Attention is increasingly focused on the potential mechanism(s) for Zika virus infection to be transmitted from an infected mother to her fetus. This communication addresses current evidence for the role of the placenta in vertical transmission of the Zika virus.Placentas from second and third trimester fetuses with confirmed intrauterine Zika virus infection were examined with routine staining to determine the spectrum of pathologic changes. In addition, immunohistochemical staining for macrophages and nuclear proliferation antigens was performed. Viral localization was identified using RNA hybridization. These observations were combined with the recent published results of placental pathology to increase the strength of the pathology data. Results were correlated with published data from experimental studies of Zika virus infection in placental cells and chorionic villous explants.Placentas from fetuses with congenital Zika virus infection are concordant in not having viral-induced placental inflammation. Special stains reveal proliferation and prominent hyperplasia of placental stromal macrophages, termed Hofbauer cells, in the chorionic villi of infected placentas. Zika virus infection is present in Hofbauer cells from second and third trimester placentas. Experimental studies and placentae from infected fetuses reveal that the spectrum of placental cell types infected with the Zika virus is broader during the first trimester than later in gestation.Inflammatory abnormalities of the placenta are not a component of vertical transmission of the Zika virus. The major placental response in second and third trimester transplacental Zika virus infection is proliferation and hyperplasia of Hofbauer cells, which also demonstrate viral infection. Following many months of intensive collaborative research, during which pathology studies played a prominent role, on April 13th, 2016 the United States Centers for Disease Control (CDC) in Atlanta issued a statement that Zika virus (ZIKV) was a cause of microcephaly and other severe fetal brain defects . Rapid pThe Zika virus is a TORCH agent  . It has Trypanosoma cruzi, and others [Listeria infections. Examination of the second and third trimester placentas from fetuses having intrauterine infection with the Zika virus, together with recently reported observations, has determined that the inflammatory and necrotic lesions of the placenta which are typical of other TORCH infections do not occur in placentas from fetuses with intrauterine Zika virus infection , funisitis , and deciduitis —examples include Group B streptococcus, The absence of villitis in transplacental Zika virus infection is unexpected, especially because the virus can cause necroinflammatory reactions when it reaches the fetal brain. Prior to the Zika virus epidemic, the most common hematogenously transmitted virus which did not produce villitis was the human immunodeficiency virus (HIV); however, this virus does not cause necroinflammatory damage to the fetal tissues.Those infectious agents which cause necrosis (irreversible cell death) in the fetus following vertical transmission typically also cause necrotic changes in the placenta. Infection or inflammation of the cells lining the blood vessels in the chorionic villi can result in endothelial necrosis, vascular obliteration, and thrombosis, with the production of avascular (scarred) villi. Infectious agents which induce villous necrosis include syphilis, cytomegalovirus, listeriosis, toxoplasmosis, and others. The Zika virus causes necrosis of cells in the fetal brain , 12; howHofbauer cells are fetal cells of monocytic origin and are a normal component of the stroma of the chorionic villi. They first appear in the chorionic villi at the 10th–18th days of gestation, and are believed to initially be of fetal mesenchymal origin, derived from monocyte progenitor cells of the hypoblast-derived yolk sac that have migrated to the mesenchymal core of the villi. As gestation progresses, it has been suggested that they are derived from a population of recruited fetal monocytes , 14. SimIn normal pregnancies, Hofbauer cells diminish in number by the 4th–5th months of gestation, and can be difficult to identify without the use of antibody staining to macrophage antigens. Hyperplasia of Hofbauer cells is abnormal, and occurs in a wide variety of pathological conditions of pregnancy. These include villitis of unknown etiology (VUE), ascending infections, and maternal blood-borne infections which cause villitis such as TORCH infections including cytomegalovirus and syphilis , 16, andReview of the second and third trimester placentas from fetuses with congenital Zika syndrome using immunohistochemistry for nuclear proliferation markers has revealed that Hofbauer cells undergo proliferation within the chorionic villous stroma in response to Zika virus infection of the placenta Fig. . As a reThe Zika virus has recently been definitively identified in Hofbauer cells from placentas of human fetuses with intrauterine infection and microcephaly. Investigators have used RNA techniques to detect Zika virus nucleic acid  Fig. 6)6) and anIn placentas examined from first trimester pregnancies, Zika virus has a much wider cellular distribution than in second and third trimester placentas. Zika virus has been identified in first trimester placentas of infected fetuses using immunohistochemistry in Hofbauer cells as well as in fetal leukocytes and endothelial cells . In a stExperimental infections using ex vivo first trimester placental organ and cell culture models have demonstrated Zika virus infection and replication in Hofbauer cells, mesenchymal cells and fibroblasts, and cytotrophoblast. Mesenchymal cells of the umbilical cord and cells from the decidua basalis including decidual fibroblasts and macrophages could also become infected . The ZikZika virus has been found to be capable of infecting and replicating within Hofbauer cells from human term placentas , 27. In The syncytiotrophoblast of the placenta covers the chorionic villi, separating it from the surrounding maternal blood in the intervillous space. Lying immediately beneath these cells is the mononuclear cytotrophoblast, which among other functions, coalesces to form the multinucleated syncytiotrophoblast. Several experimental laboratory studies have provided evidence that both the syncytiotrophoblast and cytotrophoblast are resistant to Zika virus infection , 28. HowPlacental pathology has proven valuable in situations where infections may be transmitted from pregnant women to their fetuses, providing clinically relevant information on the mechanism(s) and timing of maternal-fetal  transmission, the nature of the inflammatory response by the mother and fetus to the agent, anatomic distribution and cellular localization of the agent, and the effects of the organism on the placental tissues and the fetal circulation, such as necrosis, hemorrhage or vascular disease. Unfortunately, so far during this current Zika virus pandemic, there have been relatively few placentas from the endemic countries that have been scientifically examined and published. However, it is not surprising that only a small number of placentas have been analyzed. During the recent 2013–2015 Ebola virus epidemic in West Africa, placental analysis would have been very useful in understanding the pathophysiology of disease transmission and potential methods for clinical intervention. Unfortunately, to date, there have been no published reports of placental pathology from Ebola virus-infected mothers and their infants from this epidemic. Even during the global human immunodeficiency virus (HIV) pandemic which was initially recognized in adult men in the US in 1981, and subsequently, in infants of infected mothers in 1982, it took greater than one decade for large case-controlled studies of human placentas from women and infants with and without HIV infection to be designed, implemented and analyzed –32.Our analysis of the second and third trimester placentas from fetuses with congenital Zika virus infections does not demonstrate any significant inflammatory abnormalities, either of maternal or fetal origin. The absence of villitis, intervillositis, chorioamnionitis, and funisitis is unlike most other TORCH infections, which cause a spectrum of inflammatory abnormalities in the chorionic villi, umbilical cord, and placental membranes. It is important to communicate these observation of a lack of a maternal or fetal inflammatory response in placentas of Zika virus-infected fetuses from the second and third trimesters, because placental inflammation has been hypothesized by multiple authors in several articles as contributing to the development of placental dysfunction and insufficiency and the congenital Zika syndrome –35—our fSome placentas from fetuses with congenital Zika syndrome have been described as containing microcalcifications and fibrin deposits. Microcalcifications within the placenta are non-specific findings which are considered to be normal, and occur in non-diseased placentas from fetuses with no underlying pathological processes. Excessive microcalcifications can be found in placentas with necrotizing villitis, cytomegalovirus infections, other causes of villous necrosis, maternal floor infarction, and massive perivillous fibrin deposition; however, Zika virus-infected placentas do not exhibit these tissue-destructive or ischemic processes. Focal or “patchy” fibrin deposition in placentas is also a normal finding unless excessive; however, there have been no reports of excessive fibrin accumulations in Zika virus-infected placentas. Both microcalcifications and fibrin deposition can be seen in placentas from stillborn fetuses from any cause, and are not specific for fetal demise resulting from Zika virus infection .It has been approximately 12 months since the announcement in April 2016 of the Zika virus as a cause of fetal infections and malformations [


Potyvirus in evergreen oak forests of the Iberian Peninsula, the main wild ecosystem in this geographic region and in the entire Mediterranean basin. During this period, we have also measured plant species diversity, host density, plant biomass, temperature, relative humidity, and rainfall. Results indicated that potyviruses were always present in evergreen oak forests, with a novel virus species explaining the largest fraction of potyvirus-infected plants. We determined the genomic sequence of this novel virus and we explored its host range in natural and greenhouse conditions. Natural host range was limited to the perennial plant mountain rue (Ruta montana), commonly found in evergreen oak forests of the Iberian Peninsula. In this host, the virus was highly prevalent and was therefore provisionally named mediterranean ruda virus (MeRV). Focusing in this natural host–virus interaction, we analyzed the ecological factors affecting MeRV infection risk and population genetic diversity in its native wild ecosystem. The main predictor of virus infection risk was the host density. MeRV prevalence was the major factor determining genetic diversity and selection pressures in the virus populations. This observation supports theoretical predictions assigning these two traits a key role in parasite epidemiology and evolution. Thus, our analyses contribute both to characterize viral diversity and to understand the ecological determinants of virus population dynamics in wild ecosystems.Increasing evidence indicates that there is ample diversity of plant virus species in wild ecosystems. The vast majority of this diversity, however, remains uncharacterized. Moreover, in these ecosystems the factors affecting plant virus infection risk and population genetic diversity, two traits intrinsically linked to virus emergence, are largely unknown. Along 3 years, we have analyzed the prevalence and diversity of plant virus species from the genus  Viruses are the most frequent causal agents of emerging infectious diseases in crops , and arePlant viruses are not only highly diverse and widespread in wild ecosystems, they may also be important ecological agents. For instance, quantitative resistance of wild plants to viruses have been described , suggestAnalyzing the determinants of infection risk and population genetic diversity of native viruses in wild plant communities may be also relevant to understand their emergence. Emergent parasites are defined as those whose infection risk increases following its appearance in a new host population, or whose infection risk increases in an existing host population . ConsequQuercus ilex L.) forest. This is the most extended wild ecosystem in the Mediterranean basin, covering approximately three million ha , commonly found in evergreen oak forests of the Iberian Peninsula. We provide information on the prevalence, genetic diversity and host range of this virus, provisionally named mediterranean ruda virus (MeRV). Using this host–virus interaction, we analyze the ecological factors affecting MeRV prevalence and population genetic diversity, and we explore the potential of MeRV for dispersing into crops by determining MeRV prevalence in cultivated fields located nearby evergreen oak forests where MeRV is present.Here, we analyze the prevalence of species from the genus Figure 1 and Supplementary Table 3) by averaging values in the eight squares. We also collected from nearby weather stations information on minimal, maximal and average temperature (°C) relative humidity (%), and on rainfalls (mm) in the months when each visit was done.Six locations of evergreen oak forest located in the Iberian Peninsula were visited between the summer of 2013 and the spring of 2016. The locations were distributed among a transect of 200 km (north–south) in the center of the Iberian Peninsula (Cucumis melo L.), tomato (Solanum lycopersicum L.), and pepper (Capsicum annuum L.) located near to the sampled locations of evergreen oak forests . For each run, 10 ng of total RNA were added to the Brilliant III SYBR® Green Ultra-Fast QRT-PCR Master Mix  following the manufacturer’s recommendations. Universal primers NIb2F and NIb3R , 50°C for 20 s , and 72°C for 30 s (extension). RNA purified from turnip mosaic virus (TuMV), a well characterized potyvirus species, was always included as positive control. Amplification was confirmed in electrophoresis in 1.2% agarose gel stained with ethidium bromide in Tris-acetate-EDTA (TAE) buffer. PCR products of the expected length were purified using the StrataPrep DNA Gel Extraction Kit (Agilent Technologies) and sequenced. Sequences were assembled using MEGA 6  – polyvinyl pyrrolidone (PVP) method , which and NIb3R  for specg MEGA 6 . Nucleotcriteria , sequencis genus . Total pAmaranthaceae, Asteraceae, Boraginaceae, Chenopodiaceae, Cistaceae, Cucurbitaceae, Fabaceae, Fagaceae, Poaceae, Rutaceae, and Solanaceae were inoculated (Table 1). These species were chosen to include the most abundant species in the sampled evergreen oak forests, and the most common crops in the surrounding areas. For each plant species, four to seven plants (10–15 days old) were mechanically inoculated by applying purified virion RNA (100 ng/ul) in 0.1 M Na2HPO4 onto the first two completely expanded leaves dusted with carborundum. The inoculated plants were maintained in a greenhouse , and symptoms were weekly recorded over an 8-week period. Tissue from systemically infected leaves of symptomatic and asymptomatic plants was collected 20 days post-inoculation and tested for potyvirus infection by real-time RT-PCR using specific primers qCPFor (5′-GACTGACTATAGTTTAGCGCGC-3′) and qCPRev (5′-GCCTCTGATAGCTGCTGCTTTC-3′) that amplify a 111-bp region of the MeRV CP gene.To determine the host range and the symptoms induced by MeRV infection, different plant species of the botanical families Ruta montana L.) from El Pardo that tested positive for MeRV infection (MeRV-ParP17) was treated with TURBO DNA-freeTM Dnase . After treatment, ribosomal RNA (rRNA) was removed using Ribo-ZeroTM Plant Leaf kit . For genome sequencing, 3 μg of total RNA was used for library preparation and subjected to high-throughput NGS using the Illumina platform , generating 20 million paired-end reads. Adapters and low-quality sequences from NGS data were removed using Seqtk2. MeRV genome was assembled using a reference-guided read mapping to the phylogenetically nearest potyvirus genomes as implemented in Bowtie 2.0   . ImportaTM Reverse Transcriptase  was used for retrotranscription and PCR Phusion® High-Fidelity DNA Polymerase  for PCR amplification. The 5′ and 3′ ends of the virus were obtained by rapid amplification of cDNA ends (RACE) using AmbionTM FirstChoice® RLM RACE kit (Life Technologies). All fragments were sequenced, and assembled with MEGA 6, revealing 100% nucleotide identity between overlapping fragments and with the NGS-derived nucleotide sequence. The same nucleotide sequence was also obtained using RNA from purified virions as template.The NGS-derived MeRV genomic sequence was confirmed using the same mountain rue plant RNA preparation as template for RT-PCR, utilizing different sets of specific primers. Primer pairs were designed to produce 12 fragments in such a way that adjacent fragments overlapped by at least 100 nt, covering the potyvirus polyprotein  for nucleotides, and the Whelan and Goldman (WAG) substitution model for amino acids. All analyses were run until relevant parameters converged, with 25% of the MCMC chains discarded as burn-in. Maximum clade credibility (MCC) trees, with Bayesian posterior probability values providing a measure of the robustness of each node, were also summarized from the MrBayes tree samples.Phylogenetic relationships between MeRV and the other members of the genus Nucleotide identities (%) of the complete MeRV genome with those of the two phylogenetically closest virus species were compared using SimPlot v3.5.1 . Plots oNd) and synonymous (Sd) nucleotide substitutions per site, were estimated using the Kimura-2-parameter nucleotide substitution models implemented in MEGA 6, as this was selected as the best-fitted nucleotide substitution model in jModelTest v. 2.1.10 , and the haplotype diversity [Hd = (1 - Σix2)n/(n - 1), where xi is the frequency of an haplotype and n is the sample size (The genetic diversity of the MeRV population was estimated based on the CP gene sequence of 69 MeRV isolates (Acc No. MF953306-MF953374). For this purpose, total RNA extracts of MeRV-infected plants were used to RT-PCR amplify the viral CP gene (837 nt), utilizing specific primers CPFor (5′-CCAAAGCTTGAACAAGAGAGAATTGTTTCG-3′) and CPRev (5′-ACACCAAGCATGKTRTGCATAT-3′), and PCR products were sequenced. Virus genetic diversity (π) as average pairwise nucleotide difference between sequences, and the mean number of non-synonymous , between sampling years at each season, and between geographic locations were compared using Chi-square tests. These statistical analyses were done using SPSS 21.0 . Mixed effect multiple regression tests were used to analyze the association between ecological factors and MeRV prevalence and population genetic diversity parameters . We cons2 ≥ 5.03, P ≤ 0.025), prevalence being similar in the later two seasons .Infection by potyvirus was detected in every season during the monitored period, and prevalence was calculated as the percentage of infected individuals relative to the total number of analyzed plants. Average potyvirus prevalence in evergreen oak forests of the Iberian Peninsula ranged between 1.06 and 3.43%, depending on the season and sampling cycle. In the analyzed sampling cycles, potyvirus prevalence was higher in summer than in spring and autumn (χZucchini yellow fleck virus (ZYFV) (34/171), which was detected in six host plant species with a prevalence ranging between 4.76 and 61.54%; Clover yellow vein virus (ClYVV) (10/171), which was detected in two plant species with prevalence of 2.86–13.33%; and Endive necrotic mosaic virus (ENMV) and Turnip mosaic virus (TuMV) (2/171 each), which were found infecting individuals of a single plant species each, with prevalence of 3.51% and 4.65%, respectively. Interestingly, the remaining identified potyvirus accounted for the largest fraction of infected plants . This virus species was only detected in mountain rue (Ruta montana), a perennial plant that showed the highest potyvirus prevalence (41%) among the host plant species identified in evergreen oak forests. None of the other detected potyvirus was found in mountain rue plants. The comparison of the NIb of this rue-infecting virus with those of all known potyviruses revealed a nucleotide sequence identity of 75% with the closest species. This suggested that this was a novel species of genus Potyvirus that we provisionally named MeRV. For further characterizing the virus, tissue of one mountain rue plant sampled in El Pardo and infected with MeRV was selected. This plant tested negative for other species of the genus Potyvirus and for cucumber mosaic virus (CMV) by RT-PCR. More importantly, analyses of the NGS sequence data obtained from total RNA extracts of this field-infected mountain rue plant did not detect any other viral sequence. Tissue of the infected field plant was grinded in 0.1 M phosphate buffer pH 7, 0.02% sodium diethyldi-thiocarbamate (DIECA), and used to inoculate Nicotiana benthamiana plants. After 20 days, N. benthamiana plants developed a systemic mosaic. MeRV virions were purified from these plants according to the protocol described by N. benthamiana plants, and that this species belonged to the genus Potyvirus, discarding the possibility of mixed viral infection. This isolate was named MeRV-ParP17, and its virions were used for further characterization.Identification of the potyviruses present in the 171 infected plants indicated the presence of five virus species: Potyvirus, the MeRV-ParP17 genome has a single ORF encoding a polyprotein of 3,077 amino acids with an estimated molecular weight of 350 kDa. This ORF encodes all typical proteins, consensus cleavage sites and conserved catalytic domains present in the potyviruses . The P1 protein, a serine protease, contains the strictly conserved catalytic triad His208-(X8)-Asp217-(X30)-Ser248 with the conserved GHSG246-249 motif around the active serine site. In the HC-Pro, the protease active site residues are located at Cys344, contained in the conserved sequence GYCH342-345, and at His417. Also, a conserved cysteine-rich region was identified in the N-terminal region of HC-Pro stretching from Cys27 to Cys58. HC-Pro motifs involved in aphid transmission (RITC52-55 and PTK310-312), genome replication (IGN152-154) and systemic movement (CCC292-294)  protein cistron includes the conserved RNA helicase motifs located between residues 85 and 359, and the NIa contains the conserved His46, Asp81, Cys151, His167 cysteine protease catalytic tetrad. Conserved sequence motifs in RNA-dependent-RNA polymerases of positive strand RNA viruses were also identified in the MeRV-ParP17 NIb protein. Finally, the CP cistron contains a NAG motif, essential for aphid transmission in some potyviruses  tail. As for all members of the genus 292-294)  are alsoyviruses , and locmovement , 1995, an = 107). The percentage of nucleotide identity of the MeRV-ParP17 genome with that of the other potyviruses ranged from 50 to 66%, showing highest identity with bean yellow mosaic virus (BYMV) (65%) and clover yellow vein virus (ClYVV) (66%). Accordingly, Bayesian phylogenetic trees revealed that MeRV-ParP17 clustered together with BYMV and ClYVV . Following the approach described by Figure 2). When each cistron was analyzed individually, the MeRV-ParP17 P1 and P3 cistrons showed the lowest nucleotide identity with the same cistrons of BYMV and ClYVV . The remaining cistrons showed a sequence identity with the corresponding ones of BYMV and ClYVV ranging on average between 57 and 69%. Equivalent results were obtained using the amino acid sequences (not shown).The complete nucleotide sequence of the MeRV-ParP17 polyprotein was aligned with those of available fully sequenced potyviruses , in which virus infection was asymptomatic. Only three Solanaceae species were infected by MeRV-ParP17: N. benthamiana (5/5), C. annuum (4/6), and S. lycopersicum (6/6), with infection in these three species causing mild mosaic symptoms. There was no variation in susceptibility across hosts .The MeRV-ParP17 host range was determined by inoculating purified virions of MeRV-ParP17 into 10 wild species belonging to seven botanical families, which represent the most abundant species in evergreen oak forests, and 13 plant species of six botanical families, including 2 ≤ 2.74, P ≥ 0.098), and this factor was not considered in subsequent analyses. On the other hand, MeRV prevalence varied across seasons between 18% in spring 2015 and 74% in summer 2014 (Table 2). Average MeRV prevalence did not vary between sampling cycles. However, during the first and second cycle the prevalence was higher in summer than in spring and autumn , whereas in the third sampling cycle the highest prevalence occurred in autumn  (Table 2). Given that the MeRV host range included two crop species, tomato and pepper, commonly cultivated near the sampled evergreen oak forests, we analyzed MeRV prevalence in these crops during the same time span. We also included melon fields in our surveys, as, together with pepper and tomato, this was the major cultivated plant in the area. MeRV was never detected in melon tomato and pepper plants.We used MeRV prevalence as a measure of virus infection risk. MeRV prevalence in mountain rue plants of Iberian evergreen oak forests did not significantly varied between geographic locations (χTable 2). We included season as a covariate to account for the dependency of MeRV prevalence over time. To analyze the association between these traits and MeRV prevalence, we used multiple regression model selection analyses (Table 3). The best-ranked model contained plant density as the only predictor, such that there was a positive association between mountain rue density and MeRV prevalence  . This model closely competed with that containing rainfall as the only predictor, which showed a negative association between MeRV prevalence and rainfall  . The rest of the models showed much poorer predictive power (Δi > 2) (see footnote of Table 3).To further understand the ecological determinants of MeRV infection risk in evergreen oak forest, we considered the following factors as predictors of MeRV prevalence: host plant density and relative abundance, species richness, plant biomass, temperature and relative humidity , and rainfall in the sampled locations (Table 4). Using this sequence data set, we estimated the number of haplotypes (H), the haplotype diversity (Hd) and the average nucleotide genetic diversity (π) of the MeRV population at each visit. MeRV population haplotype number, haplotype diversity and genetic diversity greatly varied between seasons  (Table 4).Most of the MeRV sequenced isolates were collected at El Pardo, the location with the highest density of mountain rue plants. The low number of isolates from other locations prevented analyzing the genetic structure of the virus population according to geographic location. However, MCC trees indicated that isolates from location other than El Pardo did not form a monophyletic cluster (data available upon request). Hence, to test whether changes in ecological factors and MeRV prevalence across seasons affected the genetic diversity of the virus population, we only utilized the CP sequence of 69 MeRV isolates from El Pardo, collected along the three sampling cycles . The best-ranked model explaining Hd contained prevalence as the only predictor, and indicated a positive association between both traits  . This model closely competed with that including MeRV prevalence and plant species richness as main predictors , virus prevalence having much higher relative importance than plant species richness . The model containing MeRV prevalence and host relative abundance also showed Δi < 2 , with virus prevalence having again the highest relative importance (99.1%) (Table 3). In addition, the model best explaining MeRV population genetic diversity (π) contained MeRV prevalence and plant species richness as predictors , prevalence having the highest relative importance (77.2%). This model closely competed with that containing MeRV prevalence as the sole predictor  , or this variable together with rainfall . However, this later model did not show a significant association between the predictors and the response variable (Table 3). None of the tested models accurately predicted Nd (P ≥ 0.106) (Table 3). On the other hand, the best ranked models predicting Sd and Nd/Sd contained MeRV prevalence as predictor, solely  or in combination with plant species richness and rainfall. In all these models, MeRV prevalence had always much higher predictive power (relative importance ≥ 72%) than the other predictors (relative importance ≤ 25%) (Table 3).We analyzed the ecological factors affecting MeRV population HPotyvirus, for which the species demarcation criteria include: (1) different inclusion body morphology, (2) differences in host range, (3) overall nucleotide sequence identity not higher than 76%, and (4) capsid protein sequence identity not higher than 80% . Thus, in the analyzed ecosystem MeRV behaves as a specialist virus, i.e., viruses able to infect one or a few closely related host species , which could explain the poor predictive power of species diversity. Also, previous analyses identifying species diversity as a determinant of plant virus infection risk focused in viruses with a generalist strategy or at least with several hosts in the monitored ecosystem. The absence of alternative host species for MeRV in evergreen oak forests could minimize the effect of species diversity. These observations would support the idea that the applicability of the Dilution and the Amplification Effect hypotheses would depend on the plant community composition (Table 2) and density of aphids (the main vectors of potyviruses) peaks  peaks . AlthougS, both variables showing a positive association, but not of Nd. Accordingly, selection pressures, measured as Nd/Sd, were negatively associated with virus prevalence. This indicates that neutral evolution, rather than adaptive selection, might be responsible for the changes in MeRV population genetic diversity associated with fluctuations in virus prevalence. Similar results have been obtained in other plant virus populations infecting wild hosts . Fourth, although the best-ranked models explained a large proportion of the variation in MeRV infection risk and population genetic diversity, other factors not considered here could also play a role in MeRV evolution and epidemiology. For instance, host genetic diversity has been also reported as an important determinant of virus disease risk and population genetic diversity in wild ecosystems . Second, the analyses of the association between MeRV prevalence and ecological factors are based on the data from six mountain rue populations, and the analyses of the association between MeRV evolutionary parameters and ecological/epidemiological factors are based on data from a single host population. We are aware that this might be a small sample size. However, it was enough to detect significant, and in many cases strong, correlations between the studied parameters. Third, the analysis of the effect of MeRV infection on host mortality is based on the differential virus prevalence between adult and young plants. It should be noted that these results could be also explained if virus titer in adult plants would be much lower than in younger ones, such that it could lead to an underestimation of virus prevalence in adults. However, no significant differences in MeRV accumulation were observed between young and adult plants (osystems . UnfortuIn summary, our results provide evidence of the relevant role that infections of native plant viruses may play in modulating the population dynamics of their hosts in wild ecosystems. The effect of the most important ecological factors driving MeRV infection risk and population genetic diversity in its wild host partially supports theoretical predictions. Deviations from these predictions could be at least partially attributed to the behavior of MeRV as specialist parasite. Because both infection risk and population genetic diversification have been proposed to be involved in the appearance of new infectious diseases , our resCR-N collected and analyzed plant samples, obtained data on ecological factors and virus sequences, assembled the MeRV genome and wrote the manuscript. NM collected samples, performed multiple regression analyses, and participated in drafting the manuscript. IP designed and coordinated the study, helped collecting samples, and wrote 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.


Begomovirus, family Geminiviridae) and an associated betasatellite found infecting Vernonia amygdalina Delile (family Compositae) in Uganda were cloned and sequenced. Begomoviruses isolated from two samples showed the highest nucleotide sequence identity (73.1% and 73.2%) to an isolate of the monopartite begomovirus tomato leaf curl Vietnam virus, and betasatellites from the same samples exhibited the highest nucleotide sequence identity (67.1% and 68.2%) to vernonia yellow vein Fujian betasatellite. Following the current taxonomic criteria for begomovirus species demarcation, the isolates sequenced here represent a novel begomovirus species. Based on symptoms observed in the field, we propose the name vernonia crinkle virus (VeCrV) for this novel begomovirus and vernonia crinkle betasatellite (VeCrB) for the associated betasatellite. This is the first report of a monopartite begomovirus-betasatellite complex from Uganda.The complete genomes of a monopartite begomovirus (genus The online version of this article (doi:10.1007/s00705-016-3175-2) contains supplementary material, which is available to authorized users. Begomovirus is the largest of the seven genera in the plant virus family Geminiviridae  and [Uganda-Kawungera UG9-2015].Cloned ~2.8-kbp DNA from samples UG7  and UG9  had a genome organization typical of OW monopartite begomoviruses, with CP and pre-CP proteins encoded in the virion-sense strand, and Rep, TrAP, Ren and C4 proteins encoded in the complementary-sense strand. Begomoviruses from samples UG7 and UG9 showed the highest nucleotide sequence identity  to an isolate of the monopartite begomovirus tomato leaf curl Vietnam virus (EU189150). DNA of ~1.3 kb cloned from the same samples  showed the typical genome organization of betasatellites . Betasatellites from UG7 and UG9 showed the highest nucleotide sequence identity  to vernonia yellow vein Fujian betasatellite (JF733779) found in Recombination is commonly detected in begomovirus and betasatellite genomes , 10, 14.Phylogenetic analysis showed that VeCrV isolates clustered with two OW begomoviruses from Africa, tobacco leaf curl Zimbabwe virus (AF350330) and tobacco leaf curl Comoros virus (AM701760) Fig. A. In conVernonia in Africa and the first identification of a betasatellite in Uganda.This is the first report of a begomovirus-betasatellite complex infecting plants of the genus Supplementary material 1 (DOC 35 kb)Below is the link to the electronic supplementary material.


Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days.We assayed Zika virus–infected mosquitoes stored at room temperature for  Laboratory detection of Zika virus in field-collected mosquitoes is often accomplished by using molecular methods, such as reverse transcription PCR (RT-PCR) or virus isolation protocols. Sample handling conditions recommended for these assays include maintaining a cold chain from field to laboratory to preserve live virus and mitigate degradation of virus RNA.<30 days by using real-time RT-PCR to determine the rate of recovery of virus RNA and Vero cell plaque assays to determine the rate of recovery of infectious virus.Ideally, mosquitoes should be stored and shipped to a diagnostic laboratory at a temperature of −70°C or below. However, in some locations, an optimal cold chain is difficult to maintain when shipping the specimens because of unavailability of a liquid nitrogen or dry ice supplier. We tested Zika virus–infected mosquitoes that had been stored at room temperature for Aedes aegypti (REXD strain) mosquitoes were intrathoracically inoculated with 0.3 μL of ≈4.7 log10 PFU/mL of Zika virus (MR766 strain) as described  according to the manufacturer’s protocol. Real-time RT-PCR (sensitivity limit 25 RNA copies) (t) <38 were considered positive. In addition, day 0 control mosquitoes that had been stored at −80°C and all mosquitoes collected on days 2–30 were assayed for viable virus by using Vero plaque assay cell culture as described   . Viable 0 PFU/mL . The remAlthough virus titers of desiccated mosquitoes had been reduced, evidence of residual infectivity indicates that precautions, such as use of proper personal protective equipment and appropriate biosafety protocols, are required when handling field-collected samples regardless of storage conditions because these samples might contain viable virus. The large reduction of infectivity in desiccated samples collected on days 2–10 and the complete reduction of infectivity in samples beyond day 10 confirm that a cold chain is required if infectious virus retrieval is the goal of the field collection effort.t values for day 0 control samples ranged from 23.9 to 29.1 , with the exception of 1 sample from day 18 from which virus RNA could not be detected (–Maintaining a cold chain is less critical for detecting Zika virus RNA in mosquito pools. Real-time RT-PCR C to 29.1 . Althougdetected . StabiliResults from this study suggest that, although Zika virus RNA in samples kept at room temperature degrades with time, qualitative detection by real-time RT-PCR is possible for up to 30 days. Although agencies should strive to implement recommended protocols and maintain a cold chain, this is not always possible when performing field-based entomologic arbovirus surveillance, particularly in remote areas where dry ice and ultralow freezers are absent. If the goal of a surveillance program is to determine the infection rate in mosquito populations as efficiently as possible, and is not focused on recovery of live virus, storage and shipment from the field to the testing laboratory under less than optimal conditions would be sufficient for virus detection by using real-time RT-PCR.


Pathogen reduction technologies are among methods to eliminate transfusion transmitted infections. Mirasol method using riboflavin in combination with ultraviolet rays is one of them. The aims of this study were to investigate the effectiveness of Mirasol method to inactivate some model pathogens as well as examination of the sensitivity of plasma proteins after treatment.2) were employed to inactivate model pathogens. Four standard viruses were used in this study including Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus1 (HSV-1), Bovine Viral Diarrhea Virus (BVDV) and Polio Virus. 50% Tissue Culture Infectious Dose (TCID50) and Reed–Muench Methods were used to estimate viruses’ titers. E. coli and Staphylococcus aureus were used as bacterial models. Four plasma proteins including factor V, VIII, fibrinogen and antithromin were used to determine their sensitivity to pathogen inactivation treatment.Riboflavin in 50μM concentration and ultraviolet (365 nm) in three different energy doses (3.6, 7.2, and 10.8 j/cm2 that is corresponding to Log 6.10 for BVDV, Log 6.09 for HSV-1, Log 6.62 for VSV and Log 3.36 for Polio. Bacterial reduction titer was Log 6.94 for E. coli and Log 7.00 for S. aureus. Indicator proteins for plasma activity were determined to be 75% for factor V, 88% for factor VIII, 52% for fibrinogen and 94% for antithrombin.The most pathogen reduction titre was determined for 15 minutes irradiation period equal to 10.8 J/cmResults showed that the employed method can inactivate most of the pathogens in fresh frozen plasma. The acceptable activities of selected plasma proteins remained after treatment. Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) and emerging or re-emerging viruses such as West Nile Virus or Chikungunya Virus which there is no method to detect them , have been developed to prevent transfusion transmitted infections. Some methods using photosensitisers as a key character, which absorb light energy and, in the ensuing electronically excited species, undergo redox reaction with surrounding molecules. Most current methods target nucleic acids and lead to pathogen inactivation .Riboflavin in combination with long wave Ultra Violet (UV) is one of the effective PRTs. Riboflavin as Vitamin B2, is an essential nutrient in humans and it is generally recognized as Safe (GRS) . The safBovine Viral Diarrhea Virus (BVDV), Herpes Simplex Virus 1 (HSV-I), Vesicular Stomatitis Virus (VSV) and Poliomyelitis Virus (Polio), to represent enveloped, non-enveloped and both RNA and DNA V Viruses were selected for this study. All four strains obtained from virus bank of Iranian Blood Transfusion Organization (IBTO)  .2.Madin-Darby Bovine Kidney (MDBK), African Green Monkey Kidney (Vero), and Cervix Carcinoma (HeLa) cell lines were used to propagate and quantify model viruses. All cell lines were obtained from National Cell Bank of Iran (NCBI), Pasteur Institute of Iran. The cells were grown in monolayers in Dulbecco Modified Eagle Medium (DMEM)  with 5% Fetal Bovine Serum (FBS) (Gibco) and 1% Pen-Strep (Sigma) at 37°C in a humid atmosphere of 5% CO50), were used to quantify viruses. Model viruses propagated in mono-layer cell lines and their infectivity titer were determined by Reed and Muench method employing 10 fold dilution series four replicate culture per dilution (6 according to WHO guidelines for PRT (Pathogen Reduction Technologies) (50% Tissue Culture Infectious Dose (TCIDdilution . Each viologies) . Then viE. coli (ATCC:25922) and S. aureus (ATCC: 25923). They cultured in nutrient agar for 24 hours at 37°C. Then a suspension of each organism was prepared in sterile phosphate buffer saline (PBS) (pH=7.4) to reach 106 CFU/ml.With respect to the most common bacterial contamination in blood transfusion two Gram-positive and Gram-negative bacteria were selected for this study: FFP samples have been obtained from IBTO. They were negative for HIV (Human Immunodeficiency Virus), HCV (Hepatitis C Virus) and HBV (Hepatitis B Virus) and stored at −20°C until used. All of them were O positive.To determine the quality of FFP before and after riboflavin and UV treatment, 4 plasma proteins including factor V, factor VIII, antithrombin and fibrinogen were selected as indicators to examine their concentration before and after treatment.Riboflavin was obtained from Darou Pakhsh Holding Co and stored at 25°C in dark before use. After that it was suspended in PBS and used in 50 μM final concentration in FFP samples.2 respectively.The source of irradiation was a UV Irradiator from HAMAMATSU (lighting lure series LC5 model). It could irradiate UV in 200–400 nm and its optimum irradiation was in 365nm. The energy dose in samples depends on their distance from the source as 12cm distance designed to reach optimum energy and also 3 different exposure times of 5, 10 and 15 minutes were employed to obtain different energy dose to identify optimum dose for inactivation of pathogens. The final calculated energy dose for each exposure time were 3.6, 7.2, and 10.8 j/cm6 TCID50 for virus and 106 CFU for bacteria per ml. Then riboflavin in a final concentration of 50μM was added to each sample  was used for data analysis.The original titer of BVDV, HSV, VSV and Polio Viruses were 6.54, 6.82, 7.07 and 6.66 respectively. Using riboflavin in 50μM final concentration in associated with UV in 365nm and in three different times reduced BVDV log in FFP samples to 6.10 .For HSV-1 best log reduction was 6.09 for 15 minutes irradiation, and VSV with the same exposure times showed a 6.62 log reduction. Polio virus was the most resistance virus that showed only a 3.36 log reduction after 15 minutes irradiation in presence of the riboflavin. The results were compared to the controls.E. coli was 107.7 CFU/ml and for S. aureus was 107.0 CFU/ ml. E. coli showed Log 6.94 CFU/ml reduction after 15 minutes treatment and S. aureus howed more sensitivity to riboflavin and UV treatment that log 7 CFU/ml reduction has been shown after 10 minutes (The original titer for  minutes .The results showed selected plasma proteins remained in normal range after riboflavin in combination with UV treatment except fibrinogen which was the most sensitive factor to this method. Antithrombin was the most resistance one. 2 were used. Marschner and Goodrich showed VSV titer reduced 6.3 Log using the Mirasol method in comparison with current study although they use Sindbis Virus as a model for HCV that showed a 3.2 log reduction, in this study BVDV model virus for HCV recommended by WHO guidelines was used and showed a 6.10 Log reduction using 10.8 j/cm2 energy dose (Blood-borne pathogens removal or reduction to the acceptable level always considered as a critical point in blood transfusion process. Although the efficiency of present methods, including screening blood donor and testing blood samples are useful to improve blood component quality, but still remain some risks, especially because of emerging or re-emerging viruses and critical window period for detection of some viruses , 5, 15. rgy dose .In current study Poliovirus employed as a model virus for non- enveloped that showed more resistant to this PRT method, in this way, other study shows Human B19 Parvo virus 5 Log reduction after using Mirasol method. There are no published data available on HSV and VSV reporting the sensitivity to riboflavin in combination with UV blood-borne pathogens removal or reduction to acceptance level .Staphylococcus epidermis, S. aureus and E. coli sensitivity to Mirasol method. In current study the same results for E. coli and S. aureus, was observed. However, E. coli showed high sensitivity to treatment, as more than 6 Log reduction after 15 minutes was observed (The bacterial selection in this study was based on their high contamination rate in blood transfusion. Previous studies showed observed , 11.Marsh and et al. showed antitrombin can save 90% of its activity after using the Mirasol method and is the most resistant protein and fibrinogen lost 30% of its activity and is the most sensitive one. The current results were near to it and all plasma factors remained in normal range after pathogen inactivation , 17.2 is the most effective energy dose, although, 7.2 J/cm2 still is remaining as an alternative to save more plasma proteins’ activity.The results showed the PRT treatment could inactivate most pathogens including both bacterial and viral contamination at the much closed level to WHO guideline, and is a safe and acceptable method for pathogen reduction in blood transfusion centers. A 10.8 J.cm


The complete genome of Zika virus (ZIKV) strain CVD_06-274 was isolated from the serum of an infected patient in Thailand in 2006. Phylogenetic analysis showed that this strain belongs to the Asian lineage and also high titers in Vero cells (RCB 10-87). It has potential for development as an inactivated ZIKV vaccine. Flavivirus, family Flaviviridae. This family also includes dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) (Zika virus (ZIKV) is an icosahedral enveloped arbovirus. ZIKV is a member of the genus us (YFV) . The ZIKus (YFV) . The strus (YFV) . This phToxorhynchites splendens mosquitoes for 3 passages . The serum sample was inoculated into passages  and ZIKVhttps://www.ncbi.nlm.nih.gov/BLAST). The whole-genome sequence of the CVD_06-274 isolate and other ZIKV strains from GenBank were used for phylogenetic tree analysis. A neighbor-joining tree was constructed with 1,000 bootstrap replicates using MEGA6  and converted to cDNA using the Maxima H Minus first-strand cDNA synthesis kit . cDNA was amplified using the Phusion Flash high-fidelity PCR master mix (Thermo Scientific). A set of ZIKV whole-genome sequencing primers  was usedng MEGA6 .10 PFU/ml in WHO-certified Vero cells, on a par with inactivated-ZIKV vaccine strain PRVABC59 (GenBank accession number KU501215), which was developed by the Walter Reed Army Institute of Research   and SK403/13AS strain . A phylogenetic tree revealed that the CVD_06-274 isolate belongs to the Asian lineage. CVD_06-274 produces a medium plaque size (2 to 3 mm) in LLC-MK2 cells. This virus showed a high level of replication at a peak titer of 8 logIR, USA) . An inacMG645981.The assembled complete genome sequence of the Zika virus CVD_06-274 was submitted to GenBank under the accession number


Hepadnaviridae, tentatively named domestic cat hepadnavirus, was discovered in a lymphoma sample and its complete 3187 bp genome characterized. Phylogenetic analysis placed the domestic cat hepadnavirus as a divergent member of mammalian orthohepadnaviruses that exhibits no close relationship to any other virus. DNA extracted from whole blood from pet cats was positive for the novel hepadnavirus by PCR in 6 of 60 (10%) FIV-infected cats and 2 of 63 (3.2%) FIV-uninfected cats. The higher prevalence of hepadnavirus viraemia detected in FIV-infected cats mirrors that seen in human immunodeficiency virus-infected humans coinfected with hepatitis B virus. In summary, we report the first hepadnavirus infection in a carnivore and the first in a companion animal. The natural history, epidemiology and pathogenic potential of domestic cat hepadnavirus merits additional investigation.High-throughput transcriptome sequencing allows for the unbiased detection of viruses in host tissues. The application of this technique to immunosuppressed animals facilitates the detection of viruses that might otherwise be excluded or contained in immunocompetent individuals. To identify potential viral pathogens infecting domestic cats we performed high-throughput transcriptome sequencing of tissues from cats infected with feline immunodeficiency virus (FIV). A novel member of the  Orthohepadnavirus, family Hepadnaviridae, are partially double-stranded DNA viruses that infect a variety of mammals including primates, bats and rodents. The type species, hepatitis B virus (HBV), is a major public health problem; chronic HBV infection increases the risk of liver diseases including cirrhosis and hepatocellular carcinoma, and an estimated 257 million people are currently living with HBV [Viruses of the genus with HBV .We identified a new hepadnavirus in a domestic cat from Australia. In 2016, a seven year-old male-neutered domestic shorthair cat was presented for vomiting and weight loss. On physical examination a large mid-abdominal mass was palpated. Fine needle aspirate cytology of the mass was consistent with large cell lymphoma. The owners requested euthanasia and necropsy and consented to tissue collection approved by the University of Sydney animal ethics committee, (2014/626). Multicentric, large cell, high-grade, B-cell lymphoma was diagnosed on immunohistopathology of formalin-fixed paraffin embedded tissue. Infection with the immunosuppressive lentivirus, feline immunodeficiency virus (FIV), was diagnosed by serology for anti-FIV antibody  and PCR of tumour DNA confirmed by sequencing as described previously [Total RNA extracted from frozen lymphoma was enriched for non-ribosomal RNA and Illumina TruSeq paired-end libraries were prepared. Briefly, 30 mg of lymphoma was homogenised using 1.4 mm ceramic beads , total RNA was extracted using the Qiagen RNeasy mini kit  followed by on-column DNA removal to remove genomic DNA. RNA quality, examined using an Agilent Bioanalyzer 2100 , demonstrated RNA integrity numbers greater than eight. Library preparation was performed using a TruSeq RNA library preparation kit  and ribosomal RNA was removed using a Ribo-Zero gold epidemiology kit . RNA sequencing of 100 bp pair-end libraries on an Illumina HiSeq 2500 platform  yielded 321,297,408 total reads. Sequencing reads were uploaded onto the NCBI Sequence Read Archive (SRA) database under the BioProject submission SUB3718346. −10 and against the non-redundant protein database (nr) using Diamond version 0.8.28.90 with an E-value threshold of 1 × 10−4 [To identify potential viral transcripts, sequencing reads were assembled de novo using the Trinity software (version 2.1.1) . The resOrthohepadnavirus, which we tentatively named domestic cat hepadnavirus. The complete genome sequence of domestic cat hepadnavirus has been deposited in GenBank, accession number MH307930.To confirm the presence of the hepadnavirus, DNA was extracted from frozen lymphoma and examined with a panhepadnavirus PCR assay targeting the polymerase ORF . A 250 bHepadnaviridae family downloaded from GenBank. To show the phylogenetic diversity of vertebrate hepadnavirusues in the P protein tree, homologous sequences representative of the viral diversity in other vertebrates [To determine the evolutionary relationships of domestic cat hepadnavirus, we compared it to reference sequences of P proteins from the entire tebrates ,7 were atebrates . Howevertebrates , and alltebrates . This prPhylogenetic trees of all three data sets were inferred using the maximum likelihood approach available in PhyML 3.0, employing the LG + Γ model of amino acid substitution and a SPR branch-swapping algorithm . In the Hepadnaviruses are typically hepatotropic, although viraemia is common in immunosuppressed hosts . To inveIn conclusion, we report the first hepadnavirus infection in a member of the mammalian order Carnivora and the first report of a hepadnavirus in a companion species, with hepadnavirus viraemia commonly detected among FIV-infected cats. Future investigation of the natural history and epidemiology of domestic cat hepadnavirus and its impact on feline health is clearly warranted.


Shortening of the telomeres occurs during genome replication, thereby limiting the replication potential of somatic cells. To counteract this shortening, vertebrates encode the telomerase complex that maintains telomere length in certain cell types via de novo addition of telomeric repeats. Several herpesviruses, including the highly oncogenic alphaherpesvirus Marek’s disease virus (MDV), harbor telomeric repeats (TMR) identical to the host telomere sequences at the ends of their linear genomes. These TMR facilitate the integration of the MDV genome into host telomeres during latency, allowing the virus to persist in the host for life. Integration into host telomeres is critical for disease and tumor induction by MDV, but also enables efficient reactivation of the integrated virus genome. In addition to the TMR, MDV also encodes a telomerase RNA subunit (vTR) that shares 88% sequence identity with the telomerase RNA in chicken (chTR). vTR is highly expressed during all stages of the virus lifecycle, enhances telomerase activity and plays an important role in MDV-induced tumor formation. This review will focus on the recent advances in understanding the role of viral TMR and vTR in MDV pathogenesis, integration and tumorigenesis.Telomeres protect the ends of vertebrate chromosomes from deterioration and consist of tandem nucleotide repeats (TTAGGG) Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes vast economic losses in the poultry industry worldwide . MDV, alIn infected chickens, MDV productively replicates in B-cells that transfer the virus to T-cells ,17. The S and UL) that are flanked by terminal (TRL and TRS) and internal (IRL and IRS) inverted repeat regions  flanking the unique short region (US). Finally, five class E herpesviruses harbor TMR arrays  that are present in the a-like sequences at the ends of the virus genome and the IRL-IRS junction as described for MDV.Aside from MDV, a number of herpesviruses genomes harbor TMR arrays identical to the host telomere sequences . To date genomes , as revi genomes . The onl genomes . Three hSo far, integration into host chromosomes has been shown for six of the telomere herpesviruses, namely: MDV, HHV-6A, HHV-6B, HHV-7, GaHV-3 and MeHV-1 ,25,30,31roseola infantum), while the level of HHV-6A infection and pathogenic burden remain less well understood [HHV-6A and HHV-6B are two closely related betaherpesviruses. HHV-6B infects most humans during early childhood, causing a febrile illness with a rash  can integrate into host chromosomes . GaHV-3 inst MDV . Upon ininst MDV . Similarinst MDV . Future Several cellular and viral factors have been proposed to be involved in the integration process. Two viral factors that could facilitate MDV integration are UL12 and UL29. They are homologues of the herpes simplex virus 1 (HSV-1) genes UL12 and UL29 (ICP8), which encode a 5′-3′ exonuclease and a single-strand DNA binding protein respectively. Both of these viral proteins have been shown to facilitate recombination during replication of the HSV-1 genome and act through a single strand annealing (SSA) DNA repair pathway ,54,55. WAnother viral protein that could facilitate integration of HHV-6A and HHV-6B is the putative viral recombinase U94. U94 is an orthologue of the adeno-associated virus 2 (AAV-2) integrase (Rep68), and is highly conserved amongst all HHV-6 strains . ExpressAvian and mammalian cells encode two central recombinases involved in homologous DNA recombination (HR), Rad51 and DMC1. While DMC1 is restricted to meiotic cells , Rad51 iL and IRL regions that are located between the TMR and the vIL-8 gene  . These mTo investigate if the tumor promoting functions of vTR are dependent on the telomerase activity, we generated a mutant MDV where the base paring of the P6.1 stem of vTR was disrupted . MutatiovTR is highly expressed during all stages of the virus lifecycle including lytic virus replication, persistent infection and in MDV-induced tumor cells . It is tPrevious studies have revealed that mutation of the hTR template sequence led to the incorporation of incorrect telomeric repeats into telomeres, resulting in telomere instability and ultimately apoptosis . MoreoveA number of viruses can integrate their genome into host chromosomes during their virus lifecycle.Some viruses such as adeno-associated viruses (AAV) integrate into human genome at a specific site (AAVS1), while others such as retroviruses can integrate across the entire host genome . MDV andIn addition to the TMR, the MDV genome also encodes vTR that has a high sequence identity to the host chTR, suggesting that the gene was acquired from the host genome during evolution. vTR is expressed during lytic and latent stages of MDV infection and is the most abundant transcript in MDV-transformed cells. Furthermore, it has been shown that overexpression of vTR plays an important role in tumor formation. Intriguingly, overexpression of human telomerase RNA (hTR) has been shown to possess anti-apoptotic functions that are independent of its role in telomerase activity. Similarly, telomerase-independent functions of vTR are crucial for efficient tumor formation; however, the exact mechanism behind this remains unknown. The interaction between vTR and RpL22 could play an important role in tumor formation and should be addressed in future studies. Finally, further work remains to be done in order to understand the contribution of vTR and cellular TRs towards the development of cancer.


Zika virus, an emerging serious infectious disease, is a threat to persons living or travelling to regions where it is currently endemic, and also to contacts of infected individuals. The aim of this study was to assess knowledge about this new public health threat to persons residing in a Middle Eastern country.We conducted a survey at several international universities in Qatar to assess knowledge and awareness about this disease. An adapted version of the survey was also conducted using online channels from Qatar.poor” (66%), “basic” (27%) or “broad” (7%). Forty-five (16%) persons with poor knowledge considered themselves to be well-informed.The median age of the 446 participants, was 25 years, 280 (63%) were females, and 32% were from Gulf Cooperation Council (GCC) or other Middle East countries. Based upon their knowledge about availability of a vaccine, role of mosquitoes and other modes of transmission, and disease complications, we classified respondent’s knowledge as “This report from a sample of persons associated with Middle East educational complex, reveals inadequate knowledge about Zika virus, a serious emerging infectious disease. Although few cases have been reported from the region, future cases are possible, since this area is a transit hub connecting currently infected regions to North America, Europe and Asia. As a preventive measure, an educational program about Zika virus would be valuable, especially for individuals or family members travelling to afflicted regions. Transmission of Zika virus has been reported in multiple countries throughout the world . AlthougAedes albopictus and Aedes aegypti mosquitoes) are present [Aedes aegypti for Zika virus [Due to the threat it presents, all nations around the world need to be vigilant about Zika virus’s potential to spread to remote regions including the Middle East , where p present , 8. Qataka virus . Qatar ASuccessful disease preventive programs depend upon public awareness of risk factors and disease characteristics. This study aims to assess the knowledge about the Zika virus and its risks among residents in Qatar.We conducted a survey in Education City, which houses a multinational population of approximately 5750 students, faculty, and staff who come from countries around the world to study and work in Qatar. The sample size was estimated by contacting Student Affairs and Human Resource Divisions of the institutions housed in Education City. The sample size of 396 was estimated using a margin of error of 5% and 95% confidence interval levels to appropriately represent the population in Education City. An additional 10% of the estimated sample size was recruited to account for attrition and incomplete responses. An English language survey adapted from existing literature was used to gather socio-demographic information, and questions evaluating the knowledge and perceptions towards Zika virus, such as communicability, availability of vaccine, symptoms and treatment. No personally identifiable information was collected. Trained personnel from the Division of Global and Public Health at Weill Cornell Medicine – Qatar approached potential subjects in person to conduct the self-administered survey. All recruited participants were 18 years or above. Data was collected from August – September 2016 and entered in an ad-hoc database.QatarLiving.com and iLoveQatar.net to reach a wider sample of the general public. Participants were also recruited via online advertising on Google search results and through the social media channel of Facebook. As part of the survey, all participants had to check off that they were above 18 years of age and resident in Qatar at the time of participation. The online recruitment was open for the same period as the self-administered survey version.An online version of the survey was posted on the internet along with an Arabic translation on the Qatar-based websites The self-administered survey questionnaires  included a series of questions about participant demographic characteristics , general information about Zika virus , personal knowledge about Zika and questions related to participants’ travel to endemic countries.poor” when responders didn’t know that there is currently no vaccine for Zika virus and/or that the disease is transmitted by infected mosquitoes and/or that anyone could get Zika virus; “basic” when responders knew that there is currently no vaccine for Zika virus, that the disease is transmitted by infected mosquitoes and that anyone could get Zika virus; and “broad” when in addition, responders knew that the disease could be transmitted through sexual intercourse, blood transfusion or during pregnancy, and that if a pregnant woman has Zika virus, there are risks for her baby/fetus.Based on the correctness of responses to questions related to facts about Zika virus, we classified participants’ general knowledge about Zika virus as: “p-values were two-sided. P-values <0.05 were considered statistically significant.We used simple descriptive statistics (frequencies and percentage) to describe responses to the questionnaire. The Chi-square test was used to test for differences between groups. We used univariate and multivariable logistic regression to investigate participants’ characteristics that are associated with “basic” or “broad” knowledge about Zika virus, taking those with “poor” knowledge as reference group. Only factors associated with knowledge status at univariate analysis were included in the multivariable logistic regression model, to identify independent predictors. For nationality, we used participants from GCC and other middle east countries as reference group, we grouped participants from Asia and Africa, and those from Europe, North America, Australia and New Zealand. For participants’ role, we used students who represent the largest group as reference. For institution, a preliminary analysis (data not shown) revealed that knowledge was similar for all those non affiliated to a school of medicine or art, and this joint group was used a reference. Analyses were performed with SAS version 9.2 . All . Fifty (11.2%) participants responded to the online survey and 396 (88.8%) to the survey administered in the education city, including 191 (42.8%) students, 162 (36.3%) staff and 42 (9.4%) faculty members. The median age of the participants was 25 years, ranging from 18 to 71 years; 256 (57.4%) were single. Sixty five (14.6%) participants are originating from a GCC country including Qatar, 76 (17.0%) from other Middle East countries; 30 (6.7%) from Africa, 131 (29.4%) from Asia, 31 (7.4%) from Europe and 81 (18.2%) from North America, Australia or New Zealand.The response rate for the online survey was 94%. Demographic characteristics of the 446 participants, 161 (36.1%) males and 280 (62.8%) females, are presented in Table Responses to a first series of questions about the Zika virus are given in Table Sixty eight (15.2%) participants never heard about Zika virus. The most common source of information for those who heard about Zika virus is internet (60.8%), followed by social media (43.7%), television (42.8%), friends or family (16.8%), radio (10.8%) or doctor's office (1.8%).Ninety seven (21.7%) participants thought that a vaccine to prevent Zika virus exists, while 9.6% did not answered this question. Majority (68.6%) knew that no vaccine is currently available.Bite from an infected mosquito (73.3%) was the most frequently reported source of infection with the Zika virus. 192 (43.0%) participants knew that Zika virus could be transmitted from mother-to-child during pregnancy, 126 (28.3%) through sexual intercourse and 103 (23.1%) from a blood transfusion. Against the current evidence, 52 (11.7%) participants thought that Zika virus could also be spread through coughing and sneezing, 37 (8.3%) through breastfeeding, 31 (7.0%) drinking polluted water and 21 (4.7%) eating contaminated food.Three hundred and sixty six (82.1%) participants thought that Zika virus could be transmitted to pregnant women, 304 (68.2%) to children, 269 (60.3%) to adult women and 253 (56.7%) to adult men. In addition, 345 (77.4%) participants knew that if a pregnant woman has Zika virus, there are risks for her baby or fetus. Ninety eight (22.0%) participants were uncertain about this last question and only 3 (0.7%) disagreed.broad” general knowledge about Zika virus (i.e. responded correctly to 10 questions about Zika virus), 122 (27.4%) a “basic” knowledge and 295 (66.1%) no or only “poor” knowledge about Zika virus.When we summarized responses to all items related to the general knowledge about Zika virus, only 29 (6.5%) participants demonstrated to have a “At univariate analysis, males , older participants , those originating from Asia or Africa  or Europe, North America, Australia or New Zealand  compared to those originating from GCC or other Middle East countries, staff , faculty members  or responders to the online survey  compared to students and those at a school of medicine (WCM-Q)  were more likely to have higher general knowledge about Zika virus, while those at an artistic school (VCU-Q)  had lower general knowledge about Zika virus  of 114 who reported not being knowledgeable about the risk of Zika virus and 65 (28.3%) of 230 with neutral response (P < 0.0001).One hundred and two (23.1%) participants recognized to be knowledgeable about the risk of Zika virus, 114 (25.6%) disagree and 230 (51.6) were not sure. Participants’ self perception correlated with their actual knowledge about Zika virus: 57 (55.9%) of the 102 participants who reported being knowledgeable about the risk of Zika virus have a “poor” knowledge about Zika virus. The frequency of participants with such an erroneous perception of knowledge about the risk of Zika virus was not associated with gender, age, nationality, marital status, education or participants group. It was however significantly higher (25.0%) among the 48 participants from artistic school (VCU-Q) (P < 0.0001).In total, 45 (10.1%) participants reported to be knowledgeable but actually had a “they can control their exposure to Zika virus”, 140 (31.4%) reported that “the thought of Zika virus makes them feel anxious or worried”, 96 (21.5%) thought that “Zika virus can be treated”, 225 (50.4%) that “Zika virus could be prevented” and 108 (24.2%) that “exposure to Zika virus always leads to sickness”  participants thought that “Only 22 (4.9%) participants reported having travelled to any of the listed countries, where Zika virus is endemic, in the past year.The aim of this study was to determine the level of knowledge about Zika virus--an emerging potentially serious viral infection in a sample of educated persons living in Qatar, one of the GCC nations. Understanding the level of disease knowledge in the general population is an important public health measure for prevention or control of any disease. Although not a region where the disease is prevalent, there are many flights from the Middle East to countries or areas where the disease is widespread, raising the real possibility that returning passengers who have contracted Zika virus, could unknowingly transmit this disease. Returning Zika virus-infected patients have already been documented in the Middle East .Our survey was designed to capture basic knowledge covering areas such as mode of disease transmission, disease prevention, susceptible populations, and availability of a protective vaccine. We also included several self-assessment questions to determine if there was a correlation between respondents actual knowledge and their perception of what they knew about this disease.Although not a representative population sample, we believe that the education level of the respondents is higher than the background population, implying that the knowledge level about Zika virus in the general population is likely to be lower than in our sample. We have no information about Zika virus awareness in regional health care workers, but in the sample of medical students the responses indicated that about a third of this group were poorly informed and none had a broad knowledge of basic facts about Zika virus.We carried out the study at about the time of the 2016 Olympics in Brazil, a period when there was great international interest about the threat of Zika virus to athletes and to travelers. Despite the widespread global media interest at the time of the survey, the questionnaire revealed that many respondents are unaware of critical information concerning Zika virus. Fifteen percent of respondents had never heard of Zika virus, 27% were unaware that mosquitoes were involved in disease transmission, and 22% believed there is already a protective vaccine. Another disturbing finding is that of the 102 respondents who considered themselves knowledgeable about Zika virus, the survey revealed that nearly half proved to be poorly informed. These findings indicate a need for disseminating information about this disease, especially for persons who will be travelling to areas where the disease is prevalent.P = 0.0001). We do not have a precise explanation for this difference, but expatriate students may be more knowledgeable about Zika than local students, since they would have been likely to be exposed to information about Zika from local sources when travelling to or from their native country. This should be explored further since it is important for public health reasons to understand why a local endemic population does not understand or know much about an emerging infection like Zika virus. Since there was no local outbreak of Zika in the country, the locals may not have been interested as such in learning about the Zika virus. Public health authorities can play a role in increasing awareness and disseminating information about emerging infections within the local population. In addition to student source, type of training was also a significant predictor: students, faculty or staff associated with a medical school performed significantly better (P = 0.009) than other groups, while the equivalent group at an artistic school did significantly worse (P = 0.04).In multivariable analysis, respondents born in  had superior knowledge about Zika virus than respondents born in other countries  which can transmit Zika virus are inhabitants of the region [Is Zika virus a current threat to persons living in the Middle East? Although the prevalence is always likely to remain low, person to person transfer of infection could occur in one of two ways. The first method would be via mosquito transmission from an infected to an unaffected person. This is likely to be rare in this generally arid region, although two primary vector species  and it is likely that a more representative sample of less well-educated persons from this region would have a lower knowledge level than observed in this sample. Another strength is that the survey included respondents from many different countries, thus providing regional comparative data about health knowledge for this disease. The main study limitation is the non-representative population sampling strategy. Since the in-person survey was administered only in Education City, Qatar, this data has local significance. However, we believe that information gathered from this privileged sample could be important to healthcare givers, identifying an area where health promotion and disease prevention programs could be established. Furthermore, even though this study is essentially single-site as may be the case for previously published studies, these findings can be extrapolated to other populations elsewhere.This study reveals a lack of the local population engaging with information about Zika virus--a potentially serious emerging infectious disease—in an educated sample of individuals residing in a Middle East Country to date. There have been few reported patients with Zika virus in this region, but there is the real potential for persons to contract the disease from previously infected persons returning to the region. Information programs could be especially important, especially for individuals planning to travel to any one of the many countries where Zika virus is now prevalent.


Sentinel, enhanced passive, and syndromic surveillance in 2016 did not identify any evidence of transmission. Aedes albopictus mosquitoes prompted the New York City Department of Health and Mental Hygiene to conduct sentinel, enhanced passive, and syndromic surveillance for locally acquired mosquitoborne Zika virus infections in New York City, NY, USA, during June–October 2016. Suspected case-patients were those >5 years of age without a travel history or sexual exposure who had >3 compatible signs/symptoms . We identified 15 suspected cases and tested urine samples for Zika virus by using real-time reverse transcription PCR; all results were negative. We identified 308 emergency department visits for Zika-like illness, 40,073 visits for fever, and 17 unique spatiotemporal clusters of visits for fever. We identified no evidence of local transmission. Our experience offers possible surveillance tools for jurisdictions concerned about local mosquitoborne Zika virus or other arboviral transmission.A large number of imported cases of Zika virus infection and the potential for transmission by  Flavivirus, has spread rapidly across Latin America and the Caribbean region after an epidemic was identified in Brazil in early 2015 , NY, USA; however, the less-efficient mosquito vector Ae. albopictus is present throughout the city  interim response plan for Zika recommends enhanced surveillance in areas with Ae. albopictus mosquito breeding, or areas with large adult Ae. albopictus mosquito populations.DOHMH identified the first travel-associated Zika cases in NYC in January 2016 ; no history of travel to a Zika-affected area in the previous 4 weeks; no history of sex with a person who traveled to a Zika-affected area in the previous 4 weeks; and urine specimen collected within 14 days after illness onset.Enhanced passive surveillance is an amplification of standard passive surveillance in which public health agencies send notifications to healthcare providers and facilities to remain alert for suspected cases of a particular disease or condition . During these interviews, investigators asked patients whether they had any nonsexual household contacts who developed Zika-like signs/symptoms and whether the contact had traveled to a Zika-affected area. Any reports of symptomatic, nonsexual household contacts without travel were assessed for risk and referred to testing if appropriate.>3 mL of urine in sterile tubes. According to CDC guidelines, urine must have been obtained within 14 days after illness onset to be eligible for testing , cluster size relative to the estimated <200 m range of the Ae. albopictus mosquito, and any geographic overlap with areas at high risk for Zika virus importation  were reported from 5 sentinel sites and 7 (47%) from 6 nonsentinel sites , includiThe median diagnosis time for all submissions was 46.3 hours (IQR 27–98 hours), and the median testing time was 6.2 hours (IQR 6–26 hours). No specimens had Zika virus RNA detected by rRT-PCR. Ae. albopictus mosquitoes . We detected 17 unique spatiotemporal fever syndrome clusters. Upon investigation, we discarded 2 clusters with low recurrence intervals, because these clusters would not have signaled after applying the final signaling threshold. We examined 14 other clusters that we identified as artifacts resulting from 17 hospitals transitioning during the analysis period to using HL7 international reporting standards. The transition resulted in longer fields for the chief complaint, and thus more opportunity to include a fever keyword, causing localized increased syndrome capture compared with the baseline. The remaining cluster did not overlap with areas classified as high risk for Zika importation and had a radius of 11.1 km, inconsistent with the estimated <200 m range of We identified 308 ED visits for Zika-like illness during June 1–October 31, 2016 ; daily mBased on the case finding pilot, we established that follow-up case investigation was not necessary when the visit text had one of the following: mention of recent travel to a country or territory with local Zika virus transmission, an alternate diagnosis listed, or a chief complaint containing “Zika.” Following this protocol, during August 5–October 31, 2016, a total of 12 of 163 Zika-like illness visits qualified for additional follow-up: 8 were found to have traveled to Zika-affected countries, but this information was not noted in the original chief complaint or travel history data fields; 1 patient had already been tested for Zika virus; 1 patient was ruled out because medical records noted allergic reaction with no fever; 1 patient was admitted to labor and delivery from the ED and the ED medical record was inaccessible to the hospital staff contact; and 1 patient could not be matched to hospital records on the basis of information available in the syndromic record.During the peak period of travel-associated Zika cases and the mosquito season in NYC in 2016, DOHMH used case investigation, sentinel, enhanced passive, and syndromic surveillance to monitor for the presence of local mosquitoborne Zika virus transmission. None of the systems detected any local mosquitoborne transmission.During the sentinel surveillance period, healthcare providers identified 15 NYC residents without recent travel history or sexual exposure who met the sentinel case definition for Zika virus; all tested negative for Zika virus. Only 21% of sentinel sites reported any suspected cases, which might indicate a lack of engagement of some sites, inappropriate selection of site locations, or a true lack of patients who met the criteria. Future iterations of a sentinel surveillance program should include an evaluation component, such as the deployment of a mock patient with locally acquired Zika virus infection or a review of medical records, to evaluate provider awareness and effectiveness of the sentinel sites.Although DOHMH targeted efforts to identify suspected locally acquired cases at presumed higher-risk sentinel sites, 47% of patients tested for Zika virus were seen at nonsentinel sites. We believe this might have been because Zika virus infection is a reportable disease in New York and healthcare facilities were already sending specimens from suspected travel-associated Zika virus cases to PHL for testing , followed by fever (82%) and arthralgia (66%)  of ED visits for Zika-like illness met protocol criteria for DOHMH staff to follow up with hospital infection control practitioners regarding patient travel history. Follow-up activities would be challenging to sustain with a higher volume of cases. The pilot study highlighted the importance of collecting travel history data and led DOHMH to request during the study period that clinical facilities include an additional travel history field in the daily ED data transmission to improve capture of patient travel. Continued collaboration between DOHMH and EDs to improve travel data will facilitate surveillance for Zika virus as well as for other travel-associated diseases.A limitation of the syndromic surveillance system was the incomplete transition of all hospitals to HL7 international reporting standards, resulting in differences in the average number of terms in the ED chief complaint per hospital and precluding use of a more specific, multiple-symptom definition for cluster detection. For the Zika-like illness linelist, requiring at least 3 signs/symptoms of Zika virus infection biased case detection toward hospitals that provide more detailed chief complaints, which might not have corresponded to areas where the risk of local mosquitoborne transmission was highest in NYC.,The major limitation of all surveillance for local transmission of Zika virus is that 80% of Zika virus infections are asymptomatic; therefore, any surveillance system reliant on the presence of symptoms will underestimate the true incidence of infection (Ae. aegypti mosquitoes, the multipronged surveillance approach taken by NYC DOHMH during the first year of the epidemic was considered a robust plan to detect local mosquito transmission of Zika virus. The likelihood that another vectorborne infection will emerge somewhere in the world is high, and surveillance is a critical tool for the detection and evaluation of control measures (,Despite the stated limitations, our experience suggests that enhanced passive surveillance, with frequent outreach to providers in communities with large numbers of travel-associated human cases or habitats conducive to potentially competent mosquito vectors, was an efficient and manageable method to monitor for locally transmitted mosquitoborne Zika virus infection in NYC. Given that NYC does not have


Using 2012 data on fee-for-service Medicare claims, we documented regional and county variation in incremental standardized costs of heart disease  by type of service . Absolute incremental total costs varied by region. Although the largest absolute incremental total costs of heart disease were concentrated in southern and Appalachian counties, geographic patterns of costs varied by type of service. These data can be used to inform development of policies and payment models that address the observed geographic disparities. Treatment of heart disease is one of the largest drivers of health care costs in the United States, accounting for approximately $273 billion annually . AlthougInternational Classification of Diseases, Ninth Revision, Clinical Modification) codes for heart disease are included in the HCC except codes 390–398 (acute and chronic rheumatic fever). We calculated total costs by summing Medicare payments, beneficiary payments, and third-party payments for the following service categories: inpatient, outpatient, post-acute care, hospice, physician, procedure/imaging, and other. Because calculating costs incurred directly for heart disease care is not possible, we calculated incremental costs incurred among beneficiaries with heart disease above and beyond the costs incurred by beneficiaries without heart disease. Incremental costs were calculated as the difference between mean annual costs per capita for beneficiaries with heart disease and beneficiaries without heart disease in 2 ways: 1) as the absolute difference, by subtracting the cost per beneficiary without heart disease from the cost per beneficiary with heart disease, and 2) as the relative difference, by dividing the cost per beneficiary with heart disease by the cost per beneficiary without heart disease. Incremental costs were age-standardized (2000 standard US population age 65 years or older) and analyzed by region, county, and type of service. We generated county-level maps of the prevalence of heart disease and the age-standardized absolute incremental values for total costs, inpatient costs, and outpatient costs of heart disease. Subnational cost analyses were adjusted to eliminate geographic differences in payment rates  (We used 2012 data from the Centers for Medicare & Medicaid Services’ (CMS) Chronic Conditions Data Warehouse, a database with 100% of fee-for-service Medicare enrollment and claims data . We limiTotal costs among beneficiaries with heart disease was approximately $96 billion in 2012. The absolute and relative incremental total costs of heart disease per beneficiary were $10,345 and 2.6 per capita, respectively. Inpatient, post-acute care, and outpatient services accounted for the vast proportion of total absolute incremental costs . Relative incremental costs of heart disease varied minimally by region  . HoweverWe found substantial absolute incremental costs of heart disease among fee-for-service Medicare beneficiaries. Inpatient, post-acute care, and outpatient services contributed most to incremental costs. Although the highest absolute incremental total costs of heart disease were concentrated in counties in the Southeast and Appalachia, geographic patterns of cost varied by type of service. For example, counties in the South and southern Appalachia generally had the highest absolute incremental inpatient costs but the lowest absolute incremental outpatient costs.Researchers disagree on which factors drive the regional variation in health care costs among Medicare beneficiaries. Among these factors are the need for medical care  and the provision of care ,7. Our fOne limitation of this study was the use of claims data rather than clinical data to identify beneficiaries with heart disease. However, claims data are reliable in assessing cardiovascular outcomes . Also, sGeographic variation in the incremental costs of care for beneficiaries with heart disease and geographic differences in use of services suggest a non-uniform use of resources to prevent and treat heart disease. Public health, health care systems, and health plans can use these findings to inform development of policies and payment models that address geographic disparities in service use and overall costs for care among Medicare beneficiaries with heart disease.