteolysis. More than 300 cases of GSD have been described in the literature, but the underlying cause remains unknown , 9.Various strategies are used to treat GSD –11, but Using an autologous bone graft for the reconstruction of the shoulder glenoid was a critical step in the present case that provided a basis for the installation of the shoulder prosthesis. This method of operation has important clinical significance and provides an operative reference for the treatment of GSD or similar conditions. The cause of GSD, duration of osteolysis, and the identification of factors that prevent or slow down development of the disease require further study.


Viral infection involves a large number of protein-protein interactions (PPIs) between virus and its host. These interactions range from the initial binding of viral coat proteins to host membrane receptor to the hijacking the host transcription machinery by viral proteins. Therefore, identifying PPIs between virus and its host helps understand the mechanism of viral infections and design antiviral drugs. Many computational methods have been developed to predict PPIs, but most of them are intended for PPIs within a species rather than PPIs across different species such as PPIs between virus and host.In this study, we developed a prediction model of virus-host PPIs, which is applicable to new viruses and hosts. We tested the prediction model on independent datasets of virus-host PPIs, which were not used in training the model. Despite a low sequence similarity between proteins in training datasets and target proteins in test datasets, the prediction model showed a high performance comparable to the best performance of other methods for single virus-host PPIs.http://bclab.inha.ac.kr/VirusHostPPI.Our method will be particularly useful to find PPIs between host and new viruses for which little information is available. The program and support data are available at  There are many types of viruses that cause a wide variety of viral infections or viral diseases. For example, more than 11,000 deaths were reported in Africa during the outbreak of Ebola virus disease in 2014 and 2015 [H. sapiens and M. tuberculosis H37Rv. Cui et al. [Many computational methods have been developed to predict PPIs, but most of them are intended for PPIs within a same species rather than for PPIs across different species. Methods for predicting intra-species PPIs do not distinguish interactions between proteins of the same species from those of different species, and thus are not appropriate for predicting inter-species PPIs. Motivated by a recent increase in data of virus-host PPIs, a few computational methods have been developed to predict virus-host PPIs using machine learning methods. For instance, a homology-based method  and domai et al.  developei et al. . Amino ahttp://www.ebi.ac.uk/Tools/webservices/psicquic/view/main.xhtml). We extracted virus-host PPIs from four databases, APID, IntAct, Mentha and UniProt, which use same protein identifiers. The sequences of the proteins involved in any of the PPIs were obtained from the UniProt database (http://www.uniprot.org). As of December 2016, there are a total of 12,157 PPIs between 29 hosts and 332 viruses  from UniProt, and removed those with a sequence similarity higher than 80% to any positive data using CD-HIT-2D .Training (TR) and test (TS) sets for assessing the applicability to new viruses TR1: PPIs between human and any virus except H1N1 TR2: PPIs between human and any virus except Ebola virus TR3: PPIs between any host and any virus except H1N1 TR4: PPIs between any host and any virus except Ebola virus TS1: PPIs between human and H1N1 virus TS2: PPIs between human and Ebola virusTraining (TR) and test (TS) sets for assessing the applicability to new hosts TR5: PPIs between human and any virus TS5.1: PPIs between non-human animal and any virus TS5.2: PPIs between plant and any virus TS5.3: PPIs between bacteria and any virus TS5.4: PPIs between any non-human host and any virusWe constructed several datasets to examine the applicability of our prediction method to new viruses or hosts. The datasets are classified into two types: To examine the applicability of the prediction method to new viruses, we constructed a training dataset with 10,955 PPIs between human and any virus except H1N1 virus . The prediction method was later tested on a test dataset with 381 PPIs between human and H1N1 virus , which were not used in training the method. We constructed another training dataset TR2 with 11,341 PPIs between human and any virus except Ebola virus. The prediction method trained with TR2 was tested on a test dataset TS2, which contains 150 PPIs between human and Ebola virus  For training the SVM model, we tried to select as many virus proteins as possible which have known interactions with host proteins. (2) For testing the SVM method on new viruses, we selected H1N1 and Ebola virus because the viruses caused a large number of deaths recently but no specific vaccine or effective treatment is available yet.The applicability of the prediction method to new hosts was evaluated using training dataset TR5 and test datasets TS5.1–TS5.4. TR5 contains 11,491 PPIs between human and any virus. The prediction method trained with TR5 was tested on PPIs of non-human hosts with virus, which were not used in training the method. The test datasets include TS5.1 , TS5.2 (PPIs of plant with virus), TS5.3 (PPIs of bacteria with virus) and TS5.4 (PPIs of any non-human host with virus) .To assess the independence of the test data from the training data, we analyzed the sequence similarity between the training datasets and test datasets using EMBOSS Needle tool . As showFeature selection and representation are critical to the success of prediction of PPIs. In particular, one of the challenges in sequence-based prediction of virus-host PPIs is to represent two types of proteins of variable lengths into a feature vector of a fixed length. Several encoding schemes have been used to represent protein sequences for predicting PPIs. For instance, Shen et al.  clustereIn this study, we represent six different features of a protein sequence in a feature vector. For representation, we fist clustered twenty amino acids into seven groups, {AGV}, {C}, {FILP}, {MSTY}, {HNQW}, {DE}, and {KR} based on the dipoles and volumes of the side chains of amino acids. The classification of amino acids is the same as that of Shen et al.  and othei-th amino acid triplet is defined by Eq. fi, avgF, and maxF denote the frequency of the i-th amino acid triplet, the average, and the maximum frequency of amino acid triplets in the protein sequence, respectively. For each pair of host and virus proteins, we represent the relative frequency of amino acid triplets (RFAT) as a feature vector with 686 elements (343 for a host protein and 343 for a virus protein). The RFAT of the fhi is the frequency of the i-th amino acid triplet in the host protein of the host-virus pair, and fvi is the frequency of the i-th amino acid triplet in the virus protein of the same host-virus pair. avgFD and maxFD denote the average and the maximum frequency difference of amino acid triplets in a host-virus pair, respectively. Another feature is the frequency difference of amino acid triplets (FDAT) between virus and host proteins, which is defined by Eq. i is the frequency of the i-th amino acid present in a host-virus pair divided by the maximum frequency of an amino acid in the pair. We also represent amino acid composition (AC) in each pair of host and virus proteins Eq. . AC i isThe above three features, RFAT, FDAT and AC were developed in our previous study for inter-species PPIs of a single type . HoweverAs additional features, we used composition, transition and distribution of amino acid groups . Composiγ were found by running the grid search of LIBSVM on training datasets. Unless specified otherwise, the results shown in this paper were obtained with C = 32, γ=0.03125. The SVM models take a pair of virus and host protein sequences as input. As output, the SVM models classify whether or not the virus protein interacts with the host protein. The SVM models and supporting data are available at http://bclab.inha.ac.kr/VirusHostPPI.We built several support vector machine (SVM) models using LIBSVM  to prediThe performance of the prediction models were evaluated by several measures: sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), Matthews correlation coefficient (MCC) and the area under the ROC curve (AUC), which are defined as follows In Eqs. http://bclab.inha.ac.kr/VirusHostPPI.We performed 10-fold cross validation of the SVM model with several datasets which contain different ratios of positive to negative data . Due to the randomness of selecting negative data, we constructed three different datasets for each ratio of positive to negative data. Table We also examined the contribution of features to the prediction performance of our SVM model. Table http://bclab.inha.ac.kr/VirusHostPPI.Table In order to examine the applicability of our prediction model to new hosts, we tested it on PPIs of viruses with new hosts, which were not used in training the model. As described earlier, the model trained with human-virus PPIs was tested on PPIs of viruses with non-human . As shown earlier in Table The difference seems ascribed to the difference in the number of target proteins in test datasets and to the difference in the number of partner proteins of the target proteins, which are shared by training and test datasets. Test datasets TS1 and TS2 have 381 interactions of 11 H1N1 virus proteins and 150 interactions of 3 Ebola virus proteins with human proteins, respectively /2=266 PPIs and a test dataset for new hosts has (488+17+143)/3=216 PPIs. Thus, the difference in the average number of PPIs of the two types of test datasets is not large. However, there is a big difference in the number of target proteins in the test datasets and in the number of proteins common to training and test datasets. The average number of virus proteins in a test dataset for new viruses is only (11+3)/2=7, whereas the average number of host proteins in the test datasets for new hosts is (368+13+106)/3=162. Thus, virus-host PPIs in the test datasets for new viruses share many host proteins in the training datasets  even though no virus proteins are shared by the test and the training datasets. In contrast, virus-host PPIs in the test datasets for new hosts share a much smaller number of virus proteins in the training datasets .This is a known problem with pair-input methods, which was first reported by Park and Marcotte , but nothttp://bclab.inha.ac.kr/VirusHostPPI.We compared our method with two other methods, DeNovo  and Barmhttp://bclab.inha.ac.kr/VirusHostPPI.In Barman’s study  three maMost computational methods of predicting PPIs are intended for interactions within a species rather than for interactions across different species such as interactions between virus and host cell proteins. A small number of computational methods which were recently developed for predicting PPIs between virus and host are limited to interactions of single virus or single host, and therefore a separate prediction model is required to predict PPIs of new viruses or hosts. However, proteins of new viruses or hosts often exhibit quite a low sequence similarity to proteins of known viruses or hosts, and little information is available for new viruses or hosts.In this study, we developed a prediction model of virus-host PPIs, which is applicable to new viruses and hosts. We tested the prediction model on independent datasets of virus-host PPIs, which were not used in training the model and have a very low sequence similarity to any protein in training datasets of the model. Despite a low sequence similarity between proteins in training datasets and target proteins in test datasets, the prediction model showed a high performance comparable to the best performance of other methods for single virus-host PPIs. Our prediction model will be useful in finding potential PPIs of new viruses with new hosts, for which little information is known.


Alpha-, Beta-, and Gammapapillomavirus, with a strong phylogeny-tropism association as observed in HPVs. Using a Bayesian Markov Chain Monte Carlo framework, we demonstrated ancient intra-host divergence of primate PVs in which multiple ancestors had split and adapted to specific host ecosystems at least 41 million years ago, prior to the speciation events of primate host species. Following subsequent divergence and niche adaptation, distinct but phylogenetically related PV types were transmitted to similar host ecosystems by closely related host animals when host speciation occurred, which may explain in part the origin of carcinogenicity of HPV type 16 (HPV16) and Macaca fascicularis PV type 3 (MfPV3) that evolved from a most recent common ancestor containing the determinants for cervicovaginal colonization and cervical cancer. The findings identifying evolutionary and biological relatedness between human and non-human primate PVs lay a genetic foundation for research on parasite-host interactions and carcinogenic outcomes, which will prove useful in further study of viral pathogenesis and host specificity.Given high genetic diversity of papillomaviruses (PV) and complex scenario of virus-host interaction, the genetic basis underlying the mechanisms of HPV carcinogenicity is not well understood. In an effort to evaluate the origin and evolution of PV pathogenicity, we collected paired oral, perianal, and genital swabs from a wild macaque population. Of the 117 surveyed macaques, 88 (75.2%) were positive for PV DNA in one or more sites, mostly common from genital swabs, followed by oral and perianal sites. All putative macaque PV types phylogenetically clustered into the genera Papilloamviridae in general and the primate papillomaviruses in specific in relevant to virus-host interactions should provide important implications for viral pathogenesis and disease prevention.To better understand the origin and evolution of PV carcinogenicity associated with cervical cancer, we applied a combination of phylogenetic and bioinformatic analyses to investigate the genetic diversity of macaque papillomaviruses, and estimate divergence times of human and non-human primate PVs. The majority of both human and non-human primate PVs cluster into α-, β-, and γ-PVs, sharing similar evolutionary histories and biological properties to each other. The strong phylogeny-tropism association of primate PVs indicates an important role of niche adaptation and virus-host codivergence shaping the diversity of viral genomics, host specificity, immune exposure, and pathogenic property. Understanding the evolution of the family  Papillomaviridae comprised a large family of double-stranded circular DNA viruses of about 8,000 nucleotides in size. Papillomavirus (PV) genome contains three regions, including an upstream regulatory region (URR), an early region of open reading frames (ORFs)  encoding functional proteins, and a late region of ORFs encoding structural proteins (L1 and L2) (Alphapapillomavirus (α-PV), Betapapillomavirus (β-PV), and Gammapapillomavirus (γ-PV) ( and L2) . PVs inf and L2) . To date and L2) . The cli and L2) . For exa and L2) . The majs (γ-PV) . All gens (γ-PV) .Alphapapillomavirus 12 (α12) isolated from the cervicovaginal region of macaques have been associated with cervical cancers  inhabiting a protected nature reserve in Hong Kong. Genetic diversity and evolutionary relationship among macaque PVs as well as among HPVs were characterized, with divergence time estimated using Bayesian molecular clock model to trace the divergence of primate PVs within niche-specific clades. The findings identifying biological and evolutional relatedness between human and non-human primate PVs lay a genetic foundation for research on parasite-host interactions and carcinogenic outcomes, which will prove useful in further study of viral pathogenesis and host specificity.Papillomaviruses are ancient DNA viruses that have co-evolved with their hosts over millions of years, resulting in a high level of diversity at host specificity, tissue tropism, viral genetics, prevalence and pathogenesis . The impM. mulatta), including 84 females and 33 males, were collected from Kam Shan Country Park, Hong Kong, China  sites of 117 non-captive wild rhesus macaques  with 200 μl STM suspension and eluted in 200 μl elution buffer (pH 8.0) for PCR-based papillomavirus genotyping. Two recently developed next-generation sequencing (NGS) amplicon assays targeting the consensus regions of PV L1 open reading frame of the full spectrum of primate papillomaviruses including α-, β-, and γ-PVs were used . SuccessFollowing the demultiplexing, the paired short reads passing the quality filter (≥Q20 and ≥50-bp) were merged into single reads using FLASh v1.2.11  and blasM. mulatta papillomaviruses (MmPV2 to 7) were characterized using virus-enriched shotgun sequencing, as previously reported  at 49 (95% CI of 41 – 58) million years ago (mya) matching the divergence between Old World and New World monkey ancestors (Pan paniscus PV 1 (PpPV1) at 7 (6 – 8) mya matching the split between hominin and chimpanzee ancestors (Alphapapillomavirus tree) and >2000 (HPV variant trees of each type), indicating that all Bayesian chains were well sampled and have converged. The divergence times of non-human primate PVs partially sequenced by MY and FAP were estimated using the L1 substitution rate and the evolution models as mentioned above.We used a Bayesian MCMC method implemented by BEAST v2.4.5  and the ncestors , (2) thencestors , and (3)ncestors . For β- M. mulatta). A total of 88 macaques (75.2%) had detectable PV DNA in one or more body sites, with similar infection rate between female  and male animals  (p = 0.8123). As expected, genital swabs had the highest infection rate , followed by oral , and perianal  sites  swabs from 117 wild rhesus macaques (7) sites .Cross body site infection was common in the surveyed macaques . At leasn = 142), 77 (54.2%) were infected with at least one α-PV type, 6 (6.2%) with a β-PV type, and 77 (54.2%) with a γ-PV type (p < 0.001). In consistence with the tropism of body site between macaque PV genera, all 32 putative α-PV types were able to be detected in genital samples while only 2 types in oral swabs. We found 25 and 19 γ-PV types in oral and genital swabs, respectively, with 8 types in both sites (p < 0.001)  . The ana< 0.001) .n = 19), OTUfA3 (n = 17), MfPV5 (n = 14), MfPV11 (n = 14) and MmPV6 (n = 11), commonly found in genital and/or perianal sites were absent in oral cavity. OTUfA21 (n = 11), OTUfA5 (n = 9) and OTUfA34 (n = 8) phylogenetically clustered into the γ-PV genus, however, were more common in oral samples, although they might infect perianal and/or genital sites occasionally  . MfPV7, sionally .Multiple-PV type infection in a same sample was common. 54.2% of PV-containing samples (77/142) were infected with at least two or more types, mostly common in genital swabs , followed by perianal , and oral  samples.n = 12), followed by oral-perianal pairs (n = 6), and oral-genital pairs (n = 2). OTUmA12 and OTUmA28, two α-PV types, were mostly detected in 4 and 3 perianal-genital pairs, respectively. It is worth to note that infection of a same PV type crossing body sites could be from different sources but the close anatomic locations between genital and perianal areas likely facilitated the viral transmission from one body site to another more easily.Eighteen macaque animals shared same PV type(s) between body sites. Identical types were found more commonly in perianal-genital pairs  isolated from the surveyed animals have been previously characterized by the authors’ group. As shown in The complete genomes of six macaque papillomaviruses  of 145 PV types representing 136 species from unique host species , Gorilla gorilla (GAA2 to 4), Ateles fusciceps (SMAA1), and Macaca fascicularis (MfAA1 to 26) phylogenetically located within β- and γ-PV genera  and α-PVs split from a MRCA approximately 41 mya   , coincid 44 mya) –S6. Chim 44 mya) .The tree topologies and divergence times support a model of ancient intra-host divergence of primate PVs in which multiple viral ancestors had split and adapted to specific host ecosystems  within an ancestral host animal lineage , prior to the speciation events of host species . FollowiIn this study, we surveyed a broad spectrum of α-, β-, and γ-PV infection from paired oral, perianal and genital swab samples collected from 117 non-captive healthy rhesus macaques using Next-Gen sequencing amplicon assays. Overall, high infection prevalence and genetic diversity of macaque PVs were detected. All identified macaque PV sequences clustered into the genera α-, β-, and γ-PV, with similar phylogenetic topologies as that of HPVs. Significant tissue tropism of macaque PVs were observed; the majority of PV types isolated from genital regions phylogenetically clustered into the genus α-PV, whereas the oral cavity harbored more γ-PVs. In consideration of low mutation rate of PV evolution and rare interspecies transmission, the close relationship between macaque PVs and HPVs isolated from similar anatomic sites  and/or sharing similar pathogenic properties  suggests a model of ancient divergence of primate PV ancestors adapting to specific host ecosystems prior to the host speciation. This phylogenetic topology was supported by a number of previously reported PV sequences from a wide range of apes and monkeys exclusively clustering into the genus α-, β-, and γ-PVs, in accordance to host anatomic sites where these viruses were originally isolated . AlthougUsing Bayesian molecular clock models, we estimated ancient intra-host divergence of primate PV ancestors within an ancestral host animal lineage occurred at least 41 million years ago, the era prior to the speciation between New World and Old World monkeys . FollowiVirus-host codivergence was generally responsible for the large evolutionary distances between PVs of hosts belonging to different order and/or class . However, this term does not explain the distribution of primate PVs infecting a single host species into different phylogenetic clades. Ancient intra-host viral divergence undergoing slow genomic mutations in respond to selection pressure by host immune responses and viral genetic drift may have properties beneficial for virus strategies, such as improved infectivity, regulation and pathogenesis within specific host niches. For example, all α-PVs but not β-/γ-PVs contain a short region in length of 300 – 500 bp between E2 and L2 genes, including an E5 ORF sharing high homology between genital high-risk HPVs and MfPVs . The E5 Ovis aries PVs, Camelus dromedaries PVs, and Cervus elaphus PVs, suggesting an ancient intra-host viral divergence history occurred in the ancestors of artiodactylous PVs. Following virus-host codivergence within specific host ecosystems, artiodactylous PVs infecting a single host species might form distinct clade that was phylogenetically related to viruses from other closely related host species. We found the similar evolutionary pattern in canine and feline PVs that distributed into the genera Chi-, Lambda- and Tau-PV, although these viruses were mainly isolated from the oral cavity and their pathogenesis warrants further investigation.Diversification of PV genomes independent from host speciation can occur in non-primate animals harboring many PV types belonging to different genera, such as bovine, canine and feline PVs. At least 27 distinct BPV types have been characterized and were phylogenetically assigned into five genera including Delta- , Xi- , Episilon- , Dyoxi- , and Dyokappa-PV  . Bovine It is unclear whether viral transmission occurred between human and non-human primate ancestors. Although there is no report of any HPV DNA being detected in an animal or of any animal PV DNA in a human, the extreme species restriction is probably exclusively operative in primate PVs. Given close cutaneous or mucosal contacts between hominid and macaque ancestors in the early stage of host speciation, host interbreeding and switching via horizontal transmission may be responsible in part for a recent split of MmPV5 and CgPV2 from their closest HPV relatives, as estimated around 13 – 14 mya.Non-human primates share various behavioral, physiological and genetic similarities with humans, making monkey PVs as ideal animal models to understand the origin and evolution of HPV carcinogenicity. A number of macaque PV types within the species α12 isolated from mucosal lesions of rhesus and cynomolgus macaques have been associated with cervical neoplasia . The evoOne of the limitations of this study is the short of cytological and/or histological information to define the potential pathogenicity of macaque PVs in the surveyed macaque population. The most predominant genital macaque PV was MfPV7 found in 19 animals (16%), a member type of the species α12 containing MfPV3 associated with cervical cancer. However, the oncogenic potential of MfPV7 and/or other closely related types within the species α12 is not clearly known. Although the L1-based amplicon sequencing assays were able to detect a broad spectrum of primate PVs, the implementation of whole genome metagenomic sequencing may provide a deep genomic coverage to further explore viral mutation and diversity . We onlyIn conclusion, this study establishes a comprehensive theoretical framework on the evolution of primate PVs, suggesting an ancient intrahost viral divergence model upon niche adaptation followed by virus-host codivergence. Our model provides a genetic foundation to better understand the origin and evolution of HPV pathogenesis and carcinogenicity associated with cervical cancer, and the underlying contribution of host niche adaptation to viral fitness. The oncogenic outcomes of human and non-human primate PVs imply an evolutionary convergence of virus-host interaction involving complex interaction between host and pathogens in association with different genotypes, phylotypes and phenotypes.MG837557, MG837558, MG837559, MH745747, MH745748, and MH745749.Accession numbers for the sequences determined in this study are available in GenBank under the accession numbers The animal use protocol has been approved by the Agricultural and Fishery Department and presented to the Animal Welfare Advisory Group (AWAG). All experiments were performed in accordance with the relevant guidelines and regulations.All authors were involved in the conception and design of the study, and approved the final manuscript. ZC, PC, and RB contributed to the study design and supervision. TL, PW, and WH processed the samples and performed the sequencing. ZC and TL contributed to the data analysis and wrote the original draft of the manuscript. PC and RB reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dicksonia tree ferns world-wide, and none are well documented. In this study we examine the relationship of a newly-discovered Dicksonia tree fern from Whirinaki, New Zealand, which does not fit the current taxonomy of the three species currently recognized in New Zealand. Our microsatellite genotyping and ddRAD-seq data indicate these plants are F1 hybrids that have formed multiple times between D. fibrosa and D. lanata subsp. lanata. The Whirinaki plants have intermediate morphology between D. fibrosa and D. lanata subsp. lanata and their malformed spores are consistent with a hybrid origin. The Whirinaki plants–Dicksonia fibrosa × D. lanata subsp. lanata–are an example of hybridization between distantly related fern lineages, with the two parent species estimated to have diverged 55–25 mya. Our chloroplast sequencing indicates asymmetric chloroplast inheritance in the Whirinaki morphology with D. lanata subsp. lanata always contributing the chloroplast genome.Hybridization is common in many ferns and has been a significant factor in fern evolution and speciation. However, hybrids are rare between the approximately 30 species of  Cyathea Sm. (Cyatheaceae) and Dicksonia L’Hér (Dicksoniceae) [Tree ferns  are often a locally dominant feature of wet temperate Southern Hemisphere forests. Within New Zealand’s forests tree ferns often represent a significant proportion of the forest community based on the number of individuals, biomass and basal area . Two genoniceae) , 3. The oniceae) .Dicksonia contains around 30 species that are found in Central and South America, Southeast Asia, eastern Australia, New Zealand and the Pacific [Dicksonia species are recognized in New Zealand; all are endemic [Dicksonia fibrosa Colenso (whekī-ponga) and D. squarrosa (G.Forst) Sw. (whekī) have trunked rhizomes up to 6m tall and 7m tall, respectively. Both are found throughout the North Island in lowland to montane forest and are largely confined to lowland and coastal areas in the South Island [D. lanata Colenso ex Hook.  [Dicksonia lanata subsp. hispida (Colenso) Perrie & Brownsey has a trunked rhizome to 2m tall and is found in Northland and Auckland regions in the North Island. Dicksonia lanata subsp. lanata has a prostrate rhizome and is found mostly in montane habitats south of Auckland in the North Island and in coastal and lowland forest in north-western South Island. Pacific ,4. Three endemic . Dicksonh Island . Two alltūōkura) . DicksonDicksonia. Dicksonia lanata and D. squarrosa grouped in a clade with species from the Pacific Islands, whereas D. fibrosa formed another clade with species from Australia, Timor and South America. Molecular dating estimated these clades diverged 55–25 mya [A global chloroplast phylogeny of Dicksoniaceae  placed t5–25 mya .Dicksonia plants were discovered whose morphology did not fit with any of the existing species descriptions. They appeared to have fronds similar to D. lanata but had a stout trunk resembling that of D. fibrosa. Both D. lanata subsp. lanata and D. fibrosa grow in the immediate vicinity. Our subsequent searches located around 80 so-called Whirinaki plants over 3 km that could not be placed within the existing taxonomy. They were growing in sympatry with D. lanata subsp. lanata, D. squarrosa and D. fibrosa.During a survey in 2015 of a remote area of Whirinaki Forest, eastern North Island, New Zealand, Using morphological and genetic  data we examine whether the Whirinaki morphology is:D. antarctica Labill., which is sometimes confused with D. fibrosa [the Australian species  fibrosa , includian undescribed species;D. fibrosa and D. lanata subsp. lanata that has formed once and has spread vegetatively (both D. squarrosa and D. lanata produce underground stolons from their rhizomes [a homoploid hybrid between rhizomes );D. fibrosa and D. lanata subsp. lanata;a recurrent homoploid hybrid between D. fibrosa and D. lanata subsp. lanata.a fertile allopolyploid between D. antarctica (hypothesis 1) or an undescribed species (hypothesis 2) then, given its restricted distribution, it would likely be considered threatened under the New Zealand Threat Classification System [Dicksonia, with only unconfirmed reports of wild Dicksonia hybrids [D. arborescens L’Hér. from St Helena and D. antarctica [If the Whirinaki morphology is either n System . Hypothe hybrids ,8 and a tarctica .Dicksonia specimens were collected for this study .Dicksonia samples. The following loci and primers were trialled: rps4-trnS with T1 [trnG-trnR with trnG1F and trnR22R [psbA-trnH with psbA [matK with matKF_Dicksonia and matK_1R, rpl16 with rpL16F_ferns and rpL16R_ferns and trnL-trnF with uv2 and uv4 [Six chloroplast loci were tested for variation in a subset of the New Zealand  with T1  and R1 [ with T1 , trnG-tr trnR22R , psbA-trith psbA  and trnHith psbA , matK wiPCR amplifications were performed in 12 μl reactions with 1× Mytaq mix , 5 ρmol of each primer and 1 M betaine. PCR thermocycling conditions followed . PCR protrnL-trnF region demonstrated the most variation and was sequenced for a total of 33 Dicksonia specimens, as described above. The trnL-trnF sequences contained only two unambiguous indels and these were aligned by eye. A median-joining network [trnL-trnF sequences was produced using PopArt [Sequence files were edited with Sequencher version 5.2 . The  network  for the g PopArt  with indDicksonia sellowiana Hook. [Dicksonia. Primers to a further ten microsatellite loci developed from sequences obtained from a preliminary ddRAD-seq run on four Dicksonia  were also screened. Primers were designed for these extra loci (Eleven microsatellite loci isolated from na Hook.  were testra loci  using PrTGTAAAACGACGGCCAGT) was added to the 5’ end of the forward primer of each locus. Loci were amplified individually in 10 μL PCR reactions that contained 1 μL of diluted template DNA, 0.2 μM forward primer, 0.8 μM reverse primer, 0.8 μM M13 primer  and 1× MyTaq mix (Bioline). PCR thermocycling conditions were an initial denaturation of 94°C for 5 min followed by 35 cycles of 94°C for 20 s, 52°C for 20 s, and 72°C for 20 s and a final extension at 72°C for 15 min. For loci that amplified, genotyping was performed on an ABI 3730 DNA sequencer at the Massey Genome Service . Alleles were sized using the internal size standard GeneScan 500 LIZ (Applied Biosystems) and scored using Geneious version 10.2.6 .An M13 tag (dx.doi.org/10.17504/protocols.io.zgyf3xw). The library was sequenced across a third of a lane using the Illumina HiSeq 2500 to generate 2 × 125 bp reads.Double-digest restriction site associated sequencing (ddRAD-Seq) libraries  were preRaw paired-end reads were demultiplexed with ipyrad v0.7.28 . All FasD. antarctica specimen produced a high number of raw reads but these were assembled into few loci compared with the other samples so this sample was excluded from the final dataset used for the analyses described below.A preliminary NeighborNet network (see below) was constructed from this initial assembly to confirm that the eight technical replicates clustered with their duplicate. The two duplicated sets of reads were then pooled and assembled as described above. The To examine whether there was conflicting phylogenetic signal in the ddRAD data, which may indicate a history of hybridization, a network was constructed with the NeighborNet algorithm , implemeK) was determined by calculating the ΔK statistic [K and the results visualized graphically with STRUCTURE PLOT [We used STRUCTURE v2.3.4 , 27 to etatistic  in STRUCtatistic . We alsotatistic . CLUMPP tatistic  was usedURE PLOT .D. fibrosa and D. lanata, based on published descriptions [D. fibrosa and D. lanata subsp. lanata.The morphology of the Whirinaki plants was compared to riptions , 5. The trnL-trnF alignment was 972 bp in length and contained 17 substitutions and two unambiguous indel events. The relationships between the trnL-trnF sequences, with the indels coded, are shown in the median-joining network . The haplotypes detected in D. antarctica and D. fibrosa differed from the D. lanata haplotypes by a 6 bp insertion plus at least 12 substitutions. The D. antarctica trnL-trnF sequence was identical to one of the D. fibrosa sequences with the other two D. fibrosa sharing a haplotype that differed by one substitution.The  network . The mosDicksonia species (D. fibrosa (n = 6) nor D. antarctica (n = 1) showed any variation at the three loci. Dicksonia lanata (n = 14) only varied at the DicMic01 locus, with six alleles detected. A number of D. lanata subsp. lanata samples failed to amplify at this locus suggesting that null alleles may be present in this taxon. Dicksonia squarrosa (n = 4) was invariant at the DicMic109 locus, had two alleles at the DicMic104 locus and failed to amplify at the DicMic01 locus. At locus DicMic109 all the individuals of the Whirinaki morphology (n = 14) were heterozygous for the 131 bp and 125 bp alleles. The 131 bp allele was the only allele detected at this locus in D. fibrosa and D. antarctica and the 125 bp allele was the only allele found in D. lanata and D. squarrosa. At locus DicMic104 the Whirinaki variant was fixed for a 118 bp allele, with this allele also fixed in D. lanata and also observed in D. squarrosa. At this locus D. fibrosa and D. antarctica only exhibited alleles 114 bp and 113 bp in size, respectively. At the third microsatellite locus, DicMic01, the Whirinaki variant displayed five alleles, all of which were also found in D. lanata. Every Whirinaki individual had at least one allele 316 bp in length and this allele was fixed in D. fibrosa and D. antarctica. Dicksonia squarrosa failed to amplify at this locus. Five different genotypes were found at this locus in the Whirinaki morphology.Three microsatellite loci amplified and were variable within and/or between New Zealand  species . NeitherDicksonia ddRAD libraries for all 38 individuals plus 8 duplicates resulted in 303.48M reads after initial quality filtering. Excluding the four low coverage samples, 668K to 7.9M reads per individual were obtained (mean±SD = 3.43M±2.10M reads per sample) (Illumina sequencing of  sample) .D. antarctica sample is shown in D. antarctica sample is closely related to D. fibrosa. The duplicates were combined for subsequent analyses and the D. antarctica sample was excluded owing to low coverage.The preliminary NeighborNet including the duplicate samples and the D. fibrosa and D. squarrosa. In contrast both D. lanata subsp. lanata and D. lanata subsp. hispida exhibited greater variation between individuals and while there was not strong evidence for the separation of these two subspecies, they were somewhat separated in the network.The final dataset comprised 1244 loci across 33 specimens. The NeighborNet analysis of this dataset clustered each species together with strong support . There wD. fibrosa and D. lanata.Individuals of the Whirinaki morphology grouped together in an intermediate position between ΔK indicated that the optimal K was 2 , its wider fronds and the obtuse apices of its lamina segments  because they differ in their chloroplast sequences and do not cluster together in the NeighborNet of the ddRAD-Seq data. Instead our analyses indicate that the Whirinaki morphology plants are hybrids between D. fibrosa and D. lanata. The NeighborNet of the ddRAD-Seq data placed samples of the Whirinaki morphology at an intermediate position between these two species and the STRUCTURE analyses assigned them approximately equally to the clusters containing D. fibrosa and D. lanata. Two of the microsatellite loci  also provided support that the Whirinaki morphology plants are hybrids between D. fibrosa and D. lanata. Only the third microsatellite locus (DicMic104) did not show a pattern consistent with hybridization because D. lanata and the Whirinaki morphology were fixed for the same single allele and D. fibrosa was fixed for a different allele. None of the Dicksonia individuals genotyped were heterozygous at this locus and it is possible that this locus derives from a uniparentally-inherited organelle.Our genetic results indicate that the plants with the Whirinaki morphology are not Dicksonia lanata are only weakly separated in the ddRAD-Seq analysis, and not at all in the STRUCTURE analysis. This may reflect the make-up of the sample set, particularly the inclusion of hybrids. The AFLP analysis of a larger sample set by Lewis cited in [D. lanata subsp. lanata occurs in close proximity, and morphologically the Whirinaki plants show characters of both D. lanata subsp. lanata (cf. subsp. hispida) and D. fibrosa . Furthermore no more than two alleles were detected per locus at the microsatellite loci, as would be expected for diploid hybrids. The high level of variation observed in the Whirinaki morphology in the ddRAD NeighborNet and at microsatellite locus DicMic04 indicates that this form has not arisen from a single hybridization event (hypothesis 3) but from repeated crosses between genetically differentiated parent individuals (hypothesis 4). The three chloroplast haplotypes found in the Whirinaki morphology also supports recurrent hybridization.The abnormal spores observed in the Whirinaki morphology indicate that these plants are infertile F1 hybrids and the genetic analyses largely also support this conclusion. Particularly informative was the DicMic109 microsatellite locus where all individuals of the Whirinaki morphology were heterozygous for the same two alleles, one of which was fixed in D. lanata, indicating that this species was the chloroplast donor. This 15:0 ratio is statistically different from the 1:1 expectation if D. lanata and D. fibrosa were equal contributors of chloroplasts to the hybrids . Asymmetric hybridization has been noted previously in ferns [The chloroplast haplotypes sequenced from the 15 hybrids were identical or very similar to the haplotypes detected from in ferns –36 but tin ferns . Factorsin ferns .Dicksonia fibrosa × D. lanata subsp. lanata plants are the first wild hybrids confirmed by genetics in the genus but they were locally common . Despite being such a large plant, it is possible that Dicksonia fibrosa × D. lanata subsp. lanata plants occur elsewhere but have been overlooked. However, although the distributions of D. fibrosa and D. lanata broadly overlap over a wide area [D. fibrosa and D. lanata (either subspecies) are recorded from 1006 and 791 plots, respectively, but only occur together in 15 of these plots. Differences in ecological preferences may account for this lack of local overlap, with D. lanata subsp. lanata preferring hillsides and D. fibrosa more likely to be found on gully floors. The western Whirinaki area may be unusual in that the two species occur together over a large area, possibly because it is flatter, colder and drier forest than many other sites [D. fibrosa and D. lanata are sympatric may reveal further hybrids and provide insight into the environmental conditions that lead to hybridization between these two species.Our finding that the Whirinaki morphology comprises recurrent F1 hybrids was surprising for several reasons. Firstly, these ide area , the Newide area , D. fibrer sites . ExaminiDicksonia from chloroplast clades that have been estimated to have diverged 55–25 mya [Cystopteris and Gymnocarpium  and Asp (45 mya ). Dickso0 mya  anDicksonia fibrosa × D. lanata subsp. lanata exhibited remarkably conserved morphologies given the considerable differences between the morphologies of the parents.Lastly, although recurrently formed hybrids, Asplenium [Dicksonia, hybridize only rarely, leading to limited opportunities for polyploidy. Understanding the situations where the barriers preventing hybridization break down in rarely hybridizing genera such as Dicksonia may provide insight into the dynamics of fern evolution and a contrast to genera with frequent reticulate evolution.Polyploidy is thought to be a significant process in the generation of fern diversity with an estimated 31% of fern speciation events accompanied by an increase in ploidy . Howeversplenium , 42, othDicksonia fibrosa × D. lanata subsp. lanata.We are not making a nothospecies name because the New Zealand practice for naming fern hybrids is to use a hybrid binomial i.e., S1 FigDuplicates are indicated by ‘dup’ after sequence name and were combined for subsequent analyses.(TIF)Click here for additional data file.S2 FigAccording to the ΔK, K = 2 represents the optimal structure partition in our dataset.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(TXT)Click here for additional data file.


Parkinson's disease is a progressive, disabling neurodegenerative disorder associated with significant economic burden for patients and caregivers. The objective of this study was to compare the direct and indirect economic burden of Parkinson's patients’ caregivers with demographically matched controls in the United States, in the 5 years after first diagnosis of Parkinson's disease.Policyholders (18‐64 years old) linked to a Parkinson's disease patient  from January 1, 1998 to March 31, 2014, were selected from a private‐insurer claims database and categorized as Parkinson's caregivers. Eligible Parkinson's caregivers were matched 1:5 to policyholders with a non‐Parkinson's dependent (controls). Multivariable regression adjusted for baseline characteristics estimated direct costs  and indirect costs . Income progression was also compared between cohorts.P < 0.01) and higher prescription costs  and total out‐of‐pocket costs ($1259‐1585 vs $902‐$1192) in years 1‐5 . Parkinson's caregivers had significantly higher adjusted indirect costs in years 1‐3  and higher cumulative income loss over 5 years .A total of 1211 eligible Parkinson's caregivers  were matched to 6055 controls. In adjusted analyses, Parkinson's caregivers incurred significantly higher year 1 total all‐cause insurer costs ($8999 vs $7117) and medical costs ($7081 vs $5568) (both Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.Parkinson's caregivers exhibited higher direct and indirect costs and greater income loss compared with matched controls. © 2018 International Parkinson and Movement Disorder Society © 2018 The Authors.  Parkinson's disease (PD) is a chronic, progressive neurodegenerative disease with physical symptoms manifesting as tremor at rest, rigidity, bradykinesia, and postural instability.PD may present a substantial burden to the caregivers of PD patients. PD patients may need help with many aspects of life, including personal safety, mobility, transportation, medication compliance, daily activities, and social involvements.There is a lack of literature comprehensively assessing the burden of PD on PD caregivers. Publications on PD caregivers’ burden have largely focused on the noneconomic aspects of burden or the impact on quality of life (QoL). In addition, to date, no studies have assessed the lost earnings of PD caregivers over time. The indirect cost of caregiving has been primarily estimated as the monetary value of work loss associated with caregiving rather than actual earnings lost.Data for this study, spanning January 1, 1998, to March 31, 2014, were obtained from OptumHealth Care Solutions, Inc., a deidentified private‐insurer claims database. This database contains administrative claims for >18 million privately insured individuals covered by 84 self‐insured Fortune 500 companies with locations across the United States. Medical and drug claims as well as eligibility data are available for all beneficiaries , whereas measures of work loss (ie short‐ and long‐term disability claims) were available for primary policyholders  in 43 companies. Employer‐reported income data were available for a subgroup of employees. Data were anonymized and compliant with the requirements of the Health Insurance Portability and Accountability Act; thus, no institutional board review was required. This study was conducted in accordance with the Declaration of Helsinki.The study consisted of 2 cohorts: (1) PD caregivers and (2) matched controls who were not PD caregivers. For the PD caregiver cohort, the index date was the date of the PD patient's first PD diagnosis. For the control cohort, the index date was randomly assigned. PD patients were identified as having ≥2 diagnosis for PD . Subjects were eligible for inclusion in the PD caregiver cohort if they were primary policyholders linked to a nonprimary policyholder PD patient, had continuous eligibility of at least 6 months before (baseline period) and at least 12 months following the index date, and were aged 18‐64 years during the baseline and the study periods (see definitions below). Controls were selected from primary policyholders if they did not have a family member with PD, had continuous eligibility of at least 6 months before (baseline period) and at least 12 months following the index date, and were aged 18‐64 during the baseline and study periods. Cases were matched 1:5 to controls who were randomly selected from the control pool that satisfied the requirement described above.For the direct cost analysis, all PD caregivers and controls meeting the above criteria were included. For the indirect cost analysis, PD caregivers and their controls were also required to be employed and have work loss data during the baseline and 12 months post–index date. For the income progression analysis, PD caregivers and their controls were also required to be employed and have income data during the calendar year of the index date  and for at least 1 calendar year of follow‐up. For each of the analysis, controls were randomly matched to PD caregivers in a 5:1 ratio exactly on sex, age, region, and index year.In addition, PD patients linked to the PD caregivers and the oldest dependent of each of the matched control patients were selected if they had at least 6 months of continuous eligibility prior to the index date (baseline period).The study period was defined as the period of time following the index date until the earlier of the following: (1) the patient turned 65 years old or (2) the patient lost continuous eligibility. Each patient's study period was at least 1 year (per sample selection) and was capped at 5 years. For the indirect cost and income progression analyses, patients were also required to have continuous employment during the study period.Information collected for the PD caregivers and matched controls during the baseline period included demographic characteristics , index year, insurance type, the Charlson Comorbidity Index (CCI), and caregiving‐related comorbidities identified in previous studies.Direct insurer and out‐of‐pocket costs were estimated during each year in the study period. Direct insurer costs were based on reimbursements from third‐party payers to health‐care providers and pharmacies. Direct insurer cost outcomes included all‐cause medical costs, prescription drug costs, total direct costs , and comorbidity‐related medical costs . Direct out‐of‐pocket costs were based on payments from beneficiaries to health‐care providers and pharmacies and were calculated as the sum of deductible, coinsurance, and copayment amounts. Out‐of‐pocket cost outcomes included all‐cause medical, prescription drug, and total out‐of‐pocket costs.Indirect costs among patients with work loss data and were estimated during each year in the study period. Costs were estimated based on medically related absenteeism costs  and disability costs .Income progression, or how income changed over time, among patients with income data was estimated during the index calendar year and each calendar year in the follow‐up period following the index calendar year. Income included wages and salary, excluding variable components such as equity options.Baseline characteristics were described using means and standard deviations (SDs) for continuous variables and frequencies and percentages for categorical variables. Characteristics were compared between PD caregivers and controls using generalized estimating equations (GEEs) to account for matching.P values were computed for mean differences in costs between PD caregivers and controls.Direct and indirect costs were estimated and compared between PD caregivers and controls during each year of the study period, both descriptively and following adjustment for baseline characteristics . In the descriptive analysis, mean costs were compared using GEEs to account for matching. In the adjusted analysis, longitudinal generalized linear mixed models with a Tweedie distribution and a log link function were used to account for the correlation between the PD caregivers and their matched controls as well as the correlation within patients because of the repeated measures of costs. Bootstrapped 95% confidence intervals (CIs) and 2‐sided Annualized income was estimated and compared between PD caregivers and controls during each calendar year of the study period. Income progression was assessed using a longitudinal generalized linear mixed model with a gamma distribution and log link. The model adjusted for baseline characteristics including age, sex, region, health plan, CCI, and index year. Both costs and income were inflated to 2014 US dollars using the Consumer Price Index from the US Bureau of Labor Statistics.P < 0.05 was used to determine significance during comparisons.All analyses were conducted in SAS 9.3. A The sample selection for PD and control caregivers included in the 3 analyses  is illustrated in Figure P < 0.05) compared with matched controls . The PD patients were older compared with the control dependent cohort  and had a significantly higher mean ± SD CCI score .The demographic characteristics of the PD patients (n = 1146) and control dependents (n = 3063) are shown in Supplemental Table P < 0.01), but comparable costs in years 2‐5 , mean all‐cause medical costs ($7081 vs $5568), and mean comorbidity‐related medical costs ($1589 vs $1228) compared with matched controls in the first year of the study period  and medical costs  compared with controls in all years.In all 5 years, PD caregivers had significantly higher mean adjusted total out‐of‐pocket costs compared with controls , and costs were comparable for years 4 and 5 .The regression‐adjusted cumulative income loss for PD caregivers compared with controls is displayed in Figure This study estimated the 5‐year economic burden of caregivers of newly diagnosed PD patients in terms of direct costs, indirect costs, and cumulative income loss to assess the comprehensive burden of PD caregivers over time. The results of this study indicate that caregivers of newly diagnosed PD patients incurred higher total direct health‐care burden  and total indirect costs than controls during the first year after PD diagnosis. In addition, many direct cost components  were significantly higher among PD caregivers than controls over all 5 years, and total indirect costs were significantly higher among PD caregivers during the first 3 years of the study period. Last, greater cumulative income loss was observed among PD caregivers compared with controls over the 5 years of the study period.Compared with control caregivers, PD caregivers displayed higher rates of several general caregiving comorbidities at baseline , as well as numerically higher caregiving‐related comorbidity costs in all years of the study period. These results are consistent with the finding from a case‐control study of early‐onset PD in Ireland, which found that PD caregivers were observed to have an increase in chronic illness compared with noncaretaking spouses.The present study results indicate that both direct and indirect cost contributors drive the higher economic burden among caregivers of PD patients compared with controls. In terms of direct costs, the finding that out‐of‐pocket medical costs, but not insurer medical and total costs, were significantly higher among PD caregivers compared with controls during all 5 years of the study period may be explained by 2 reasons. First, there was large variation in insurer costs across patients. Second, unlike insurer costs, out‐of‐pocket costs are more closely related to the frequency of health resource utilization (HRU), and therefore higher out‐of‐pocket costs may be a direct reflection of the increased HRU among PD caregivers. In terms of indirect costs, PD caregivers experienced significantly higher total indirect costs in the first 3 years of the study period, but comparable indirect costs during the last 2 years of the study period. The total indirect costs consisted of up to approximately 35% of disability costs and 65% of medical‐related absenteeism costs. The fluctuation of total indirect cost results over the study period might be because of the small sample size in the later years of the study period  and uneven attrition between the 2 cohorts.Both the PD caregivers and matched controls experienced a loss of income during the study period, although PD caregivers experienced twice the income loss of controls based on actual income reported by the employers . The universal loss of income by both cohorts could be because of a trend of general wage stagnation in the United States during the years assessed There is a lack of literature on the direct and indirect costs of caring for PD patients, as prior studies have mainly focused on the noneconomic burden among PD caregivers or the impact on QoL. According to a review of the PD caregiver literature conducted in 2014, the financial strain for caregivers was assessed in terms of missed work days and qualitative reporting of financial issues; other previously studied aspects of caregiver burden have included physical (daily activities and mobility) and psychosocial  functions.Because of the high costs associated with PD‐related care,A strength of this study is the 5‐year time horizon for cost analysis of PD caregivers. The economic burden associated with caregiving was already evident among newly diagnosed PD patients, which provides new insights into the caregiver burden and highlights the incremental burden of the caregivers in the early phase of PD patients’ disease journey. However, PD caregiver costs in this study may be lower than those incurred by caregivers of an average PD patient because PD severity has been identified as a determinant of a caregiver's overall burden and PD caregiver costs are likely to increase as PD advances.This study should be considered in light of several limitations, some of which are inherent to claims database analyses. An important limitation of this study is the high loss‐to‐follow‐up rate over time and the lack of longitudinal follow‐up . Although this is similar to other insurance claims databases, this high loss‐to‐follow‐up rate may have influenced the present findings in an unpredictable way. For example, it is possible that the lack of a linear trend for direct and indirect costs over time was partially because of the loss to follow‐up of highly burdened disabled PD caregivers, resulting in an underestimation the burden among the PD caregiver cohort. In addition, because of the high loss‐to‐follow‐up rate, the study was not able to evaluate PD caregivers’ incremental economic burden over a longer time frame such as 10 years. PD is a progressive disease, although the progression rate varies across patients, so future studies using databases with longer follow‐up times are warranted to demonstrate how the burden of caregiving progresses over periods greater than 5 years.Another limitation of the study is that because PD caregivers were required to be working adults who were privately insured primary policyholders and under the age of 65, the study results may not reflect the economic burden among all PD caregivers in the United States. Although if the elderly nonworking population were studied, the indirect cost could not be assessed. Thus, future studies should assess the direct costs and comorbidities associated with caring for PD patients if focusing on an elderly population. It is possible that the incremental burden is greater in this more vulnerable population. However, it is also possible that the older caregivers are less likely to bear the full caregiving burden , resulting in a lesser incremental burden compared with the working‐age and under age 65 study population of this study.In addition, it was also assumed that the primary policyholder linked to a PD patient was the primary caregiver. Thus, if PD patients were primarily taken care of by other family members, PD caregivers’ economic burden may have been underestimated in this study. Finally, PD patients may have also received professional care not covered by insurance or could have been institutionalized during the course of the study.This is the first study to provide comprehensive information on the 5‐year economic burden of PD on working‐age caregivers. The current results suggest that the economic burden associated with caregiving is already evident among caregivers of newly diagnosed PD patients and remains high over the 5 years after PD diagnosis.A synopsis of the current research was presented in poster format at the 19th International Congress of Parkinson's Disease and Movement Disorders meeting, which took place in San Diego, California, June 14‐June 18, 2015.T. Marshall, Y. Jalundhwala, K. Sail, D. Macaulay, F. Mu, E. Ohashi, and P. Martinez‐Martin) participated in the design of the study and contributed to the manuscript development. Data were analyzed by Analysis Group, Inc.  and interpreted in collaboration with all other authors . Manuscript drafts were prepared by a professional medical writer ultimately paid by the sponsor, AbbVie, Inc., in collaboration with all authors. All authors take responsibility for the integrity of the data and the accuracy of the data analysis.All authors (This study and manuscript were funded by AbbVie. The design, study conduct, and financial support for the study were provided by AbbVie. AbbVie participated in the study design, research, interpretation of data, writing, reviewing, and approving the manuscript.T. Marshall, Y. Jalundhwala, and K. Sail are employees of AbbVie, Inc. and own stock/stock options. D. Macaulay, F. Mu, and E. Ohashi are employees of Analysis Group Inc., which has received consultancy fees from AbbVie, Inc. P. Martinez‐Martin has received honoraria from Editorial Viguera and AbbVie for lecturing, International Parkinson and Movement Disorder Society for management of the Program on Rating Scales, and HM Hospitales de Madrid for advice in a clinic‐epidemiological study; and license fee payments for the King's Parkinson's Disease Pain scale.Supplemental Table 1: Yearly Sample Size and Attrition Rates During the Five‐Year Study Period1Supplemental Table 2. Demographic Characteristics and Comorbidity Profile of PD Patients and Control Dependents during the Baseline Period1,2Supplemental Table 3. Descriptive Mean Insurer Costs During the 5‐Year Study Period1,2Supplemental Table 4. Descriptive Mean Out‐of‐Pocket Costs During the 5‐Year Study Period1,2Supplemental Table 5. Descriptive Mean Indirect Costs During the 5‐Year Study PeriodClick here for additional data file.


Dicistroviridae family have attracted a great deal of attention from scientists owing to their negative impact on agricultural economics, as well as their recent identification as potential aetiological agents of febrile illness in human patients. On the other hand, some Dicistroviruses are also studied for their potential biopesticide properties. To date, Dicistrovirus characterized in African mainland remain scarce. By using High-Throughput Sequencing technology on insectivorous bat faeces (Hipposideros Caffer) sampled in a cave used by humans to collect bat guano (bat manure) as fertilizer in Zimbabwe, we characterized the full-length sequences of three Dicistrovirus belonging to the Cripavirus and Aparavirus genus: Big Sioux River Virus-Like (BSRV-Like), Acute Bee Paralysis Virus (ABPV), and Aphid Lethal Paralysis Virus (ALPV). Phylogenetic analyses of ORF-1 and ORF-2 genes showed a complex evolutionary history between BSRV and close viruses, as well as for the Aparavirus genus. Herewith, we provide the first evidence of the presence of Dicistrovirus in Zimbabwe and highlight the need to further document the impact of such viruses on crops, as well as in beekeeping activities in Zimbabwe which represent a crucial source of income for Zimbabwean people.Viruses belonging to the  Dicistroviridae is a family of non-enveloped viruses with a linear ssRNA genome of approximately 7–10 kb. The Dicistrovirus RNA genome contains two non-overlapping open reading frames (ORFs) separated by an Inter-Genic Region (IGR) internal ribosome entry site (IRES) .Cripavirus’ IGRs belonged to the class I. The predicted base pairing was adapted from Jan et al. [The IGRs were grouped into two classes, I and II. The n et al. . Bulges Dicistroviridae family have been described worldwide, only a few studies have been carried out in African mainland [Cripavirus (BSRV-like and ALPV) and one Aparavirus (ABPV) obtained from the faeces of insectivorous bat species (Hipposideros caffer) collected in a cave dwelling in Zimbabwe.Although viruses belonging in the mainland ,45,46,47ALPV full genome has been characterized only in South Africa and Kenya [BSRV-like virus was first described in Kenya [dicistrovirus, very few data exist on ABPV in Africa, and only three full genomes are available in GenBank—one from South Africa [Up to now, in African mainland, the nd Kenya ,48, wherin Kenya . As for h Africa , and twoh Africa .Dicistrovirus can cause dramatic damage to the agriculture sector. For example, numerous Dicistrovirus, likely combined with other factors, have been associated with honeybee colony losses (Collapse Disorder Colony (CDD)) [ABPV is one such virus associated with this syndrome. In Zimbabwe, beekeeping is a growing farming activity. According to the Beekeepers Association of Zimbabwe, the country reached nearly 50,000 beekeepers who produced more than 1000 tons of honey per year . This activity represents a life-sustaining source of income and livelihood for small-scale farmers [ABPV in Zimbabwe’s insectivorous bats suggested a broader circulation of this virus in the country and probably in the rest of southern Africa. Additional studies need to be carried out in Zimbabwe to assess the prevalence of ABPV in beehives and to identify concomitant factors likely involved in CCD, such as Varroa’s mite infections [Dicistrovirus could be a major concern for the agricultural sector in future.y (CDD)) . The ABP farmers . The chafections ,52, consDicistrovirus are studied for their use as potential bio-pesticides [ALPV is one of the recognized dicistroviruses which are pathogenic to insect pests of agricultural importance [On one hand, some sticides . Aphids sticides , and theportance ,54. In Zportance . ActuallBSRVs have been described in diverse arthropod hosts worldwide [BSRV taxonomic classification by the ICTV, as many other dicistroviruses, is still pending [BSRV-like CR-026 seemed to be a new Cripavirus based on the first part (ORF-1) of its genome, whereas the second part (ORF-2) of its genome suggests that it is close to the BSRVs previously characterized. Nonetheless, according to the ICTV, the species demarcation criteria within the Cripavirus is defined by the sequence identity at the amino-acid level between the capsid proteins of isolates and strains. Isolates with AA identities above 90% should be considered as the same species. Regarding this criterion, our new BSRV should be fully considered as a BSRV strain. However, considering the ORF-1 analyses, the taxonomic classification of BSRV-like CR026 remains difficult to establish. Besides the WIV and AGLV showed 100% of AA identities in both ORF-1 and ORF-2 (Capsid proteins) and have 91% of AA identities with the BSRV strains. Obviously, these two viruses, WIV and AGLV, are identical and belong to the BSRV strains (see orldwide ,18,21,22ains see . Dicistrovirus has been reported in blood samples of human and bat species [Recently, the  species ,30,31. AHipposideros caffer bat species is considered a moth specialist. Its diet consists predominantly of Lepidoptera [H. caffer in our study site. Our result confirmed the previous reports with a majority of Lepidoptera found in the faeces, but also of dipters (see Dicistrovirus, such as the Acute Bee Paralysis Virus, can be hosted by other arthropods, and highlighted the need to further document the potential insect reservoirs of such viruses.Finally, this study showed the potential of insectivorous bat faeces for the study of arthropod viromes. Insectivorous bats are generalized predators and they feed on a large number of insect species, following their availability as prey within the range of the insectivorous bat species ,57. As aidoptera ,58,59. Iters see . The abs