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SARS-CoV-2 (CoV2) infected, asymptomatic individuals are an important contributor to COVID transmission. CoV2-specific immunoglobulin (Ig)—as generated by the immune system following infection or vaccination—has helped limit CoV2 transmission from asymptomatic individuals to susceptible populations (e.g. elderly). Here, we describe the relationships between COVID incidence and CoV2 lineage, viral load, saliva Ig levels (CoV2-specific IgM, IgA and IgG) and inhibitory capacity in asymptomatic individuals between Jan 2021 and May 2022. These data were generated as part of a large university COVID monitoring program and demonstrate that COVID incidence among asymptomatic individuals occurred in waves which mirrored those in surrounding regions, with saliva CoV2 viral loads becoming progressively higher in our community until vaccine mandates were established. Among the unvaccinated, infection with each CoV2 lineage (pre-Omicron) resulted in saliva Spike-specific IgM, IgA and IgG responses, the latter increasing significantly post-infection and being more pronounced than N-specific IgG responses. Vaccination resulted in significantly higher Spike-specific IgG levels compared to unvaccinated infected individuals, and uninfected vaccinees’ saliva was more capable of inhibiting Spike function. Vaccinees with breakthrough Delta infections had Spike-specific IgG levels comparable to those of uninfected vaccinees; however, their ability to inhibit Spike binding was diminished. These data demonstrate that COVID vaccines achieved hoped-for effects in our community, including the generation of mucosal antibodies that inhibit Spike and lower community viral loads, and suggest breakthrough Delta infections were not due to an absence of vaccine-elicited Ig, but instead limited Spike binding activity in the face of high community viral loads.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly to become a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g. shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g. pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n=76), mink (n=57), multiple species of bats (n=65), and other wildlife in addition to domestic cats (n=275) and pigs (n= 184). In total, we tested 800 animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, due to lack of validated tests for animals, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing re-emergence of SARS-CoV-2 in humans or animals.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/coronavirus disease 2019 (COVID-19) pandemic has highlighted an important role for efficient surveillance of microbial pathogens. High-throughput sequencing technologies provide valuable surveillance tools, offering opportunities to conduct high-resolution monitoring from diverse sample types, including from environmental sources. However, given their large size and potential to contain mixtures of lineages within samples, such genomic data sets can present challenges for analyzing the data and communicating results with diverse stakeholders. Here, we report MixviR, an R package for exploring, analyzing, and visualizing genomic data from potentially mixed samples of a target microbial group. MixviR characterizes variation at both the nucleotide and amino acid levels and offers the RShiny interactive dashboard for exploring data. We demonstrate MixviR's utility with validation studies using mixtures of known lineages from both SARS-CoV-2 and Mycobacterium tuberculosis and with a case study analyzing lineages of SARS-CoV-2 in wastewater samples over time at a sampling location in Ohio, USA.

Summary An important problem in modern forensic analyses is identifying the provenance of materials at a crime scene, such as biological material on a piece of clothing. This procedure, which is known as geolocation, is conventionally guided by expert knowledge of the biological evidence and therefore tends to be application specific, labour intensive and often subjective. Purely data-driven methods have yet to be fully realized in this domain, because in part of the lack of a sufficiently rich source of data. However, high throughput sequencing technologies can identify tens of thousands of fungi and bacteria taxa by using DNA recovered from a single swab collected from nearly any object or surface. This microbial community, or microbiome, may be highly informative of the provenance of the sample, but data on the spatial variation of microbiomes are sparse and high dimensional and have a complex dependence structure that render them difficult to model with standard statistical tools. Deep learning algorithms have generated a tremendous amount of interest within the machine learning community for their predictive performance in high dimensional problems. We present DeepSpace: a new algorithm for geolocation that aggregates over an ensemble of deep neural network classifiers trained on randomly generated Voronoi partitions of a spatial domain. The DeepSpace algorithm makes remarkably good point predictions; for example, when applied to the microbiomes of over 1300 dust samples collected across continental USA, more than half of geolocation predictions produced by this model fall less than 100 km from their true origin, which is a 60% reduction in error from competing geolocation methods. Moreover, we apply DeepSpace to a novel data set of global dust samples collected from nearly 30 countries, finding that dust-associated fungi alone predict a sample's country of origin with nearly 90% accuracy.

Humans have infected a wide range of animals with SARS-CoV-2 viruses(1-5), but the establishment of a new natural animal reservoir has not been observed. Here, we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2 virus, are exposed to a range of viral diversity from humans, and are capable of sustaining transmission in nature. SARS-CoV-2 virus was detected by rRT-PCR in more than one-third (129/360, 35.8%) of nasal swabs obtained from Odocoileus virginianus in northeast Ohio (USA) during January-March 2021. Deer in 6 locations were infected with 3 SARS-CoV-2 lineages (B.1.2, B.1.582, B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. Probable deer-to-deer transmission of B.1.2, B.1.582, and B.1.596 viruses was observed, allowing the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are infrequently seen in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have the capacity to transmit in US wildlife, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive “One Health” programs to monitor deer, the environment, and other wildlife hosts globally.

With the advent of Next-Generation Sequencing technology, sequencing of short tandem repeats (STRs) allows for a more detailed analysis when compared to size-based fragment methods (capillary electrophoresis-CE). The implementation of high-throughput sequencing can help uncover deeper genetic diversities of different populations. Subjects from the South region of Brazil present a particular and more homogeneous ancestry background when compared to other regions of the country. Both autosomal and Y- STRs have been analyzed in these individuals; however, all analyses published to date encompass data from CE-based fragment analysis. In this study, a genetic analysis of 59 individuals from Southern Brazil was performed on STR sequences. Forensically relevant STRs were PCR-enriched using a prototype of the PowerSeq™ AUTO/Y system (Promega Corp.). Next-generation sequencing was performed on an Illumina MiSeq instrument. The raw data (FASTQ files) were processed using a custom designed sequence processing tool, Altius. Isoalleles, which are sequence-based allelic variants that do not differ in length, were observed in nine autosomal and in six Y- STRs from the core global forensic marker set. The number of distinctive alleles based on sequence was higher when compared to those based on length, 37.3% higher in autosomal STRs and 13.8% higher in Y-STRs. The most polymorphic autosomal locus was D12S391, which presented 38 different sequence-based alleles. Among the loci in the Y chromosome, DYS389II presented the highest number of isoalleles. In comparison to CE analysis, Observed and Expected Heterozygosity, Polymorphic Information Content (PIC) and Genetic Diversity also presented higher values when the alleles were analyzed based on their sequence. For autosomal loci, Polymorphic Information Content (PIC) was 2.6% higher for sequence-based data. Diversity was 9.3% and 6.5% higher for autosomal and Y markers, respectively. In the analysis of the repeat structures for the STR loci, a new allele variant was found for allele 18 in the vWA locus. The STR flanking regions were also further investigated and sixteen variations were observed at nine autosomal STR loci and one Y-STR locus. The results obtained in this study demonstrate the importance of genetic analysis based on sequencing and highlight the diversity of the South Brazilian population when characterized by STR sequencing.

Next-generation Sequencing (NGS) is a rapidly evolving technology with demonstrated benefits for forensic genetic applications, and the strategies to analyze and manage the massive NGS datasets are currently in development. Here, the computing, data storage, connectivity, and security resources of the Cloud were evaluated as a model for forensic laboratory systems that produce NGS data. A complete front-to-end Cloud system was developed to upload, process, and interpret raw NGS data using a web browser dashboard. The system was extensible, demonstrating analysis capabilities of autosomal and Y-STRs from a variety of NGS instrumentation (Illumina MiniSeq and MiSeq, and Oxford Nanopore MinION). NGS data for STRs were concordant with standard reference materials previously characterized with capillary electrophoresis and Sanger sequencing. The computing power of the Cloud was implemented with on-demand auto-scaling to allow multiple file analysis in tandem. The system was designed to store resulting data in a relational database, amenable to downstream sample interpretations and databasing applications following the most recent guidelines in nomenclature for sequenced alleles. Lastly, a multi-layered Cloud security architecture was tested and showed that industry standards for securing data and computing resources were readily applied to the NGS system without disadvantageous effects for bioinformatic analysis, connectivity or data storage/retrieval. The results of this study demonstrate the feasibility of using Cloud-based systems for secured NGS data analysis, storage, databasing, and multi-user distributed connectivity.

The North Atlantic right whale is one of only three species within the Eubalaena genus, a group heavily targeted by the whaling industry prior to the twentieth century. All three species (Eubalaena australis, Eubalaena japonica and Eubalaena glacialis) are CITES listed (Appendix I), with the latter two species listed as endangered (IUCN, v 2017-1). The mitochondrial genome of the first two species have been sequenced and are publicly available, while E. glacialis was unavailable. Here we present the complete mitochondrial genome sequence of E. glacialis, while also detailing a straight forward mitochondrial enrichment method that facilitates analysis using next generation sequencing. This method simultaneously allows for efficient sequencing of the mitochondrial genome, while demonstrating quality sequence coverage that is even across the genome. This enrichment illustrates a marked improvement in sequence depth compared to that achieved when performing whole genome sequencing using a standard DNA extraction.

The forensic science community is poised to utilize modern advances in massively parallel sequencing (MPS) technologies to better characterize biological samples with higher resolution. A critical component towards the advancement of forensic DNA analysis with these technologies is a comprehensive understanding of the diversity and population distribution of sequence-based short tandem repeat (STR) alleles. Here we analyzed 786 samples of individuals from different population groups, including four of the mostly commonly encountered in forensic casework in the USA. DNA samples were amplified with the PowerSeq™ Auto/Y System Prototype Kit (Promega Corp.), and sequencing was performed on an Illumina® MiSeq instrument. Sequence data were analyzed using a bioinformatics processing tool, Altius. For additional data analysis and profile comparison, capillary electrophoresis (CE) size-based STR genotypes were generated for a subset of individuals, and where possible, also with a second commercially available MPS STR assay. Autosomal STR loci were analyzed and frequencies were calculated based on sequence composition. Also, population genetics studies were performed, with Hardy-Weinberg equilibrium, polymorphic information content (PIC), and observed and expected heterozygosity all assessed. Overall, sequence-based allelic variants of the repeat region were observed in 20 out of 22 different STR loci commonly used in forensic DNA genotyping, with the highest number of sequence variation observed at locus D12S391. The highest increase in allelic diversity and in PIC through sequence-based genotyping was observed at loci D3S1358 and D8S1179. Such detailed sequence analysis, as the one performed in the present study, is important to help understand the diversity of sequence-based STR alleles across different populations and to demonstrate how such allelic variation can improve statistics used for forensic casework.

Sequencing short tandem repeat (STR) loci allows for determination of repeat motif variations within the STR (or entire PCR amplicon) which cannot be ascertained by size-based PCR fragment analysis. Sanger sequencing has been used in research laboratories to further characterize STR loci, but is impractical for routine forensic use due to the laborious nature of the procedure in general and additional steps required to separate heterozygous alleles. Recent advances in library preparation methods enable high-throughput next generation sequencing (NGS) and technological improvements in sequencing chemistries now offer sufficient read lengths to encompass STR alleles. Herein, we present sequencing results from 183 DNA samples, including African American, Caucasian, and Hispanic individuals, at 22 autosomal forensic STR loci using an assay designed for NGS. The resulting dataset has been used to perform population genetic analyses of allelic diversity by length compared to sequence, and exemplifies which loci are likely to achieve the greatest gains in discrimination via sequencing. Within this data set, six loci demonstrate greater than double the number of alleles obtained by sequence compared to the number of alleles obtained by length: D12S391, D2S1338, D21S11, D8S1179, vWA, and D3S1358. As expected, repeat region sequences which had not previously been reported in forensic literature were identified.

The use of environmental trace material to aid criminal investigations is an ongoing field of research within forensic science. The application of environmental material thus far has focused upon a variety of different objectives relevant to forensic biology, including sample provenance (also referred to as sample attribution). The capability to predict the provenance or origin of an environmental DNA sample would be an advantageous addition to the suite of investigative tools currently available. A metabarcoding approach is often used to predict sample provenance, through the extraction and comparison of the DNA signatures found within different environmental materials, such as the bacteria within soil or fungi within dust. Such approaches are combined with bioinformatics workflows and statistical modelling, often as part of large-scale study, with less emphasis on the investigation of the adaptation of these methods to a smaller scale method for forensic use. The present work was investigating a small-scale approach as an adaptation of a larger metabarcoding study to develop a model for global sample provenance using fungal DNA signatures collected from dust swabs. This adaptation was to facilitate a standardized method for consistent, reproducible sample treatment, including bioinformatics processing and final application of resulting data to the available prediction model. To investigate this small-scale method, 76 DNA samples were treated as anonymous test samples and analyzed using the standardized process to demonstrate and evaluate processing and customized sequence data analysis. This testing included samples originating from countries previously used to train the model, samples artificially mixed to represent multiple or mixed countries, as well as outgroup samples. Positive controls were also developed to monitor laboratory processing and bioinformatics analysis. Through this evaluation we were able to demonstrate that the samples could be processed and analyzed in a consistent manner, facilitated by a relatively user-friendly bioinformatic pipeline for sequence data analysis. Such investigation into standardized analyses and application of metabarcoding data is of key importance for the future use of applied microbiology in forensic science.

Massively parallel sequencing (MPS) is gaining attention as a new technology for routine forensic casework, including paternity testing. Recently released MPS multiplex panels provide many more loci compared to CE methods, plus provide sequence-based alleles that together improve the statistical power of the genetic testing. Here, an MPS system (PowerSeq™ AUTO/Y) was applied for STR sequencing in the study of first-degree STR sequence allele inheritance from families in Southern Brazil. In 29 trios (mother-child-father) analyzed, the paternity index values generally increased when data from sequence-based analysis were used in comparison to length-based data. Further, allele inconsistencies (e.g., single repeat mutation events) between child and parents could be resolved with MPS by assessing the core repeat and flanking region sequences. Lastly, the sequence information allowed for identification of isoalleles (alleles of the same size, but different sequence) to determine specific paternal and maternal inheritances. The results from this study showed advantages of implementing sequence-based analysis, MPS, in paternity testing with improved statistical calculations and a greater resolution for the trios/families tested.

The development of molecular tools to detect and report mitochondrial DNA (mtDNA) heteroplasmy will increase the discrimination potential of the testing method when applied to forensic cases. The inherent limitations of the current state-of-the-art, Sanger-based sequencing, including constrictions in speed, throughput, and resolution, have hindered progress in this area. With the advent of next-generation sequencing (NGS) approaches, it is now possible to clearly identify heteroplasmic variants, and at a much lower level than previously possible. However, in order to bring these approaches into forensic laboratories and subsequently as accepted scientific information in a court of law, validated methods will be required to produce and analyze NGS data. We report here on the development of an optimized approach to NGS analysis for the mtDNA genome (mtgenome) using the Illumina MiSeq instrument. This optimized protocol allows for the production of more than 5 gigabases of mtDNA sequence per run, sufficient for detection and reliable reporting of minor heteroplasmic variants down to approximately 0.5–1.0% when multiplexing twelve samples. Depending on sample throughput needs, sequence coverage rates can be set at various levels, but were optimized here for at least 5000 reads. In addition, analysis parameters are provided for a commercially available software package that identify the highest quality sequencing reads and effectively filter out sequencing-based noise. With this method it will be possible to measure the rates of low-level heteroplasmy across the mtgenome, evaluate the transmission of heteroplasmy between the generations of maternal lineages, and assess the drift of variant sequences between different tissue types within an individual.

The ability to determine the tissue source of biological materials from evidence samples can be highly informative for interpreting forensic data. In this study, a previously published CE-based method to probe locus-specific DNA methylation was modified to accommodate detection using next-generation sequencing (NGS) to perform tissue source attribution. DNA samples (1 ng) from each of four different tissue types were digested with the methylation sensitive restriction endonuclease Hha1 and PCR was used to amplify an optimized subset of ten methylated loci, including positive and negative control loci. The products were prepared as NGS libraries, pooled in a multiplex assay with sample-specific barcodes, sequenced with an Illumina MiSeq, and analyzed using a k-Nearest Neighbor algorithm. With this initial effort a concordance rate of 15/16 was demonstrated from samples of varying types: semen, saliva, skin epidermis, and blood. This method also was designed to be compatible with the workflows published to date for NGS of STRs. Thus, the methylation approach described here is highly accurate and upon further validation and testing may be potentially used in practice as a confirmatory test in conjunction with other NGS protocols used in forensic laboratories.

The rhesus macaque (RM) model has the potential to be an invaluable tool for studying B cell populations during pathogenic infections, however, to date, there has been no definitive delineation of naïve and memory B cell populations in the RM. This has precluded a rigorous analysis of the generation, persistence and resolution of a pathogen-specific memory B cell response. The present study utilized multiple analyses to demonstrate that CD27 expression on B cells is consistent with a memory phenotype. Compared to CD20+CD27− B cells, CD20+CD27+ B cells were larger in size, and preferentially accumulated at effector sites. Direct sequence analysis revealed that CD20+ CD27+ B cells had an increased frequency of point mutations that were consistent with somatic hypermutation and at a functional level, CD40 ligation improved CD20+CD27− but not CD20+ CD27+ B cell survival in vitro. These data provide definitive evidence that the naïve and memory B cell populations of the RM can be differentiated using surface expression of CD27.

Achieving humoral immunity against human immunodeficiency virus (HIV) is a major obstacle in AIDS vaccine development. Despite eliciting robust humoral responses to HIV, exposed hosts rarely produce broadly neutralizing antibodies. The present study utilizes simian immunodeficiency virus (SIV) to identify viral epitopes that conferred antibody neutralization to clone SIV/17E-CL, an in vivo variant derived from neutralization resistant SIVmac239. Neutralization assays using rhesus macaque monoclonal antibodies were performed on viruses engineered to express single or multiple amino acid mutations. Results identified a single amino acid mutation, P334R, in the carboxy-terminal half of the V3 loop as a critical residue that induced neutralization while retaining normal glycoprotein expression on the surface of the virus. Furthermore, the R334 residue yielded neutralization sensitivity by antibodies recognizing diverse conformational and linear epitopes of gp120, suggesting that neutralization phenotype was a consequence of global structural changes of the envelope protein rather than a specific site epitope.

Despite eliciting a robust antibody response in humans, several studies in human immunodeficiency virus (HIV)-infected patients have demonstrated the presence of B-cell deficiencies during the chronic stage of infection. While several explanations for the HIV-induced B-cell deficit have been proposed, a clear mechanistic understanding of this loss of B-cell functionality is not known. This study utilizes simian immunodeficiency virus (SIV) infection of rhesus macaques to assess B-cell population dynamics beginning at the acute phase and continuing through the chronic phase of infection. Flow cytometric assessment demonstrated a significant early depletion of both naïve and memory B-cell subsets in the peripheral blood, with differential kinetics for recovery of these populations. Furthermore, the altered numbers of naïve and memory B-cell subsets in these animals corresponded with increased B-cell activation and altered proliferation profiles during the acute phase of infection. Finally, all animals produced high titers of antibody, demonstrating that the measurement of virus-specific antibody responses was not an accurate reflection of alterations in the B-cell compartment. These data indicate that dynamic B-cell population changes in SIV-infected macaques arise very early after infection at the precise time when an effective adaptive immune response is needed.

To study hydrocarbon biodegradation in marsh sediments impacted by Macondo oil from the Deepwater Horizon well blowout, we collected sediment cores 18-36 months after the accident at the marshes in Bay Jimmy (Upper Barataria Bay), Louisiana, United States. The highest concentrations of oil were found in the top 2 cm of sediment nearest the waterline at the shorelines known to have been heavily oiled. Although petroleum hydrocarbons were detectable, Macondo oil could not be identified below 8 cm in 19 of the 20 surveyed sites. At the one site where oil was detected below 8 cm, concentrations were low. Residual Macondo oil was already highly weathered at the start of the study, and the concentrations of individual saturated hydrocarbons and polycyclic aromatic hydrocarbons continued to decrease over the course of the study due to biodegradation. Desulfococcus oleovorans, Marinobacter hydrocarbonoclasticus, Mycobacterium vanbaalenii, and related mycobacteria were the most abundant oil-degrading microorganisms detected in the top 2 cm at the oiled sites. Relative populations of these taxa declined as oil concentrations declined. The diversity of the microbial community was low at heavily oiled sites compared to that of the unoiled reference sites. As oil concentrations decreased over time, microbial diversity increased and approached the diversity levels of the reference sites. These trends show that the oil continues to be biodegraded, and microbial diversity continues to increase, indicating ongoing overall ecological recovery.

DNA-based methods for human identification principally rely upon genotyping of short tandem repeat (STR) loci. Electrophoretic-based techniques for variable-length classification of STRs are universally utilized, but are limited in that they have relatively low throughput and do not yield nucleotide sequence information. High-throughput sequencing technology may provide a more powerful instrument for human identification, but is not currently validated for forensic casework. Here, we present a systematic method to perform high-throughput genotyping analysis of the Combined DNA Index System (CODIS) STR loci using short-read (150 bp) massively parallel sequencing technology. Open source reference alignment tools were optimized to evaluate PCR-amplified STR loci using a custom designed STR genome reference. Evaluation of this approach demonstrated that the 13 CODIS STR loci and amelogenin (AMEL) locus could be accurately called from individual and mixture samples. Sensitivity analysis showed that as few as 18,500 reads, aligned to an in silico referenced genome, were required to genotype an individual (>99% confidence) for the CODIS loci. The power of this technology was further demonstrated by identification of variant alleles containing single nucleotide polymorphisms (SNPs) and the development of quantitative measurements (reads) for resolving mixed samples.

Resveratrol inhibits herpes simplex virus (HSV) replication by an unknown mechanism. Previously it was suggested that this inhibition may be mediated through a cellular factor essential for HSV replication [Docherty, J.J., Fu, M.M., Stiffler, B.S., Limperos, R.J., Pokabla, C.M., DeLucia, A.L., 1999. Resveratrol inhibition of herpes simplex virus replication. Antivir. Res. 43, 145-155]. After examining numerous cellular factors, we report that resveratrol suppresses NF-kappaB (NF-kappaB) activation in HSV infected cells. Reports have indicated that HSV activates NF-kappaB during productive infection and this may be an essential aspect of its replication scheme [Patel, A., Hanson, J., McLean, T.I., Olgiate, J., Hilton, M., Miller, W.E., Bachenheimer, S.L., 1998. Herpes simplex type 1 induction of persistent NF-kappa B nuclear translocation increases the efficiency of virus replication. Virology 247, 212-222; Gregory, D., Hargett, D., Holmes, D., Money, E., Bachenheimer, S.L., 2004. Efficient replication by herpes simplex virus type 1 involves activation of the IkappaB kinase-IkappaB-RelA/p65 pathway. J. Virol. 78, 13582-13590]. Electromobility shift assays determined that resveratrol, in a dose dependent and reversible manner, suppressed activation of NF-kappaB in Vero cells infected with HSV-1, HSV-2 and acyclovir resistant HSV-1. Furthermore, resveratrol did not protect IkappaBalpha, a cytoplasmic NF-kappaB inhibitor, from degradation in HSV-1 infected cells. Immunohistochemical studies demonstrated that RelA/p65, a component of the dimeric NF-kappaB complex, translocated to the nucleus of HSV-1 infected cells in the presence of resveratrol. Finally, direct effects on viral transcription and DNA synthesis were evaluated. Real-time RT-PCR analysis showed that resveratrol treatment of infected cells resulted in reductions of mRNA for ICP0, ICP4, ICP8 and HSV-1 DNA polymerase by 2.1-, 3.3-, 3.8- and 3.1-fold, respectively. Plus, mRNA for glycoprotein C, an HSV late gene, was completely absent in the presence of resveratrol. Lastly, quantitative PCR showed that resveratrol significantly blocked HSV DNA synthesis. Cumulatively, these data indicate that resveratrol (i) suppresses HSV induced activation of NF-kappaB within the nucleus and (ii) impairs expression of essential immediate-early, early and late HSV genes and synthesis of viral DNA.

The ability to determine the tissue source of biological materials from evidence samples can be highly informative for interpreting forensic data. In this study, a previously published CE-based method to probe locus-specific DNA methylation was modified to accommodate detection using next-generation sequencing (NGS) to perform tissue source attribution. DNA samples (1 ng) from each of four different tissue types were digested with the methylation sensitive restriction endonuclease Hha1 and PCR was used to amplify an optimized subset of ten methylated loci, including positive and negative control loci. The products were prepared as NGS libraries, pooled in a multiplex assay with sample-specific barcodes, sequenced with an Illumina MiSeq, and analyzed using a k-Nearest Neighbor algorithm. With this initial effort a concordance rate of 15/16 was demonstrated from samples of varying types: semen, saliva, skin epidermis, and blood. This method also was designed to be compatible with the workflows published to date for NGS of STRs. Thus, the methylation approach described here is highly accurate and upon further validation and testing may be potentially used in practice as a confirmatory test in conjunction with other NGS protocols used in forensic laboratories.

Human saliva is clinically informative of both oral and general health. Since next generation shotgun sequencing (NGS) is now widely used to identify and quantify bacteria, we investigated the bacterial flora of saliva microbiomes of two healthy volunteers and five datasets from the Human Microbiome Project, along with a control dataset containing short NGS reads from bacterial species representative of the bacterial flora of human saliva. GENIUS, a system designed to identify and quantify bacterial species using unassembled short NGS reads was used to identify the bacterial species comprising the microbiomes of the saliva samples and datasets. Results, achieved within minutes and at greater than 90% accuracy, showed more than 175 bacterial species comprised the bacterial flora of human saliva, including bacteria known to be commensal human flora but also Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Gamma proteobacteria. Basic Local Alignment Search Tool (BLASTn) analysis in parallel, reported ca. five times more species than those actually comprising the in silico sample. Both GENIUS and BLAST analyses of saliva samples identified major genera comprising the bacterial flora of saliva, but GENIUS provided a more precise description of species composition, identifying to strain in most cases and delivered results at least 10,000 times faster. Therefore, GENIUS offers a facile and accurate system for identification and quantification of bacterial species and/or strains in metagenomic samples.

In refining methodology to develop a mouse model for inhalation of Francisella tularensis, it was noted that both relative humidity and growth media impacted the aerosol concentration of the live vaccine strain (LVS) of F. tularensis. A relative humidity of less than 55% had a negative impact on the spray factor, the ratio between the concentration of LVS in the aerosol and the nebulizer. The spray factor was significantly higher for LVS grown in brain heart infusion (BHI) broth than LVS grown in Mueller-Hinton broth (MHb) or Chamberlain’s Chemically Defined Medium (CCDM). The variability between aerosol exposures was also considerably less with BHI. LVS grown in BHI survived desiccation far longer than MHb-grown or CCDM-grown LVS (~70% at 20 minutes for BHI compared to

Resveratrol was found to inhibit varicella-zoster virus (VZV) replication in a dose-dependent and reversible manner. This decrease in virus production in the presence of resveratrol was not caused by direct inactivation of VZV or inhibition of virus attachment to MRC-5 cells. The drug effectively limited VZV replication if added during the first 30 h of infection. Western blot analysis and real-time RT-PCR studies demonstrated that protein and mRNA levels of IE62, an essential immediate early viral protein, were reduced when compared to controls. These results demonstrate that VZV replication is adversely affected by resveratrol which is negatively impacting IE62 synthesis.