Aquatic viruses have been extensively studied over the past decade, yet fundamental aspects of freshwater virus communities remain poorly described. Our goal was to characterize virus communities captured in the >0.22 µm size-fraction seasonally and spatially in a freshwater harbour. Community DNA was extracted from water samples and sequenced on an Illumina HiSeq platform. Assembled contigs were annotated as belonging to the virus groups  Caudovirales, Mimiviridae, Phycodnaviridae, and virophages (Lavidaviridae), or to other groups of undefined viruses. Virophages were often the most abundant group, and discrete virophage taxa were remarkably stable across sites and dates despite fluctuations in Mimiviridae community composition. Diverse Mimiviridae contigs were detected in the samples and the two sites contained distinct Mimiviridae communities, suggesting that Mimiviridae are important algal viruses in this system. Caudovirales and Phycodnaviridae were present at low abundances in most samples. Of the 18 environmental parameters tested, only chlorophyll a explained the variation in the data at the order or family level of classification. Overall, our findings provide insight into freshwater virus community assemblages by expanding the documented diversity of freshwater virus communities, highlighting the potential ecological importance of virophages, and revealing distinct communities over small spatial scales. Viruses can modify and control the structure and function of ecosystems and, in turn, influence global biogeochemical cycles and the evolution of organisms . HistoriIn the first viral metagenomics study, over 65% of sequences recovered from surface seawater samples were not significantly similar to any sequence in existing databases, highlighting the lack of knowledge of environmental viruses . Since t4–105 viruses per mL in the photic zone and that the so-called “Megavirales” and Phycodnaviridae were the most common NCLDVs in the epipelagic oceans [The Tara Oceans Expedition was conducted from 2009–2013 with the aim of globally sampling a wide range of organismal and functional diversity in the surface oceans, while the Malaspina Circumnavigation Expedition sailed from December 2010 to July 2011 with a focus on deep ocean microbiology. The Tara Oceans Expedition resulted in several important discoveries related to diverse groups of marine planktonic taxa, including viruses  viruses, as well the as cell- and particle-associated virus community, the data were mined for viral sequences. This allowed us to determine seasonal and spatial patterns of diversity and abundance in this unique freshwater environment. More research is required to better characterize freshwater virus diversity, community structures, and patterns of abundance, and the factors that drive these phenomena. This research aims to address some of these knowledge gaps by providing a detailed view of the >0.22 µm size-fraction virus community in Hamilton Harbour.Water samples were collected from two long-term Environment Canada monitoring sites in Hamilton Harbour (referred to as stations 1001 and 9031 in previous publications). One site (station 1001) is located at the deepest and most central part of the harbour (43°17′17.0″ N 79°50′23.0″ W) with a water depth of 24 m and a 1.2 km distance from the shoreline. The other site (station 9031) is located less than 0.5 km from the shoreline (43°16′50.0″ N 79°52′32.0″ W), with a water depth of 12 m. This “nearshore” site is influenced by effluents from the Cootes Paradise watershed on the west end of the harbour, while the “mid-harbour” site is closer to the Burlington Shipping Canal and Lake Ontario. In 2015, water samples for metagenomic analyses were collected on 30 July 30, 13 August, 27 August, 10 September, and 24 September from both sites at approximately 1 m below the surface using a Van Dorn bottle sampler; in total, 10 samples were collected, 5 from each of the nearshore and mid-harbour sites. Water samples of 500 mL were filtered through 0.22 μm pore-size Sterivex capsule filters , and the filters were sealed and stored at −80 °C until further analysis. For each sample collected, physiochemical parameters including pH, dissolved oxygen, temperature, redox potential, and chlorophyll a were measured in situ using a YSI 58 . Secchi depth was also measured with each sample.g for a total of 15 min. DNA was extracted from the pelleted material using a FastDNA SPIN Kit , beginning with the addition of 500 μL of CLS-Y solution. The manufacturer’s protocol was followed, except the wash step was repeated three times to maximize the removal of environmental contaminants. Following extraction, DNA concentrations were estimated using a NanoDrop ND-1000 UV-Vis Spectrophotometer , and were standardized to 1.5 μg of DNA per sample before being sent for library preparation and shotgun metagenome sequencing by MR DNA .Within a month of each sample collection date, samples were removed from storage at −80 °C and allowed to thaw at room temperature prior to DNA extraction from the Sterivex filters. Biomass was recovered from the filters by adding 2 mL of molecular grade nuclease-free water to each filter and vortexing for 5 min. All resuspended material was sequentially transferred to a 1.5 mL microcentrifuge tube under sterile conditions and centrifuged at 6000× Sample DNA libraries were prepared in December 2015 using a Nextera DNA Sample Preparation Kit , following the manufacturer’s protocol. DNA concentrations were measured using the Qubit dsDNA HS Assay Kit  before and after library preparation . Prior tftp://ftp.ncbi.nih.gov/blast/db/FASTA/nr.gz. DIAMOND version 0.9.19 [−6 e-value cut-off. The March 2018 MEGAN protein accession mapping file was downloaded from http://ab.inf.uni-tuebingen.de/data/software/megan6/download/welcome.html. Quality-controlled reads were mapped back to assembled contigs using Bowtie 2 [After sequencing and base-calling, adapters and barcodes were removed by MR DNA . Read quality parameters were verified using FastQC version 0.11.5  prior ton 0.9.19  was usedn 0.9.19  was usedBowtie 2  in very Bowtie 2 . Table 2Bowtie 2 . The DNAContig relative abundances were estimated after accounting for differences in contig lengths and sequencing depth  per sample. More explicitly, the number of reads that mapped to each contig was divided by the contig length. Then, the relative proportion of each contig within a sample was calculated by dividing the normalized number of reads for that contig by the normalized number of reads for all contigs in the sample. All virus contigs were sorted into one of the following categories based on the NCBI taxonomic classifications: Caudovirales, Mimiviridae, Phycodnaviridae, virophages (Lavidaviridae), Iridoviridae, Poxviridae, other dsDNA viruses, ssDNA viruses, unclassified bacterial viruses, and unclassified viruses. Because the assigned taxonomic classifications for some contigs in the “unclassified bacterial viruses” and “unclassified viruses” categories were less specific than information provided in the literature within which they were originally reported, some contigs in these categories were manually curated and assigned to more specific groups based on published information .As well as assigning some contigs to more specific categories, some of the more specific assignments in the dsDNA viruses group were reassigned to the “other dsDNA viruses” group if they were observed in less than half of the samples and at <0.5% abundance. For example, contigs annotated as Marseilleviridae were only observed in 2 of the 10 samples at <0.2% abundance. Because there was not enough representation to making meaningful comparisons, the Marseilleviridae were grouped together with the “other dsDNA viruses”. As a final note, in one sample, a single contig was annotated as an RNA virus and reassigned to the “unclassified viruses” category .Aureococcus anophagefferens virus (AaV), Chrysochromulina ericina virus (CeV), Phaeocystis pouchetii virus (PpV), Pyramimonas orientalis virus (PoV), and Group I Phaeocystis globosa viruses (PgVs) [There is evidence that some viruses previously and tentatively considered phycodnaviruses  are more closely related to Mimiviridae than Phycodnaviridae, and in fact form a separate subfamily “Mesomimivirinae” within the Mimiviridae family ,41. Props (PgVs) ,43,44. HA boxplot of the overall virus community was generated using the “ggplot2” package  in RStudDiverse virus contigs were identified in Hamilton Harbour and some were classified within the virus groups Caudovirales, Mimiviridae, Phycodnaviridae, and virophages (Lavidaviridae), while others could only be classified as belonging to “unclassified bacteriophages”, “other dsDNA viruses”, and “ssDNA viruses”. Henceforth, for the sake of brevity, we name virus groups only when referring to contigs annotated as viruses from those groups. Overall, virophages were the most abundant , but were also the most variable (from 0.1% to 74.5% abundance). The Mimiviridae were the second most abundant group, comprising an average of 21.1% of the virus community across all samples. Interestingly, though they were intimately associated, the abundances of Mimiviridae (ranging from 8.2% to 36.0% abundance) did not fluctuate to the same extent as the virophages . At the In general, the abundance of different groups of viruses was more variable in the mid-harbour compared to the nearshore samples. Sample similarity based on community composition was assessed using UPGMA clustering of a Bray–Curtis dissimilarity matrix, which reinforced the contrast of the nearshore and mid-harbour sites A. ThoughChrysochromulina ericinia viruses (CeV) were the most abundant Mimiviridae in all mid-harbour samples, except on 10 September  was performed to assess the influence of pH, temperature, redox potential, dissolved oxygen, Secchi depth, and chlorophyll a on the viral groups at each site. The CCA model explained 47.8%  = 0.005) of the variability in the data. A test of 10,000 permutations revealed that the chlorophyll a concentration was the only significant environmental parameter of those assessed. Temperature, pH, dissolved oxygen, Secchi depth, and redox potential were not significant explanatory factors of changes in virus community composition and relative abundances between samples. However, there was a strong inverse relationship of Caudovirales and chlorophyll a concentration. The 27 August and 24 September mid-harbour samples, which were dominated by Caudovirales (>70%), were similar in community composition A and werftp://ftp.ncbi.nlm.nih.gov/refseq/release/release-statistics/). An estimated <1% of the Earth’s virome has been discovered [https://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi; retrieved December 2018). This bias could lead to a higher chance of detecting Caudovirales than virophages, which are more likely to be unannotated and discarded in downstream analyses. In other words, it is likely that many virophages exist that have not yet been discovered and these will be missed in our analyses. On the other hand, annotation is also dependent on diversity within a virus group, as well as contig size. Contig size is directly influenced by genome size, hence, viruses with smaller genomes are more likely to generate longer contigs that are more likely to be annotated. In any case, the remarkably high abundance and dynamic nature of virophages detected in Hamilton Harbour suggests their important ecological role in this system.In this study, we observed diverse virus communities that were both spatially and seasonally variable, and often dominated by virophages. However, as usual for environmental metagenomes, and particularly for viruses, a large portion of sequences remained unclassified and were discarded at the annotation step of the data processing pipeline. Viruses have much fewer sequenced representatives in databases than prokaryotes or eukaryotes and are more likely to remain unannotated. For example, in May 2018, RefSeq released 88 contained >7500 viruses with >325,300 associated accessions . In contrast, there were >50,400 bacteria with >100,583,400 associated accessions  . RelateThe importance of viruses in aquatic environments is well documented. These viruses are estimated to kill approximately 10% of the phytoplankton population and up to 50% of the bacterial population in surface marine waters, with greater impacts in high nutrient environments  virus community composition was very different over the 3 km distance between the sites, while on other dates , the community composition was very similar. Relative abundances of virus orders and families fluctuated at the mid-harbour site much more than the nearshore site, highlighting the high diversity of viruses on local scales and the impact of small-scale environmental differences.Since the recent discoveries of the Mimiviridae and their virophages, few studies have looked at the relative abundances of these groups over the duration of a season. We captured fluctuations in the Mimiviridae community over the summer and observed vastly different communities on the same date at different locations in the harbour. While the nearshore and mid-harbour samples generally contained distinct Mimiviridae communities, the virophage community composition remained consistent. This highlights the complexity of these ecological relationships and the gaps in our understanding of how these intimately associated viruses interact. The stability of virophage community composition, despite the fluctuating Mimiviridae community, appears to support the hypothesis that virophages and their Mimiviridae hosts are not connected solely by infection. The recent discovery that some virophages frequently enter eukaryotic host cells independently, remaining latent until infection by a giant virus where they then compete with the giant virus for its replication machinery , is a poHamilton Harbour is known to support high algal biomass in the summer and early autumn months. Given the large differences in community composition between the nearshore and mid-harbour sites, Mimiviridae appear to be important and dynamic algal viruses in Hamilton Harbour. The most common algae-infecting Mimiviridae was CeV, which was especially abundant at the mid-harbour site. Interestingly, while our sampling of the >0.22 µm fraction may have enriched for some Mimiviridae, we would not expect this to be the case for CeV since their capsid sizes are <0.22 µm. The “other dsDNA viruses” category did not contribute more than 4% in any individual sample and included mostly Mimiviridae. The small percentage of ssDNA viruses that were detected represented only those that were replicating in cells in the dsDNA form, since only dsDNA was targeted in the library preparation and sequencing. Therefore, the ssDNA viruses may be underrepresented compared to other virus groups which were detected as virions adsorbed to and within particles and cells, in addition to viruses actively replicating within cells.Overall, Hamilton Harbour metagenomes included a diverse array of viruses ranging from large dsDNA Mimiviridae to small ssDNA viruses. Relative abundances of virus groups varied widely over relatively small spatial scales within the harbour, with higher consistency in the nearshore samples compared to the mid-harbour samples. Virophage relative abundances ranged widely across all samples, but were the most abundant virus family in most samples. A wide diversity of Mimiviridae was detected in the samples and the two sites appeared to host distinct Mimiviridae communities, suggesting their importance as algal viruses in Hamilton Harbour and likely other freshwater environments. The abundances of discrete virophage taxa were remarkably stable despite the dissimilar Mimiviridae communities at the two sites, highlighting our limited understanding of how these intimately associated viruses interact. Caudovirales and Phycodnaviridae relative abundances were low in most samples, in contrast to other studies of eutrophic freshwater environments. It is important to note the contrasting approach used in this study compared to other viromic studies. We analyzed the >0.22 µm fraction to capture large free virions, virions associated with cells and particulates, and viral genomes integrated into host genomes. Therefore, it is not surprising that the patterns of diversity we observed differed from other viromic studies. Nonetheless, these findings provide additional insights into virus community structures in freshwater environments, expanding the documented diversity of freshwater virus communities, highlighting the potential ecological importance of virophages, and revealing distinct communities over small spatial scales.


Lilium in the family Liliaceae. Most cultivated lily plants are propagated by bulbs. Therefore, numerous lily bulbs are frequently infected by diverse viruses causing viral diseases. To date, no study has examined the viromes of plants of one type with identical genetic backgrounds collected from different geographical regions.The lily is a perennial flowering plant belonging to the genus Plantago asiatica mosaic virus (PlAMV), Cucumber mosaic virus (CMV), Lily symptomless virus (LSV), Tulip virus X (TVX), Lily mottle virus (LMoV), and Tobacco rattle virus (TRV). Of them, PlAMV was the most common virus infecting the lily. Scale and flower samples possessed a high number of virus-associated reads. We assembled 32 nearly complete genomes for the six identified viruses possessing the polyadenylate tails. Genomes of all six viruses were highly conserved in the lily cultivar “Sorbonne” based on mutation analysis. We identified defective RNAs from LSV, TVX, and PlAMV localized in the triple gene block region. Phylogenetic analyses showed that virus genomes are highly correlated with geographical regions and host plants.Here, we examined different viromes of the lily cultivar “Sorbonne” using 172 gigabytes of transcriptome data composed of 23 libraries from four different projects for the cultivar “Sorbonne.” We identified 396 virus-associated contigs from all but one library. We identified six different viruses, including We conducted comprehensive virome analyses of a single lily cultivar, “Sorbonne,” using transcriptome data. Our results shed light on an array of lily virome-associated topics, including virus identification, the dominant virus, virus accumulation in different plant tissues, virus genome assembly, virus mutation, identification of defective RNAs, and phylogenetic relationships of identified viruses. Taken together, we provide very useful methods and valuable results that can be applied in other virome-associated studies.The online version of this article (10.1186/s12864-018-5138-3) contains supplementary material, which is available to authorized users. Lilium in the family Liliaceae [Lilies are tall perennial herbaceous flowering plants, which are members of the genus iliaceae . The floiliaceae . To dateiliaceae .Lily symptomless virus (LSV) in the genus Carlavirus, Lily mottle virus (LMoV) in the genus Potyvirus, and Cucumber mosaic virus (CMV) in the genus Cucumovirus [In order to keep the genetic characteristics of lily flowers, lilies are usually clonally propagated using bulbs and scaling, which involves detaching scales from the bulb and planting individual scales to make new bulbs . Due to umovirus ), more tumovirus .Tulip breaking virus (TBV) in the genus Potyvirus, can be rapidly spread by aphids [It is known that LMoV causes vein clearing, leaf mottle, mosaic, and chlorotic symptoms in infected lily plants, whereas LSV-infected plants are generally symptomless when they are grown under normal conditions . In manyy aphids , 10, 11.In order to diagnose viruses infecting lilies, many studies have developed diverse techniques. For example, a multiplex reverse-transcription (RT) polymerase chain reaction (PCR) , a multiLily yellow mosaic virus (LYMV) in the genus Potyvirus [Due to the rapid advances in next-generation sequencing (NGS), co-infected known and novel viruses can be easily identified with full genomes –21. For otyvirus . HoweverOur previous studies have shown that plant transcriptomes are very useful for plant virome studies involving the identification of known and novel viruses, the assembly of virus genomes, mutation analyses, and the comparison of different viromes –26. In thttps://www.ncbi.nlm.nih.gov/genome/viruses/). All but one library (SRR1390677 from PRJNA252055) contained virus-associated contigs. To simplify the names of the three different projects, we created the following names: Study A (PRJNA192656) containing a single library, Study B (PRJNA341300) containing 13 libraries, and Study C (PRJNA435633) containing eight libraries (Table Plantago asiatica mosaic virus (PlAMV) and Tulip virus X (TVX) in the genus Potexvirus, CMV in the genus Cucumovirus, LSV in the genus Carlavirus, LMoV in the genus Potyvirus, and Tobacco rattle virus (TRV) in the genus Tobravirus. Based on the number of virus-associated contigs, PlAMV (129 contigs) was the dominant virus followed by CMV (74 contigs), LSV (67 contigs), TVX (67 contigs), LMoV (36 contigs), and TRV (23 contigs)  datasets derived from four different projects were associated with the lily cultivar “Sorbonne” Fig. . The 23 es Table . The idegs) Fig. . The numgs) Fig. . There wgs) Fig. . Study AViral abundance can vary depending on sample conditions and library preparation methods. All libraries in the three studies were prepared by mRNA isolation for cDNA library preparation. However, the sample conditions differed. For example, Study A used a mixture of six different tissues, including basal roots, scales, leaves, epidermal tissues, tepals, and stigmas Table . Study BAs we have shown, most libraries were co-infected by different viruses, except the 12 libraries containing only PlAMV in Study B. To reveal the viral populations in each library, we focused on two different numbers: number of virus-associated reads and number of Fragments Per Kilobase of transcript per Million (FPKM) mapped reads  using assembled virus genome sequences as references. For example, the assembled viral genome of LSV isolate A was used for SNP calling of LSV genome in the library A while the assembled genome of LSV isolate C4 was used for SNP calling of LSV genome in the library C4. In the case of LSV, we identified no SNP in isolate A, seven SNPs in isolate C4, six SNPs in isolate C5, six SNPs in isolate C7, and 12 SNPs in isolate C8 Fig. . In TVX,We obtained 32 nearly complete genomes for all six identified viruses using the obtained virus-associated contigs and mapping of reads on the corresponding reference genome sequences . Thus, plants, viruses, and environments are important factors affecting the viromes of specific plants, as suggested previously .Lilium species [Our next question was focused on how many viruses can infect a single lily plant. Our study showed that up to five different viruses  can infect a single lily plant. To date, LMoV, LSV, and CMV are the most common viruses infecting lilies, while PlAMV resulting in serious damage to lily production is considered one of the rapidly spreading viruses in  species , 34. Alt species –37, theyNGS or high-throughput sequencing (HTS) is now the most important technique for plant virus diagnostics  and viruCo-infection of diverse viruses in a single plant is now common in many horticulture plants. Furthermore, it is difficult to determine which virus is the main virus causing disease symptoms. Nevertheless, it is now possible to identify a dominant virus with virus-associated reads using transcriptome data. As indicated in our previous study , 26, thiUsing virus-associated contigs and reads, we assembled nearly complete genomes of several viruses. Interestingly, all six viruses possessed the polyadenylate tails . TherefoTymovirales. Moreover, some isolates produced defective RNAs that were preferentially localized in the TGB region. A previous study demonstrated that the defective RNA produced from Potato mop-top virus (PMTV) in the genus Pomovirus interferes with virus infection [Tomato black ring virus (TBRV) in the genus Nepovirus interfered with TBRV replication [Mapping of raw sequence reads on the viral genome revealed that three viruses  produced defective RNAs. Interestingly, genomes of all three viruses had three partially overlapping open reading frames (ORFs) referred to as TGB, which is common in the order of nfection . A recenlication . Thus, wlication . HoweverWith the help of the NGS technique, numerous virus genomes are now being assembled . SimilarWe conducted comprehensive virome analyses of a single lily cultivar, “Sorbonne,” using transcriptome data. Our results shed light on an array of lily virome-associated topics, including virus identification, the dominant virus, virus accumulation in different plant tissues, virus genome assembly, virus mutation, identification of defective RNAs, and phylogenetic relationships of identified viruses. Taken together, we provide very useful methods and valuable results that can be applied in other virome-associated studies.https://www.ncbi.nlm.nih.gov/sra) using “Lily” and “Sorbonne” as queries. There were 23 RNA-Seq datasets from four different projects. Detailed information on the RNA-Seq datasets can be found in Additional file https://www.ncbi.nlm.nih.gov/sra/docs/toolkitsoft/). All downloaded SRA datasets were converted to fastq files using fastq-dump implemented in the SRA toolkit.We searched RNA-Seq data for the lily cultivar “Sorbonne” in the SRA database of the National Center for Biotechnology Information (NCBI) . The obtained virus-associated contigs were again subjected to BLASTN against NCBI’s nucleotide (NT) database using default parameters to delete lily endogenous virus-like sequences. Only pure virus-associated contigs were used for further analyses, such as virus genome assembly.In order to identify virus-associated contigs in each RNA-Seq library, bioinformatics analyses were conducted as described previously . In brieIn order to assemble complete or nearly complete genomes for identified viruses, we aligned virus-associated contigs in each library on the reference virus genome using the ClustalW program implemented in the MEGA7 program . The misFor the construction of phylogenetic trees, virus genome sequences covering all ORFs were used. In the case of the four viruses composed of a single RNA, three LSV genomes, seven LMoV genomes, five PlAMV genomes, and three TVX genomes in this study were used. In the case of TRV composed of two RNA fragments and CMV composed of three RNA fragments, two and three different phylogenetic trees, respectively, were used. Assembled genome sequences in this study and the complete genomes of each virus available in GenBank were aligned together using the ClustalW program. The aligned nucleotide sequences were subjected to phylogenetic tree construction using the MEGA7 program with the maximum likelihood method based on the Tamura–Nei model and 1000 bootstrap replicates .SNPs for six viruses in each transcriptome were analyzed, as described previously . In our Additional file 1:Table S1. Information of RNA-Seq libraries for lily cultivar “Sorbonne.” Index indicates name of individual library from three different projects, which can be further divided into A, B, and C. Raw data can be downloaded from SRA database using respective accession numbers. Table S2. Summary of virus-associated reads, contigs, and FPKM values. Raw sequence reads in SRA format were converted to FASTA format using the SRA Toolkit. MEGABLAST was conducted using raw sequence reads and assembled contigs against virus reference genomes using e-value 1e-6 as a cutoff. To calculate FPKM values, raw sequence reads were mapped on the reference virus genome using the BWA program followed by the pileup program implemented in the BBMap package. Table S3. Assembled virus genome sequences. Orange shells indicate assembled virus genomes covering ORFs, while light yellow shells indicate partial virus genome sequences. (XLSX 116 kb)


Coxsackievirus B (CVB) group belonging to the Enterovirus genus of the family Picornaviridae, have been suggested to be the cause of virus-induced type 1 diabetes (T1D). Recently, Stone et al. showed that a CVB1 vaccine can protect CVB1-induced diabetes in suppressor of cytokine signaling 1 (SOCS1)-transgenic NOD mice [Infectious agents, particularly Even diabetogenic EMC-D virus could induce overt diabetes only in limited mice strains, indicating that the combination of viral diabetogenicity and host genetics is essential to reach clinical diabetes. In addition, several strains exhibit moderate susceptibility to EMC-D, implying that minor susceptibility gene(s) contribute to the etiology.Hober and Alidjinou claimed that a multivalent vaccine for prevention of T1D could be designed to include all CVBs serotypes, and also claimed that the precise composition of multivalent vaccine is important . AlthougTyrosine kinase 2 (Tyk2) gene as a murine EMC-D virus-induced diabetes susceptibility gene [TYK2, named TYK2 promoter variant (ClinVar ID: 440728), is associated with increased risk of human T1D, most highly, among flu-like syndrome associated anti-GAD antibody negatives, indicating that this variant is likely to be a human virus-induced diabetes susceptibility gene [TYK2 promoter variant and/or other major/minor human virus-induced diabetes-associated genetic variants to animals is imperative. Thus, the development of these animal models harboring virus-induced diabetes susceptibility gene(s), to detect the diabetogenic viruses at a high sensitivity, will help us not only to understand the mechanisms of human virus-induced diabetes but also to identify diabetogenic and/or latently diabetogenic viruses. Such studies will contribute to the development of precise composed effective vaccines to eradicate virus-induced T1D.Several studies have showed that animals with human disease risk variant(s) help us to understand the etiologic mechanisms of human diseases. Based on ity gene , we coulity gene . Since v


This manuscript aims to highlight all the clinical features of the herpes virus, with a particular focus on oral manifestations and in the maxillofacial district about Herpes Simplex Virus-1 (HSV-1) and Herpes Simplex Virus-2 (HSV-2). Oral herpes virus is a very common and often debilitating infectious disease for patients, affecting oral health and having important psychological implications. The collection of relevant data comes from the scientific databases Pubmed, Embase; initially this collection obtained an extremely high number of results, 1415. After applying the inclusion and exclusion criteria, as well as a manual screening, the results included in this review were limited to 14. The results were expressed by evaluating all the signs and symptoms that this pathology entails during the study, paying attention to the characteristics linked to the quality of life and the psychological implications. This pathology has numerous therapies, which often make the healing phase of the manifestations of this viral pathology more comfortable. The therapies currently used for the treatment of this viral infection are pharmacological, topical, systemic, or instrumental, for example with laser devices. Simplexvirus. The first form, considerably diffused, is responsible for the appearance of characteristic febrile vesicles which normally affect the facial skin ; it is also called herpes simplex labial. Initially, the cold sores manifest themselves with a slight tingling and a sense of heat on a reddened point of the lip. Within a few hours, some blisters filled with clear liquid, often painful, begin to develop in the same area as the initial point: The appearance of cold sores is ascertained [Herpes viruses or herpesviruses are double-stranded DNA viruses with icosahedral symmetry, belonging to the Herpesviridae family. The main characteristic of this family of viruses is to go against replication within the host cells, without ever being completely eliminated, thus causing a latent infection. This clinical event occurs in a time that varies according to the type of the virus and to the sensitivity of the host. The virus can be reactivated from this latent state, even after many years, giving rise to a recurrence of the disease with a clinical manifestations. The stimuli that induce the “awakening” of viral activity can be heat, cold, trauma, fever, stress  and abovertained ,3,4. Theertained ,6,7,8.The purpose of this manuscript is to evaluate the recent scientific literature concerning this specific field of oral pathology, that is, infections and pathologies related to the herpes virus, with focus on herpes virus 1 and 2  . The goaA protocol including the investigation methods and the inclusion criteria for the current revision was submitted in the PROSPERO website, an international prospective register of systematic reviews. The parameters and the analytic structure of the present work can be visualized relating the CRD id and code; this systematic review has been submitted on the PROSPERO website platform. PROSPERO acknowledgement of receipt 133925 edited on 30/04/2019.The data of this systematic investigation observed the Preferred Reporting Items for Systematic Review accordingly with the (PRISMA) statement.What is the oral manifestation of a herpes virus infection, and what about new therapies?Is there an implication in Quality of Life (QoL) and psychological profile?The questions processed the following guidelines, according to PICO :The literature investigation was performed in five different electronic databases, including Ovid MEDLINE, PubMed, and EMBASE. A manual search on Dentistry and Pharmacological sources was also conducted for relevant studies published.Digital and searches by hand were performed on herpes virus and oral manifestation, with a secondary outcome in QoL and psychological implications. In-depth research of the reference lists in the recorded manuscripts was performed in order to add significant studies and to increase the sensitivity of the revision.The Medical Subject Headings (MeSH) was applied for finding the keywords used in the present revision. The selected key words: (“Herpes virus” OR “HSV”) AND  was recorded for collecting the data. The date of last search with these results is 30 April 2019.Two independent reviewers of two different universities (Messina and Catania) singularly analyzed the obtained papers in order to select inclusion and exclusion criteria as follows: Reviewers correlated their evaluations and analyzed differences through comparing the manuscripts and consulting a third experienced senior independent reviewer  when consensus could not be reached. For the stage of the full-text articles revision, a complete independent dual analysis was performed.The method of classification included all human prospective and retrospective clinical studies, split mouth cohort studies, case-control papers, and case series manuscripts, animal investigations and literature review published between February 2009 and April 2019 on herpes virus infection clinical signs and systemic implications.Herpes virus infection oral manifestations and therapies;QoL and psychological implications of this infection;Herpes virus clinical signs and therapy.The full texts of all studies related to the main revision topics were obtained comparing the inclusion parameters:Patients with other specific disease as osteoporosis, immunologic disorders, uncontrolled diabetes mellitus, or other surgical risk related systemic conditions;Not enough information regarding the topic;Animal or in vitro studies;Articles published prior to 1 February 2009;No access to the title and abstract.The following exclusion criteria were applied:After the first literature analysis, the entire manuscript titles list was highlighted to exclude irrelevant publications, case reports and non-English language publications. Then, research was excluded based on data obtained from screening just the abstracts. The final selection was performed reading the full texts of the papers in order to approve each study’s eligibility, based on the inclusion and exclusion criteria.The results and conclusions of the selected full text papers were used for assembling the data, according to the aims and themes of the present revision, as listed onwards.Author (Year)—authors and year;Sample Size—sample size number;Sample Groups—type of groups;Posology—therapy posology;Bias risk—type of study and induced risk of bias;Statistical results—statistical results.The following parameters were used as a method for assembling the data and then organized following the schemes as seen in Table 2:The grade of bias risk was independently considered, as reported in literature ,16,17,18Selection bias;Performance bias and detection bias;Attrition bias;Reporting bias;Examiner blinding, examiner calibration, standardized follow-up description, standardized residual graft measurement, standardized radiographic assessment.Potential causes of bias were investigated:Herpes simplex is a viral disease caused by the herpes simplex virus. Infections are classified accordingly to the part of the body affected: Cold sores can cause small blisters placed in groups and sore throats, whereas genital herpes presents with minimal symptoms such as small blisters that break and cause mild ulcers. The presentation of herpes symptoms occurs cyclically, with periods of active illness followed by asymptomatic periods. The first episode is often more severe and may be associated with fever, muscle pain, swollen lymph nodes and headaches. Over time the episodes of illness decrease both in frequency and in severity. Other disorders caused by herpes simplex include systemic diseases, just as it happens in other pathologies with oral and systemic correlations ,14,19,20There are two types of herpes simplex virus, type 1 (HSV-1) and type 2 (HSV-2). HSV-1 most commonly causes oral infections while HSV-2 most commonly affects the genitals. They are transmitted by direct contact with body fluids or through injuries to an infected individual. Transmission may also occur when symptoms are not present. Genital herpes is classified as a sexually transmitted disease. During delivery, herpes simplex can be transmitted to the baby. After infection, the viruses are transported along the sensory nerves to the bodies of nerve cells. The causes of recurrence can include decreased immune function, stress and exposure to the sun. Cold and genital herpes are usually diagnosed based on the presentation of symptoms. Diagnosis can be confirmed by viral culture or detection of herpes DNA in urine. Blood tests in search of antibodies against the virus may confirm a previous infection. The most effective way to avoid genital infections is to avoid vaginal, oral and anal sex. The use of condoms slightly decreases the risk. The daily intake of an antiviral drug by the infected person may reduce the spread. There is no vaccine available and, once infected, there is no cure. Paracetamol and topical lidocaine can be used to relieve symptoms. Treatments with antiviral drugs such as acyclovir or valaciclovir can reduce the severity of symptomatic episodes. Globally, rates among HSV-1 or HSV-2 adults are between 60% and 95%. HSV-1 is usually acquired during childhood. The incidence of HSV-1 is between 70% and 80% in populations of low socio-economic status and between 40% and 60% in those with a higher status. HSV infections cause several distinct medical disorders. Common infections of the skin or mucous membranes can affect the face and mouth , genital organs  or on the hands (herpetic fever). More serious disorders occur when the virus infects the eye (herpes keratitis) or invades the central nervous system, damaging the brain . People with immature or suppressed immune systems, such as newborns, transplant recipients or people with AIDS, are prone to get serious complications from HSV infections. These infections are also associated with cognitive deficits in bipolar disorder and Alzheimer’s disease, although this often depends on the genetics of the infected person. The spread of pathogens from the oral environment to the brain has recently been reported . In all Following a primary infection, the virus enters the nerves at the site of primary infection, moves towards the cellular body of the neuron and becomes latent in the ganglion. As a consequence of the primary infection, the body produces antibodies for the particular type of HSV involved, preventing a subsequent infection of this type in a different site. In individuals infected with HSV-1, seroconversion after an oral infection prevents further HSV-1 infections, such as the genital herpes, or herpes of the eye. HSV-1 seroconversion also appears to reduce the symptoms of subsequent HSV-2 infection, although HSV-2 may still be contracted. Many people infected with HSV-2 show no physical symptoms; these are called “asymptomatic”, or suffering from subclinical herpes. Herpes labialis is easily identified by a simple clinical examination of individuals with no previous history of lesions and who have had contact with an individual known to be HSV-1 positive. In these subjects, in general the vesicles are multiple, of round and superficial aspect, accompanied by an acute gingivitis. Adults with atypical presentation are more difficult to diagnose. The prodromal symptoms that occur before the appearance of herpetic lesions help to differentiate the symptoms of HSV from similar symptoms of other disorders, such as allergic stomatitis. When the lesions do not appear inside the mouth, primary oral-gold herpes are sometimes mistaken for impetigo, a bacterial infection. Even common mouth ulcers (aphthous ulcers) resemble intraoral herpes but do not present a vesicular phase.Genital herpes can be more difficult to diagnose than oral herpes, since most people infected with HSV-2 do not have the classic symptoms. Furthermore, the diagnosis can be confused with many other conditions similar to genital herpes, including fungal infection, lichen planus, atopic dermatitis, and urethritis. Laboratory tests are often used to confirm a diagnosis of genital herpes. Laboratory tests include virus culture, immunofluorescence, skin biopsy and polymerase chain reaction to verify the presence of viral DNA. Although these procedures lead to highly sensitive and specific diagnoses, their high costs and time constraints discourage their common use in clinical practice. Until 1980, serological tests for HSV antibodies were rarely useful for diagnosis and were not routinely used in clinical practice. The old IgM serological tests were unable to distinguish between antibodies generated in response to HSV-1 or HSV-2 infections. However, a G-specific glycoprotein (IgG) test for HSV, was introduced in 1980 and has been shown to discriminate between HSV-1 from HSV-2 with a reliability of 98%. Herpes labialis or genitals should not be confused with further pathologies caused by other viruses of the Herpesviridae family, such as herpes zoster, which is due to the varicella-zoster virus. Differential diagnosis involves hand-foot-and-mouth disease . The firThere is no method to eliminate the herpes virus from the body, but taking antiviral drugs can reduce the frequency, duration, and severity of outbreaks. Analgesics, such as ibuprofen and paracetamol (acetaminophen), can reduce pain and fever. Topical anesthetic treatments, such as prilocaine, lidocaine, benzocaine, or tetracaine, are also able to relieve itching and pain. Several antiviral drugs are effective for the treatment of herpes, including aciclovir, valaciclovir, famciclovir, and penciclovir. Aciclovir was the first to be discovered and is now available as an equivalent drug, as is valaciclovir. The evidence supports the use of aciclovir and valaciclovir for the treatment of cold sores, as well as for herpes infections in people with cancer. The evidence supporting the use of aciclovir in primary herpetic gingivostomatitis is weak. A number of topical antiviral drugs are effective for cold sores, including acyclovir, penciclovir, and docosanol. Lysine has been proposed for treatment based on good in vitro results; however, it has proved ineffective in humans. The cold sore virus, like other viruses encapsulated in a lipid membrane, turns out to be deactivated by long-chain saturated fatty alcohols. Of these, only docosanol has been approved by the Food and Drug Administration in the treatment of cold sores of the immunocompetent adult, having proved to be as effective as topical antivirals. Given the mechanism of action, it presents a risk of zero drug resistance minimum. The use of a trans-dermal electrical stimulator (TENS) on lesion sites has been suggested for the treatment of cold sores both in association with antiviral drugs and as a replacement ,24. AlthThe results obtained from a careful analysis of the literature, after applying the correct inclusion and exclusion criteria, were further evaluated by the authors. An independent analysis of the authors was carried out and the results compared further to evaluate their suitability. The initial research led to a high number of articles, 1415. Subsequently, applying the exclusion criteria, articles older than 10 years were removed first. At this point the remaining 261 articles were further skimmed by evaluating only the studies on humans (209), and subsequently only those available in full text format (63), so as to be able to consult all the available results and statistics. Only randomized clinical trials (RCTs) were included. At the end of the research 16 articles were obtained. Of these, only 14 papers contained sufficient information on the oral health of the patients considered, proposed innovative therapies, and spoke of herpes virus infections. The results obtained from the single articles have been summarized in According to Polansky et al.  the herbAll the international literature about this infectious disease has been screened following the inclusion criteria. The main causes of pain, burning and swelling which are incapacitating, with consequences at the systemic level and in the quality of life of the patients have been also recorded. Surely this pathology makes some dental practices difficult. Therefore, even simple maneuvers such as taking a dental impression are not feasible in the case of herpes simplex labialis or mucous in the active phase ,42. The From the study conducted it is easy to see what the most efficient therapeutic strategies for this infectious disease are. Unfortunately, in the studies taken into consideration, few assessed the efficacy of the same drug, and therefore it was not possible to perform a statistical analysis. The most effective methods involve systemic or topical therapies in agreement with the authors. Also, the low-level laser therapy is not to be underestimated, as it can improve both healing time and all factors related to the quality of life of patients.


Achatina, also appear to be widespread. Yet tracking their appearance and distribution is difficult because LSS beads are often intentionally or unintentionally lumped with OES beads, there are no directly dated examples, and bead reporting in general is highly variable in the archaeological literature. Nevertheless, Achatina and other potential cases of LSS beads are present at over 80 archaeological sites in at least eight countries, spanning the early Holocene to recent past. Here, we collate published cases and report on several more. We also present a new case from Magubike Rockshelter in southern Tanzania with the first directly dated LSS beads, which we use to illustrate methods for identifying LSS as a raw material. Despite the long history of OES bead production on the continent and the abundance of land snails available throughout the Pleistocene, LSS beads appear only in the late Holocene and are almost exclusively found in Iron Age contexts. We consider possible explanations for the late adoption of land snails as a raw material for beadmaking within the larger context of environmental, economic, and social processes in Holocene Africa. By highlighting the existence of these artifacts, we hope to facilitate more in-depth research on the timing, production, and distribution of LSS beads in African prehistory.Shell beads are well established in the archaeological record of sub-Saharan Africa and appear as early as 75,000 BP; however, most research has focused on ostrich eggshell (OES) and various marine mollusc species. Beads made from various land snails shells (LSS), frequently described as  Shell beads have a long history of production in sub-Saharan Africa and are one of the first indicators of early modern human symbolic behavior, appearing by 75,000 years ago alongside other forms material culture such as utilized ochre and portable/parietal art  beads to complement reporting convention for OES beads without implying taxonomic homogeneity of molluscs.Although land snail shell beads are most often described as Swainson  such as a Albers , Burtoa Land snails are some of the most pervasive and dense archaeological residues found at sites across Africa. However, interpretation on a case-by-case basis is typically limited to whether their presence is owed to taphonomic “natural” causes (such as self-introduction through burrowing) vs. human agency (such as subsistence behavior or harvesting). Walz , p. 90 aBeyond general inattentiveness to snail shells as forms of material culture, LSS beads are likely underreported for several reasons. Primarily, shell beads of all types are generally understudied in archaeological accounts. Whereas glass beads are frequently employed as proxies in Iron Age research for long-distance trading networks, individual and group wealth, and social stratification Bvocho , shell bWhat is becoming clear, however, is that LSS beads are hardly scarce in the sub-Saharan African archaeological record. We now seek to initiate discourse on the existence and distribution of these artifacts and their role in prehistory. This first requires a review of sites where LSS beads have been identified thus far. We then review several ecological aspects of African land snails as a vehicle for discussing how these animals enter the archaeological record, and what aspects of their physiology affect their utility as a raw material for beadmaking. Here, we build upon earlier scholarship aimed at helping archaeologists distinguish LSS beads from OES and other raw materials. Although it may be possible to assign genus or species-level identifications in some circumstances, we argue that, at minimum, describing this type of disc bead as LSS will help improve reporting and inter-site comparisons. We illustrate our methods using a new case study of LSS beads from Magubike Rockshelter in southern Tanzania, which represent the first directly dated beads of this type. The context of these finds at Magubike evokes broader questions on the nature of these artifacts, starting with when they appear and where they are found. Our ultimate objective is to consider why LSS beads begin to appear alongside, but do not replace, OES beads in many places across sub-Saharan African during the latter stages of the Holocene. We believe this can represent a starting point for more focused study of LSS beads in African archaeology, as part of a broader recognition of land snails as an important component of material culture.Reviewing the available literature, it is apparent that LSS beads are not unusual finds. Unfortunately, it also becomes apparent that there is no consistent framework for identification or reporting them. Here, we present the geographic and chronologic distribution of published cases of LSS  beads Fig. , Table 1Andara sp. at Manda , the moAchatinidae are remarkably resilient and can live in a number of environments and conditions. They are nocturnal, herbivorous creatures who can forage on up to 500 different types of plant species, making them extremely adaptable to varied rainforest and savannah environments  fulica  or humans Marlowe . WhereasP Lubell  and haveP Lubell .However, it is worth noting that evidence for snail harvesting south of the Sahara is significantly patchier  is located in the southern highlands of Iringa and possesses a stratified sequence of Middle Stone Age (MSA) to recent Iron Age/Historic deposits (Werner and Willoughby n = 58) and toward the rear of the shelter (n = 3). Those excavated from the central block in 2012 were found between 10 and 50 cm below the modern ground surface. When considering the entire collection from the central excavation, average bead diameter is 6.35 mm (4.87–7.81 mm range), with an average thickness of 1.49 mm and aperture diameter of 2.05 mm. It seems plausible that these beads were deposited around the same time and may belong to a single feature. None were in early stages of production, suggesting import to the rockshelter in a complete state. This bead concentration was associated with lithics, ceramic sherds, and iron slag. The remaining beads were excavated in 2016 toward the back of the rockshelter from a depth of approximately 25–30 cm below surface. Although there are only three beads from this area, and one is broken, they appear different from those in the main excavation area. These beads are significantly smaller in diameter (4.23 mm), with two of them having a peculiar squared shape.Potential LSS beads at Magubike were recovered in two different areas: the central excavation block   has the most apparent distinction in its layers, making it a potential candidate for the Magubike bead material; however, its pinky hue and highly coiled shell render it a mismatch. The gastropod shell is also the thinnest of the marine shells considered, measuring only 0.83 mm, while the bivalves ranged from 1.80 to 2.26 mm. Although the marine bivalves have the appropriate thickness and coloring to be potential candidates for the Magubike bead material, the distinctive laminar microstructure is absent, ruling them out as a source material.Gastropod shells all form using the same process so have similar microstructures; however, terrestrial and freshwater snails can be distinguished based on shell size and thickness. Snail shell growth is accretionary, beginning at the outer lip of the aperture, with cross-lamellar structures forming in alternating orientations that give strength to the shell structure . Throughout five seasons of survey and archaeological excavation at the site, we have not observed any living land snails or even evidence of recent snail activity. It is possible that the snail species from antiquity are no longer present in the study region. If, however, snail populations in this area remained relatively consistent over time, these MSA species are a likely candidate for those used in later bead manufacture.The shell characteristics of the whole Magubike LSS are more similar to Pfeiffer , than thWhatever their taxonomic attribution, the complete shells in the MSA levels are the best proxies available for the production of shell beads found in the rockshelter. Comparing measurements and microstructure of the shells and LSS beads supports this assertion. None of the beads have the distinctive periostracum or prismatic layers present, and none appear to have the shiny inner nacre layer. The absence of these inner and outermost layers suggests that the bead thickness would be noticeably less than that of the original shell wall. The unmodified LSS from Magubike is of sufficient thickness to have produced the recovered beads. Further, the distinctive banding of the cross-lamellar layers present in the beads is absent from the OES and marine shell samples observed.The finds from Magubike are a new instance of Iron Age LSS beads, and the first of their kind to be directly dated. Their chronology provides additional perspectives on snail bead development and spread. The range of direct radiocarbon dates and the visually distinct bead styles suggest a long-term use of LSS beads in the region, with potential for stylistic evolution. Although it is not possible at present to identify the raw material to species, clear laminations visible on bead surfaces are consistent with LSS. Furthermore, comparisons with modern and archaeological marine and LSS shell reveal consistent morphological patterns, supporting earlier work by Ward and Maggs . AlthougA review of published sources, combined with new data from Magubike Rockshelter and other previously unreported sources, presents a startlingly vast picture of LSS bead occurrence in sub-Saharan Africa. Furthermore, this is almost certainly an underestimate given variable reporting standards for land snail artifacts and disc beads respectively, as well as the challenges associated with identifying LSS as a bead raw material. We hope that by highlighting these artifacts, we can promote further discussion and the publication of new data that help refine understanding of the role LSS beads played in prehistory.why they may have been manufactured at all.The present state of this dataset is nonetheless intriguing. Analyzing the contexts in which LSS beads are found has great potential to contribute to our understanding of ecological, economic, and sociopolitical processes unfolding in the Holocene. There is already much to learn simply from examining when LSS beads occur and their geographical distribution. Ultimately, these patterns are what allow us to begin asking Investigating the chronological range of LSS bead cases is challenging because other than those from Magubike, there are no direct dates on these artifacts. At present, it is only possible to determine which LSS beads have published provenience data and correlate that with associated radiocarbon dates from charcoal and other datable material within the same level or strata. This of course only produces a rough estimate of the antiquity of LSS beads. Expectedly, the range is quite broad, spanning approximately 15,000 calBP to as recently as 160 calBP. However, the upper extent of this range is potentially a significant overestimate.Achatina beads (n = 3 of 6) and all of the broken and unfinished beads (n = 3) come from levels 1–5 ,” that are “made from the flat apex of the shell…punched at the thin centre of the spiral” , Botswana (n = 7), and Zambia (n = 6). Tanzania is also well represented (n = 7), although half the confirmed cases come from this study, suggesting further work is needed in the region. Curiously, there is no compelling evidence of LSS beads in western Africa. We found only one potential case from Kintampo Rockshelter No. 6 in Ghana; however, Stahl , suggesting gaps remain in our knowledge of their distribution. However, we cannot dismiss the fact that the four-corner region of southern Africa was the location of successive large-scale complex societies during the Iron Age, with populous capitals such as Mapungubwe, K2, and Great Zimbabwe Huffman . Since sMitchell . Social,Mitchell .Plotting the coordinates of known sites , although OES beadwork is preferred (Mitchell hxaro and larger beads for trade (cited in Mitchell hxaro.It is also interesting to consider the role interaction between hunter-gatherer and agropastoralist populations played in the appearance and proliferation of these artifacts (see Wilmsen and Denbow Mitchell . EthnogrMitchell . Hall anMitchell  hypothesMitchell , p. 71 cAt present, it appears that LSS beads are largely confined to eastern and southeastern Africa during the last 2,000 years, associated with Iron Age communities. Although this is partly biased by ongoing research foci within southeastern Africa, there is compelling evidence that this phenomenon is linked to population increase, elaboration of trade networks, and the growth of social complexity during the later Holocene. It is intriguing that LSS beads appear tens of thousands of years after the establishment of OES beadmaking, despite the much longer tenure of land snails in the same parts of the world. Although we offer some possible explanations as to why this might be, more data are required to begin formally testing hypotheses. Highlighting these artifacts is intended to promote discussion, and ultimately publication, of additional finds from scholars working in diverse regions across the continent. We hope that re-examination of many disc bead assemblages reveals some unexpected finds.This exercise in reviewing cases of LSS beads in sub-Saharan Africa has perhaps revealed more about what we do not know than what we do know. Once geographic and temporal distributions are better defined, there are many fruitful directions for further research. For example, LSS bead production remains largely a mystery. Although there are numerous ethnographic accounts of OES beadmaking, it is more difficult to find accounts using LSS. Several site reports note the presence of LSS preforms, but the lack of high-quality images, drawings, or descriptions render it impossible to assess production stages between sites or through time. Since both OES and LSS are naturally occurring convex shells of comparable thickness, both would be broken into smaller pieces, drilled and shaped, and likely followed similar production sequences (see Kandel and Conard Greater attention to distinguishing LSS beads from OES, direct dating of known and new cases, and reportage using standardized terminology should help increase visibility of these finds and pave the way for future work. To this end, we have offered a review of cases known thus far and practical approaches to identifying LSS using the easily observable microstructure of shell profiles. Recently excavated LSS beads with direct dates from Magubike Rockshelter illustrate these methods and add an important data point to the distribution of LSS beads across eastern and southern Africa. We hope this preliminary work prompts additional research questions on related topics, such as differing manufacture techniques between LSS and OES beads, the co-occurrence of these artifacts with other types of personal ornamentation and symbolic imagery, and the broader role of LSS beads in prehistoric African societies.


Respiratory viruses are common in human populations, causing significant levels of morbidity. Understanding the distribution of these viruses is critical for designing control methods. However, most data available are from medical records and thus predominantly represent symptomatic infections. Estimates for asymptomatic prevalence are sparse and span a broad range. In this study, we aimed to measure more precisely the proportion of infections that are asymptomatic in a general, ambulatory adult population. We recruited participants from a New York City tourist attraction and administered nasal swabs, testing them for adenovirus, coronavirus, human metapneumovirus, rhinovirus, influenza virus, respiratory syncytial virus, and parainfluenza virus. At recruitment, participants completed surveys on demographics and symptomology. Analysis of these data indicated that over 6% of participants tested positive for shedding of respiratory virus. While participants who tested positive were more likely to report symptoms than those who did not, over half of participants who tested positive were asymptomatic. Most observation of human respiratory virus carriage is derived from medical surveillance; however, the infections documented by this surveillance represent only a symptomatic fraction of the total infected population. As the role of asymptomatic infection in respiratory virus transmission is still largely unknown and rates of asymptomatic shedding are not well constrained, it is important to obtain more-precise estimates through alternative sampling methods. We actively recruited participants from among visitors to a New York City tourist attraction. Nasopharyngeal swabs, demographics, and survey information on symptoms, medical history, and recent travel were obtained from 2,685 adults over two seasonal arms. We used multiplex PCR to test swab specimens for a selection of common respiratory viruses. A total of 6.2% of samples  tested positive for at least one virus, with 5.6% testing positive in the summer arm and 7.0% testing positive in the winter arm. Of these, 85 (50.6%) were positive for human rhinovirus (HRV), 65 (38.7%) for coronavirus (CoV), and 18 (10.2%) for other viruses . Depending on the definition of symptomatic infection, 65% to 97% of infections were classified as asymptomatic. The best-fit model for prediction of positivity across all viruses included a symptom severity score, Hispanic ethnicity data, and age category, though there were slight differences across the seasonal arms. Though having symptoms is predictive of virus positivity, there are high levels of asymptomatic respiratory virus shedding among the members of an ambulatory population in New York City.IMPORTANCE Respiratory viruses are common in human populations, causing significant levels of morbidity. Understanding the distribution of these viruses is critical for designing control methods. However, most data available are from medical records and thus predominantly represent symptomatic infections. Estimates for asymptomatic prevalence are sparse and span a broad range. In this study, we aimed to measure more precisely the proportion of infections that are asymptomatic in a general, ambulatory adult population. We recruited participants from a New York City tourist attraction and administered nasal swabs, testing them for adenovirus, coronavirus, human metapneumovirus, rhinovirus, influenza virus, respiratory syncytial virus, and parainfluenza virus. At recruitment, participants completed surveys on demographics and symptomology. Analysis of these data indicated that over 6% of participants tested positive for shedding of respiratory virus. While participants who tested positive were more likely to report symptoms than those who did not, over half of participants who tested positive were asymptomatic. Respiratory virus infections are among the most common diseases in humans and cause significant morbidity and mortality across seasons and populations . While rSome previous studies have measured or inferred asymptomatic respiratory virus shedding prevalence levels; however, such estimates range widely across demographics, pathogens, and sampling approaches. A study carried out in Nashville, TN, and Salt Lake City, UT, recruited 238 completely asymptomatic adults (no symptoms within 14 days of recruitment), who were controls for patients with pneumonia, from among visitors to outpatient primary care clinics for routine health checks. Of these individuals, only 5 (2%) tested positive for human rhinovirus (HRV), coronavirus (CoV), or human metapneumovirus (hMPV) .Asymptomatic infection rates may vary as a function of host age and virus. In one study, 52% of asymptomatic children recruited from an Alaskan community tested positive for shedding respiratory virus in a one-time sampling scheme, with the majority having HRV and adenovirus . In anot7–2 rather than 3 nA/mm2, so the numbers differ from those reported in Shaman et al. [This wide variation in symptomatic versus asymptomatic presentations underscores the need for further studies to understand the distribution of symptoms across infection statuses. Here, we document the shedding prevalence of a number of common respiratory viruses in an ambulatory adult population, as well as the distributions of symptomatology and symptom severity. Sampling was conducted in two seasonal arms: one carried out in the late spring/early summer (summer arm), which has been previously reported , and oneWe received consent from, surveyed, and swabbed a total of 2,685 individuals 18 years of age or older from among visitors passing through a room in the tourist attraction. Some directly approached the study recruiters, while others were present while a recruiter was explaining the study and elected to join afterward. A total of 1,477 participants were enrolled between 29 April and 31 July 2016 (summer) and a total of 1,208 between 28 January and 30 April 2017 (winter) . Among aP = <0.001) though not in terms of overall positivity prevalence (P = 0.13). The majority of infections in the summer arm were HRV infections (71.0%), with 19.3% testing positive for CoV and the other 9.7% testing positive for influenza virus, hMPV, RSV, or PIV; however, the majority of infections in the winter arm were CoV infections (57.6%), with 30.6% testing positive for HRV and the remaining 11.8% testing positive for adenovirus, hMPV, influenza virus, or PIV (P < 0.001). Among all 2,685 participants across both seasons, there were a total of 168 (6.2%) individuals who tested positive for respiratory virus. Of these, 85 (50.6%) were positive for HRV, 65 (38.7%) for CoV, and 18 (10.2%) for adenovirus, hMPV, RSV, influenza virus, or PIV. There were no coinfections. In the summer arm, only 5.6% of participants tested positive . The proportion of participants testing positive who were symptomatic ranged from 3.6% to 39.4% overall, depending on symptomatic definition. There were slight differences between the seasonal arms, with 2.5% to 48.1% of positives being symptomatic during the summer arm and 4.8% to 30.1% being symptomatic for the winter arm, again depending on definition. There was some evidence of differences in the proportions of infections that were symptomatic between seasons by symptomatic definitions 1 and 2  , but there was no evidence of differences for any other definition. Rates of being symptomatic differed significantly among virus-positive and virus-negative participants, with positive-testing participants more likely to meet symptomatic criteria, though there were up to 17.6% of individuals who tested negative who were nonetheless symptomatic (depending on definition)  . This asThe best-fit logistic regression models according to minimum Akaike information criterion (AIC) values are shown in P < 0.05). There were differences between the seasonal arms for this finding, however. For the winter arm, allergies were similarly associated with quantitated score of signal intensity for all positives, whereas there was an association of signal intensity with age but not with allergies for the summer arm . Among HRV positive-testing participants (both arms combined), there was strong evidence of a negative association between signal intensity and the 50-to-64-year-old age category compared to the 18-to-29-year-old age category, weak evidence of a positive association between signal intensity and having allergies, and weak evidence of a positive association between signal intensity and being Hispanic. Again, there were seasonal differences, with the symptom score being negatively associated with signal intensity in the winter arm and with being Hispanic being positively associated in the summer arm. For those positive for CoV, signal intensity was weakly positively associated with having allergies (P < 0.1). This association held across seasons, with stronger association in the winter arm (P = 0.02). In the summer arm there were also associations with symptom score, race, and gender, with signal intensity being positively associated with symptom score, a negative association between signal intensity and all other races compared to whites, and a negative association with women compared to men.Among the positive-testing participants, signal intensity (measured continuously in nanoamps per square millimeter) was positively associated with reporting allergies (P < 0.001); participants aged 30 to 39 years, 40 to 49 years, and 50 to 64 years had lower total reported symptom scores than participants aged 18 to 29 years (ANOVA P < 0.001). These results were consistent and significant across the seasonal arms. Reporting consumption of cold and flu medicines was positively associated with higher total symptom scores across all participants and also only among positives (ANOVA P < 0.0001). This finding was also consistent across seasons. There was evidence of differences in symptom scores by month across all participants (ANOVA P < 0.001), but there were no differences seen in assessing only the participants testing positive   . There wP < 0.0001); however, there was no significant association between viral positivity and reporting a greater tendency to get sick. These results were consistent across the seasonal arms.There was a positive association between reporting a greater tendency to get sick and total self-reported symptom scores among all participants . The association between having allergies and being symptomatic among participants who tested negative was less strong for definition 2 (P < 0.05) and nonexistent for definitions 3 to 7.Interestingly, the proportions of symptomatic individuals (by the various definitions) who tested negative were quite high (66.7% to 86.9%), and this finding held across the seasonal arms. Examining this result further, we found that for symptom definitions 1 and 2 , there was a strong association among the participants who tested negative between being symptomatic and having allergies . These numbers indicate that though there may be a spectrum of symptom severity, a substantial number of infected individuals are completely asymptomatic. Regardless, the participants in this study, even if symptomatic by the definitions used , felt well enough to visit a tourist attraction. There were few individuals who reported severe symptom scores or fever, indicating that those with severe symptomatology might have stayed home and thus were not captured in this study.Conversely, a substantial percentage of individuals who were symptomatic across the definitions did not test positive. Only some of the variation appears to have been due to allergies and only for symptom definitions 1 and 2. This result may indicate that symptoms persist after an infection has cleared, that symptoms may appear before an infection is detectable, or that symptoms may be due to infections not tested for in this study.In multivariate analysis of viral positivity, we found an expected association between having worse symptoms and testing positive for respiratory virus. We also found some evidence of an association of Hispanic ethnicity with being positive, in particular, for HRV. This association has been found previously in other studies of respiratory virus , 18, espManufacturer specifications, as well as data from previous studies, indicate that there is no association between eSensor signal intensity and the amount of virus present in a sample. However, in this study, we found evidence of an association between signal intensity and having allergies among all positives combined from both study arms and also among those in the winter arm alone . There was, however, some association between signal intensity and age in the summer arm.These findings build on our previously reported summer arm study and provide an estimate of baseline prevalence of respiratory virus shedding in an ambulatory population across seasons. The results indicate that nearly 1 in 17 adults is shedding respiratory virus across seasons and that nearly 1 in 14 do so during the peak winter month. There is evidence of a difference in virus species prevalence among seasons, with HRV being the most prevalent virus species during the summer arm  and coronavirus being dominant during the winter arm . The impact of shedding prevalence on contagiousness or transmission is not yet well defined, however.There are several limitations to this study. While the sampling scheme was designed to capture individuals from a population different from those represented in studies of persons seeking medical attention, it would not capture individuals who stay home with severe symptoms. However, given that the site used in the study is frequented by tourists, who usually only have a limited amount of time in New York City, we hypothesize that many people might still visit the site despite having symptoms that would otherwise keep them home. Consequently, our sample may be less skewed in symptom distribution than a random sample from the street. Conversely, among tourist attraction visitors, those with symptoms might have been more inclined to participate in this study. Symptoms were self-reported, which can introduce error, and were reported only for the previous 48 h. For some viruses, RNA can be detected for several weeks following infection, so some seemingly asymptomatic individuals who tested positive may have been previously infected and symptomatic. Further, shedding prior to symptom development also occurs . These dThe findings presented here indicate a significant level of respiratory virus shedding in an ambulatory adult population across seasons, as well as a substantial proportion of infections that are asymptomatic. These results could help improve estimates of virus incidence and inform disease transmission modeling and forecasting, which could then be used to support control efforts. Indeed, determination of respiratory virus prevalence in nonclinical settings can help with the design of control measures in the real-world settings where most transmission occurs. Future potential work in this area could include replicating this study in other highly frequented areas in the city  and enrolling children in order to obtain estimates across a broader age range.As described in a report of a previous study  for whicParticipants were surveyed with respect to demographic attributes, including age, gender, and race, as well for as other information, including reports of recent travel, preexisting medical conditions, allergies, self-reported propensity to get sick, and flu shot status. Data were also collected, per the common cold questionnaire , on whetNasopharyngeal swabs from both the left and right nasal cavities were collected from each participant using a Minitip flocked swab . The samples were jointly stored in 2 ml DNA/RNA Shield  for up to 30 days at 4 to 25°C and were subsequently divided into 2 aliquots and stored at −80°C until assay. Nucleic acids were extracted from 200-µl samples, with the addition of 10 µl of MS2 bacteriophage as an internal control, using an easyMAG NucliSENS system . Samples were tested for infection using a multiplex PCR assay known as an eSensor XT-8 respiratory viral panel  according to the manufacturer’s instructions.2. The viruses detected by the XT-8 RVP include influenza A (any subtype), A/H1N1, A/H3N2, and AH1N1pdm2009 virus and influenza B virus; RSV A and B; PIV 1, 2, 3, and 4; hMPV; HRV; adenovirus B/E and C; and CoV 229E, NL63, OC43, and HKU1.Positive samples were identified using a threshold of 25 nA/mmThere is no standard definition of symptomatic infection, so several definitions were used so that the sensitivity of our findings could be tested. All definitions were based on self-reporting of symptom type and severity. Definition 1 consisted of reporting at least two symptoms, with at least one being moderate or severe . DefinitWe conducted a range of statistical tests to identify associations between various demographic factors and both symptom scores and viral positivity. For categorical variables, such as gender and age group, we used ANOVA and Tukey tests to determine whether there were statistical differences in the average symptom scores between categories of the variable. We used chi-square and Fisher’s exact tests to compare differences across each categorical variable with respect to positivity. We performed univariate and multivariate regression to test associations between each set of variables  and quantitated positivity scores (measured in nanoangstroms per square millimeter) for all viruses and for HRV and CoV separately. Logistic regression was used to conduct a similar analysis on associations between the demographic variables and positivity as a binary variable (positive versus negative) for all viruses and for HRV and CoV separately. The Akaike Information Criterion was used to identify the best-fitting models.


Current evidence suggests that environmental changes and interactions between wildlife, livestock, and humans contribute to the spillover of infectious agents from bats to other hosts . An incrDesmodus rotundus) can, for instance, naturally transmit rabies to other species when biting to feed on blood, particularly to livestock and sometimes to humans [Mycoplasma has also been detected in common vampire bat blood and saliva and might be transmitted between bats, for instance, during aggressive behaviors [Although bat bites may be the main transmission route coming to mind, pathogen transmission involving bat bites has been documented mostly for rabies virus (Rhabdoviridae). The common vampire bat (o humans . Mycoplaehaviors . ObligatRousettus aegyptiacus) caves before becoming sick and reported regular contacts with bats or their secretions [Contact with bat body fluids  is increasingly recognized as an important mechanism of pathogen spillover to humans. Human encroachment into bat habitats as well as increasing urbanization, which facilitates bat roosting in artificial structures, are likely to increase contact with bat body fluids. For example, Nipah virus (Paramyxoviridae) human infection cases reported in Bangladesh were associated with the consumption of raw sap from date palm trees contaminated with fruit bat saliva and urine . In the cretions .Eidolon helvum, which is the most frequently hunted and traded bat species in many African countries , has bein Ghana .Paguma larvata) got infected with a virus circulating in horseshoe bats (Rhinolophus sp.) and would have then acted as an intermediate amplifying host [Infection of wild animals such as apes, monkeys, and antelopes with bat-borne infectious agents may also play a role in the transmission chain to humans, such as for Ebola virus . In the ing host . Naturaling host ,14 but cIn addition to wild animals, the role of livestock as intermediate and amplifying hosts between wild animals and humans has been clearly demonstrated in several outbreaks, such as for Filovirus and Henipavirus ,16. IndeCamelus dromedaries) act as the natural reservoir host in which the MERS coronavirus could have circulated for more than 30 years before its first detection in humans [Lama glama) and wild boars (Sus scrofa) have shown susceptibility to MERS coronavirus infection [In contrast to rapid and short-time spillover events, long-time and silent circulation of viruses in livestock before transmission to humans may also occur, as is strongly suspected for the ongoing outbreak of Middle East respiratory syndrome (MERS). Although bats are likely to be a source of MERS-like coronaviruses, dromedary camels , although DENV transmission from bat flies to humans has never been reported [A large diversity of arthropods, such as mosquitoes, mites, flies, fleas, and ticks , can be ay occur . With thrtonella ) have bertonella . Dengue reported . To dateLeptospira could be a source of contamination to other hosts, as this bacterial genus is known to persist in moist environments [Histoplasma capsulatum). The detection of coronavirus RNA in bat guano has been demonstrated [Environmental transmission or indirect transmission through the contamination of habitats used by infected hosts has been described as a major mechanism in the epidemiology of wildlife diseases. For bat infectious agents, a limited number of experimental and field studies have been performed to assess their persistence in the environment. Henipaviruses can persist in liquids and on solid surfaces for several days under laboratory conditions and filoviruses for several weeks ,29. Bat-ronments . Transmironments . Reportsnstrated , althougTransmission of infectious agents is highly dynamic in bats and is associated with significant changes in bat population structure  . PeriodsAlthough communication and education on the risk associated with bat-borne pathogens has increased over the past decade, the benefits of bats in ecosystem functions  tend to be disregarded . Gaining


Asherman's syndrome is defined as partial or complete obstruction of the uterine cavity primarily caused by intrauterine procedures and infections. Hysteroscopic adhesiolysis is commonly used to treat Asherman's syndrome. Although the frequency of placenta accreta is known to increase with pregnancy after hysteroscopic adhesiolysis, precise data remain unknown. We report a case of placenta accreta following hysteroscopic lysis of adhesions caused by Asherman's syndrome and IVF treatment and review the literature on placenta accreta following hysteroscopic adhesiolysis. It is necessary to consider placenta accreta as a complication of pregnancies after hysteroscopic adhesiolysis for Asherman's syndrome, particularly in those conceived using IVF. Asherman's syndrome is defined as partial or complete obstruction of the uterine cavity due to damage to the basal layer of the endometrium , 2 and iHysteroscopic adhesiolysis is commonly used to treat Asherman's syndrome. Although the frequency of placenta accreta is known to increase with pregnancy after hysteroscopic adhesiolysis, precise data remain unknown.Here, we report a case of placenta accreta following hysteroscopic lysis of adhesions caused by Asherman's syndrome and IVF treatment and review the literature on placenta accreta following hysteroscopic adhesiolysis.The patient was a 40-year-old primiparous woman. She was diagnosed with submucosal fibroids by her previous gynecologist 5 years prior, based on chief complaints of atypical genital bleeding and hypermenorrhea. She underwent hysteroscopic myomectomy for one 1 cm sized and one 3 cm sized submucosal fibroid located between 2 and 3 o'clock in the uterine fundus. Asherman's syndrome was suspected after the patient exhibited secondary hypomenorrhea 10 months after surgery. Therefore, hysterosalpingography and magnetic resonance imaging (MRI) were performed. Intrauterine adhesions were suspected based on hysterosalpingography findings, while uterine cavity narrowing was identified using MRI. Hysteroscopy revealed filmy adhesions consistent with myomatous tissue at the excision site, and the patient was diagnosed with Asherman's syndrome.Eight months after diagnosis, the patient underwent hysteroscopic adhesiolysis. The filmy adhesions observed on the left side of the fundus were easily separated with Hegar cervical dilators, and an intrauterine device was inserted after dilation. The patient was diagnosed with stage I Asherman's syndrome defined by European Society for Hysteroscopy classification of intrauterine adhesions, and menstrual flow returned to normal after the operation.While the patient had a strong desire to bear children, her inability to conceive for 7 years led her to pursue in vitro fertilization (IVF). After having a miscarriage at 7 weeks of gestation, she underwent cervical dilatation and uterine curettage.Six months after the miscarriage, the patient became pregnant again through IVF and was referred to our hospital at 7 weeks of gestation. At 19 weeks of gestation, tissues with a free edge were visualized within the amniotic cavity using obstetric ultrasound and were determined to be amniotic sheets on MRI at 31 weeks of gestation . The couThe placenta adhered to the uterine wall after childbirth and could not be easily separated manually. The blood vessels on the uterine surface at the placental implantation site were engorged , leadingPlacenta increta was confirmed based on postpartum histological findings of placental villi invading the myometrium, without an interposed decidual plate.The postoperative course was uneventful, and the patient was discharged in good health on the 7th postpartum day.This case highlights two points: placenta accreta must be considered as a potential complication in pregnancies after hysteroscopic adhesiolysis for Asherman's syndrome, and IVF may further increase the risk of placenta accreta.First, we should be aware of the possibility of placental implantation disorders, placenta accrete in particular in pregnancies following hysteroscopic adhesiolysis. This patient underwent hysteroscopic adhesiolysis for intrauterine adhesions. Although approximately 90% of intrauterine adhesions are associated with intrauterine curettage in pregnant women, they can also occur in a nongravid uterus as a result of procedures like myomectomy and curettage that damage the endometrium . IntrautSecond, IVF may increase the risk of placenta accreta.The incidence of placenta accreta is significantly higher in IVF pregnancies than in spontaneous pregnancies . Esh-BroIntrauterine adhesions (IUA) severity is associated with greater reduction in fertility , 3, 17, Placenta accreta should be considered as a complication of pregnancies after hysteroscopic adhesiolysis for Asherman's syndrome, particularly in those conceived using IVF.