Identification of ancient viruses from metagenomic data of the Jomon people.

Ancient DNA studies provide genomic information about the origins, population structures, and physical characteristics of ancient humans that cannot be solely examined by archeological studies. The DNAs extracted from ancient human bones, teeth, or tissues are often contaminated with coexisting bacterial and viral genomes that contain DNA from ancient microbes infecting those of ancient humans. Information on ancient viral genomes is useful in making inferences about the viral evolution. Here, we have utilized metagenomic sequencing data from the dental pulp of five Jomon individuals, who lived on the Japanese archipelago more than 3000 years ago; this is to detect ancient viral genomes. We conducted de novo assembly of the non-human reads where we have obtained 277,387 contigs that were longer than 1000 bp. These contigs were subjected to homology searches against a collection of modern viral genome sequences. We were able to detect eleven putative ancient viral genomes. Among them, we reconstructed the complete sequence of the Siphovirus contig89 (CT89) viral genome. The Jomon CT89-like sequence was determined to contain 59 open reading frames, among which five genes known to encode phage proteins were under strong purifying selection. The host of CT89 was predicted to be Schaalia meyeri, a bacterium residing in the human oral cavity. Finally, the CT89 phylogenetic tree showed two clusters, from both of which the Jomon sequence was separated. Our results suggest that metagenomic information from the dental pulp of the Jomon people is essential in retrieving ancient viral genomes used to examine their evolution.

Predicting the response of the dental pulp to SARS-CoV2 infection: a transcriptome-wide effect cross-analysis.

Pulpitis, inflammation of the dental pulp, is a disease that often necessitates emergency dental care. While pulpitis is considered to be a microbial disease primarily caused by bacteria, viruses have also been implicated in its pathogenesis. Here, we determined the expression of the SARS-CoV2 receptor, angiotensin converting enzyme 2 (ACE2) and its associated cellular serine protease TPMRSS2 in the dental pulp under normal and inflamed conditions. Next, we explored the relationship between the SARS-CoV-2/human interactome and genes expressed in pulpitis. Using existing datasets we show that both ACE2 and TPMRSS2 are expressed in the dental pulp and, that their expression does not change under conditions of inflammation. Furthermore, Master Regulator Analysis of the SARS-CoV2/human interactome identified 75 relevant genes whose expression values are either up-regulated or down-regulated in both the human interactome and pulpitis. Our results suggest that the dental pulp is vulnerable to SARS-CoV2 infection and that SARS-CoV-2 infection of the dental pulp may contribute to worse outcomes of pulpitis.

Viral MicroRNAs Identified in Human Dental Pulp.

INTRODUCTION: MicroRNAs (miRs) are a family of noncoding RNAs that regulate gene expression. They are ubiquitous among multicellular eukaryotes and are also encoded by some viruses. Upon infection, viral miRs (vmiRs) can potentially target gene expression in the host and alter the immune response. Although prior studies have reported viral infections in human pulp, the role of vmiRs in pulpal disease is yet to be explored. The purpose of this study was to examine the expression of vmiRs in normal and diseased pulps and to identify potential target genes. METHODS: Total RNA was extracted and quantified from normal and inflamed human pulps (N = 28). Expression profiles of vmiRs were then interrogated using miRNA microarrays (V3) and the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA). To identify vmiRs that were differentially expressed, we applied a permutation test. RESULTS: Of the 12 vmiRs detected in the pulp, 4 vmiRs (including those from herpesvirus and human cytomegalovirus) were differentially expressed in inflamed pulp compared with normal pulp (P < .05). Using bioinformatics, we identified potential target genes for the differentially expressed vmiRs. They included key mediators involved in the detection of microbial ligands, chemotaxis, proteolysis, cytokines, and signal transduction molecules. CONCLUSIONS: These data suggest that miRs may play a role in interspecies regulation of pulpal health and disease. Further research is needed to elucidate the mechanisms by which vmiRs can potentially modulate the host response in pulpal disease.

Detection of HCV Persistent Infections in the Dental Pulp: A Novel Approach for the Detection of Past and Ancient Infections.

The dental pulp is a sterile highly vascularized tissue and has been commonly used as a biological material to detect the genome of infectious agents that reach the dental tissue. Indeed, the pulp is also used to reveal past and ancient infections in the field of paleomicrobiology. The present study aimed to detect the presence of Hepatitis C virus (HCV) in a small community (approximately 400 inhabitants) in the Amazon region of Brazil (Nossa Senhora do Perpetuo Socorro, Vizeu, Para, Brazil) and standardize a technique for the detection of the virus in the dental pulp. Serum samples were collected from 48 patients whose teeth were clinically recommended for surgical extraction. The group comprised an equal number of males and females, mostly agriculture workers and housewives, respectively. The majority (64.6%) received less than one minimum wage and were ill educated (less than four years of school years). An enzyme immune assay was used to detect antibodies to HCV and the 9 (18.8%) positive samples were submitted to nucleic acid extraction in the blood (using the EXTRAzol) and the pulp (QIAamp DNA Micro Kit e kit RNeasy Plus Micro). The pulp was removed using a modified protocol without the use of liquid nitrogen. Nucleic acid was found in 8 of the dental pulp, but in 7 of the blood samples. Sequencing of one of the samples showed the presence of genotype 1. CONCLUSIONS: A novel simplified methodology for the extraction and amplification of HCV nucleic acid was successful to detect the presence of persistent infections of the virus within the dental pulp tissue. The protocol may be helpful to detect past and ancient infections and to better understand the natural history of HCV.

Viruses in pulp and periapical inflammation: a review.

The presence of viruses in endodontic disease has been studied in the last decade. Their presence is associated with periapical radiolucency and with clinical findings, such as pain. The aim of this review is to analyze the scientific evidence currently published about viruses in pulp and periapical inflammation, and its possible clinical implications. A literature review was carried out using the Medline/Pubmed database. The search was performed, in English and Spanish, using the following keyword combinations: virus AND endodontic; virus AND periapical; virus AND pulpitis; herpesvirus AND periapical; papillomavirus AND periapical. We subsequently selected the most relevant studies, which complied with the search criterion. A total of 21 articles were included, of which 18 detected the present of viruses in the samples. In 3 of the studies, viral presence was not found in the samples studied. The Epstein-Barr virus was found in about 41 % of cases compared to controls, in which it was present in about 2 %. The main association between viruses and endodontic pathosis is between Cytomegalovirus and Epstein-Barr virus; these are found in 114 of the 406 samples of different endodontic pathosis. Some evidence supports that the Epstein-Barr virus is present in a significant number of endodontic diseases, without exact knowledge of their action in these diseases.

Comparison of Riboflavin and Toluidine Blue O as Photosensitizers for Photoactivated Disinfection on Endodontic and Periodontal Pathogens In Vitro.

Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections.

Different origin of adipogenic stem cells influences the response to antiretroviral drugs.

Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50 µM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in part, on their embryonal origin.

Identification of viral DNA (Anelloviridae) in a 200-year-old dental pulp sample (Napoleon's Great Army, Kaliningrad, 1812).

Ancient human remains are potential sources of biological information including traces of past infections, since previous studies have demonstrated the effective detection of several bacterial agents or host-integrated viruses in old biological remnants like tissues or teeth. Studies of skeletal dental pulp samples are of particular interest since this location is potentially exposed to bloodborne agents during life through its vascularization, and could be considered as well preserved from environment after death of the host. DNA viruses belonging to the family Anelloviridae are highly present in human populations where they harbor an extreme genetic diversity but a yet undefined implication in hosts' health. We hypothesized that anelloviruses would be detected in ancient remains and that they may also serve as tracer viruses for the study of other viral agents. We analyzed 200-year-old dental pulp samples from remains of soldiers of Napoleon's Great Army during the Russian Retreat. Successful detection of Anelloviridae DNA by PCR was obtained for 1/21 ancient samples tested. The sequence identified showed 23% nucleotide divergence with the closest group of modern isolates (genus Gammatorquevirus), and was confirmed as phylogenetically distinct from those identified in saliva samples from the two investigators in charge of the study (genera Alphatorquevirus and Betatorquevirus). PCR directed toward the human beta globin gene was also performed. Negative controls were negative. Our results demonstrate that an ubiquitary, non-integrated, DNA virus is detectable from ancient biological material, with potential developments in terms of evolution studies or subsequent molecular investigations involving further viral agents.

Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp.

Viral and bacterial associations appear to be implicated in the development of periodontal infections. Little information is available describing the periodontopathic agents in root canals with necrotic pulp. In this study, the occurrence and the combinations among herpes simplex virus type 1 (HSV-1) and Dialister pneumosintes, Tannerella forsythia, and Treponema denticola in patients with chronic periodontitis and necrotic pulp were evaluated. Clinical samples from healthy subjects and patients with periodontal or pulp infections were analyzed using a nested polymerase chain reaction PCR to detect HSV and PCR to detect the 3 periodontal bacteria. The presence of Tannerella forsythia and Treponema denticola was observed in healthy, periodontitis, and necrotic pulp patients. HSV was observed in periodontitis and necrotic pulp patients, and no healthy subject harbored D. pneumosintes or HSV. The occurrence of Tannerella forsythia was not statistically significant in patients with necrotic pulp (P = 0.704). Periodontal bacteria were observed varying from 10.3% to 20.7% in periodontitis and necrotic pulp patients. The presence of Treponema denticola - HSV association was predominant in patients showing necrotic pulp (24.1%); however, HSV alone was observed in one patient with periodontitis and in another patient with necrotic pulp. The presence of double association among bacteria or bacteria - HSV could indicate a role in both periodontitis and necrotic pulp, and Tannerella forsythia - Treponema denticola - HSV and Tannerella forsythia - D. pneumosintes - Treponema denticola - HSV associations might be important in periodontitis.

Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.

Human dental pulp contains precursor cells termed dental pulp stem cells (DPSC) that show self-renewal and multilineage differentiation and also secrete multiple proangiogenic and antiapoptotic factors. To examine whether these cells could have therapeutic potential in the repair of myocardial infarction (MI), DPSC were infected with a retrovirus encoding the green fluorescent protein (GFP) and expanded ex vivo. Seven days after induction of myocardial infarction by coronary artery ligation, 1.5 x 10(6) GFP-DPSC were injected intramyocardially in nude rats. At 4 weeks, cell-treated animals showed an improvement in cardiac function, observed by percentage changes in anterior wall thickening left ventricular fractional area change, in parallel with a reduction in infarct size. No histologic evidence was seen of GFP+ endothelial cells, smooth muscle cells, or cardiac muscle cells within the infarct. However, angiogenesis was increased relative to control-treated animals. Taken together, these data suggest that DPSC could provide a novel alternative cell population for cardiac repair, at least in the setting of acute MI.

Anterograde tracing of trigeminal afferent pathways from the murine tooth pulp to cortex using herpes simplex virus type 1.

Due to its predominantly nociceptive innervation, viral tracing from the tooth pulp provides a potential means for tracing central pain pathways. The neural pathways from the tooth pulp to cortex were determined using in situ hybridization to detect the anterograde transneuronal spread of herpes simplex virus type 1 strain H129 following inoculation into the murine mandibular incisor pulp. Virus first appeared in the brain at day 3 in the dorsomedial region of all three subnuclei of the spinal trigeminal nucleus and the principal sensory nucleus. By days 5-6 virus had spread to the contralateral medial nucleus of the medial geniculate complex, posterior thalamus, and ventroposteromedial thalamus. At days 7-8 virus was detected in laminae IV and Va of the primary somatosensory cortex and lamina IV of the secondary somatosensory cortex in regions previously shown to receive input from the lower jaw. Several mice also showed infection of laminae II/III of the ipsilateral dysgranular insular cortex, along with labeling for virus in the ipsilateral external lateral parabrachial nucleus, posterior thalamus, and posterior basolateral amygdala. Our results are highly consistent with previous tracing and electrophysiological studies utilizing the tooth pulp and with studies implicating the infected structures in nociception. Viral spread appeared to define two separate afferent systems with infection of structures which have been implicated in the sensory-discriminative aspects of pain, such as the ventroposteromedial thalamus and somatosensory cortex, as well as in the dysgranular insular cortex and related subcortical nuclei which may have a role in the affective-motivational aspects of pain.

Mechanism of neuroinvasion of Venezuelan equine encephalitis virus in the mouse.

Venezuelan equine encephalitis virus (VEE) causes a biphasic disease in mice following subcutaneous inoculation in the footpad. In the initial phase, virus replicates primarily in the lymphoid tissues and induces a high titer viremia. Subsequently, the virus invades the central nervous system (CNS) from the circulation, and an encephalitis ensues. At the earliest times that VEE specific in situ hybridization signal was observed in the CNS, it was in areas of the brain involved in olfaction, leading to the hypothesis that virus may invade the brain from the circulation through the olfactory system. The results presented in this paper define the route of CNS invasion in experimental murine VEE disease initiated by subcutaneous inoculation. Virus circulating in the blood appears to seed specific areas of the peripheral nervous system during the viremic lymphoid phase of the illness. Virus replication within olfactory and dental tissues is followed by centripetal spread of virus along neural pathways. Virus enters the brain in a pattern reflecting the proximity of the peripheral invasion site to the CNS. Specifically, virus is first found in the brain within the structures of the olfactory system, followed by areas innervated by the trigeminal nerve. Virus later disseminates along fiber tracts and connected circuits within the brain, resulting in a disseminated meningoencephalitis. Surgical or chemical interruption of the olfactory system at the level of the olfactory neuroepithelium or the main olfactory bulb inhibited entry of VEE into the CNS through the olfactory nerve. However, the olfactory route is not absolutely required for CNS invasion, as virus invaded the CNS of olfactory ablated animals through the trigeminal nerve. These observations are consistent with a model of hematogenous seeding of the peripheral nervous system, followed by invasion of the CNS by direct neural spread.