Efforts to enhance reproducibility in a human performance research project.

Background: Ensuring the validity of results from funded programs is a critical concern for agencies that sponsor biological research. In recent years, the open science movement has sought to promote reproducibility by encouraging sharing not only of finished manuscripts but also of data and code supporting their findings. While these innovations have lent support to third-party efforts to replicate calculations underlying key results in the scientific literature, fields of inquiry where privacy considerations or other sensitivities preclude the broad distribution of raw data or analysis may require a more targeted approach to promote the quality of research output. Methods: We describe efforts oriented toward this goal that were implemented in one human performance research program, Measuring Biological Aptitude, organized by the Defense Advanced Research Project Agency's Biological Technologies Office. Our team implemented a four-pronged independent verification and validation (IV&V) strategy including 1) a centralized data storage and exchange platform, 2) quality assurance and quality control (QA/QC) of data collection, 3) test and evaluation of performer models, and 4) an archival software and data repository. Results: Our IV&V plan was carried out with assistance from both the funding agency and participating teams of researchers. QA/QC of data acquisition aided in process improvement and the flagging of experimental errors. Holdout validation set tests provided an independent gauge of model performance. Conclusions: In circumstances that do not support a fully open approach to scientific criticism, standing up independent teams to cross-check and validate the results generated by primary investigators can be an important tool to promote reproducibility of results.

Metagenomic features of bioburden serve as outcome indicators in combat extremity wounds.

Battlefield injury management requires specialized care, and wound infection is a frequent complication. Challenges related to characterizing relevant pathogens further complicates treatment. Applying metagenomics to wounds offers a comprehensive path toward assessing microbial genomic fingerprints and could indicate prognostic variables for future decision support tools. Wound specimens from combat-injured U.S. service members, obtained during surgical debridements before delayed wound closure, were subjected to whole metagenome analysis and targeted enrichment of antimicrobial resistance genes. Results did not indicate a singular, common microbial metagenomic profile for wound failure, instead reflecting a complex microenvironment with varying bioburden diversity across outcomes. Genus-level Pseudomonas detection was associated with wound failure at all surgeries. A logistic regression model was fit to the presence and absence of antimicrobial resistance classes to assess associations with nosocomial pathogens. A. baumannii detection was associated with detection of genomic signatures for resistance to trimethoprim, aminoglycosides, bacitracin, and polymyxin. Machine learning classifiers were applied to identify wound and microbial variables associated with outcome. Feature importance rankings averaged across models indicated the variables with the largest effects on predicting wound outcome, including an increase in P. putida sequence reads. These results describe the microbial genomic determinants in combat wound bioburden and demonstrate metagenomic investigation as a comprehensive tool for providing information toward aiding treatment of combat-related injuries.

Gut microbiome associations with outcome following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in pigs immunized with a PRRS modified live virus vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most significant pathogens affecting swine. Co-infections are common and result in respiratory disease and reduced weight gain in growing pigs. Although PRRS modified live virus (MLV) vaccines are widely used to decrease PRRS-associated losses, they are generally considered inadequate for disease control. The gut microbiome provides an alternative strategy to enhance vaccine efficacy and improve PRRS control. The objective of this study was to identify gut microbiome characteristics associated with improved outcome in pigs immunized with a PRRS MLV and co-challenged with PRRSV and PCV2b. Twenty-eight days after vaccination and prior to co-challenge, fecal samples were collected from an experimental population of 50 nursery pigs. At 42 days post-challenge, 20 pigs were retrospectively identified as having high or low growth outcomes during the post-challenge period. Gut microbiomes of the two outcome groups were compared using the Lawrence Livermore Microbial Detection Array (LLMDA) and 16S rDNA sequencing. High growth outcomes were associated with several gut microbiome characteristics, such as increased bacterial diversity, increased Bacteroides pectinophilus, decreased Mycoplasmataceae species diversity, higher Firmicutes:Bacteroidetes ratios, increased relative abundance of the phylum Spirochaetes, reduced relative abundance of the family Lachnospiraceae, and increased Lachnospiraceae species C6A11 and P6B14. Overall, this study identifies gut microbiomes associated with improved outcomes in PRRS vaccinated pigs following a polymicrobial respiratory challenge and provides evidence towards the gut microbiome playing a role in PRRS vaccine efficacy.

Manipulation of the Gut Microbiome Alters Acetaminophen Biodisposition in Mice.

The gut microbiota is a vast and diverse microbial community that has co-evolved with its host to perform a variety of essential functions involved in the utilization of nutrients and the processing of xenobiotics. Shifts in the composition of gut microbiota can disturb the balance of organisms which can influence the biodisposition of orally administered drugs. To determine how changes in the gut microbiome can alter drug disposition, the pharmacokinetics (PK), and biodistribution of acetaminophen were assessed in C57Bl/6 mice after treatment with the antibiotics ciprofloxacin, amoxicillin, or a cocktail of ampicillin/neomycin. Altered PK, and excretion profiles of acetaminophen were observed in antibiotic exposed animals. Plasma Cmax was significantly decreased in antibiotic treated animals suggesting decreased bioavailability. Urinary metabolite profiles revealed decreases in acetaminophen-sulfate metabolite levels in both the amoxicillin and ampicillin/neomycin treated animals. The ratio between urinary and fecal excretion was also altered in antibiotic treated animals. Analysis of gut microbe composition revealed that changes in microbe content in antibiotic treated animals was associated with changes in acetaminophen biodisposition. These results suggest that exposure to amoxicillin or ampicillin/neomycin can alter the biodisposition of acetaminophen and that these alterations could be due to changes in gut microbiome composition.

Mosquito-Borne Viruses and Insect-Specific Viruses Revealed in Field-Collected Mosquitoes by a Monitoring Tool Adapted from a Microbial Detection Array.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.IMPORTANCE Viruses associated with mosquitoes have made a large impact on public and veterinary health. In the United States, several viruses, including WNV, DENV, and chikungunya virus (CHIKV), are responsible for human disease. From 2015 to 2018, imported Zika cases were reported in the United States, and in 2016 to 2017, local Zika transmission occurred in the states of Texas and Florida. With globalization and a changing climate, the frequency of outbreaks linked to arboviruses will increase, revealing a need to better detect viruses in vector populations. With the capacity of the LLMDA to detect viruses, bacteria, and fungi, this study highlights its ability to broadly screen field-collected mosquitoes and contribute to the surveillance and management of arboviral diseases.

Axiom Microbiome Array, the next generation microarray for high-throughput pathogen and microbiome analysis.

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. In this study, the Axiom Microbiome Array was evaluated to determine its sensitivity, specificity and utility in microbiome analysis of veterinary clinical samples. The array contains probes designed to detect more than 12,000 species of viruses, bacteria, fungi, protozoa and archaea, yielding the most comprehensive microbial detection platform built to date. The array was able to detect Shigella and Aspergillus at 100 genome copies, and vaccinia virus DNA at 1,000 genome copies. The Axiom Microbiome Array made correct species-level calls in mock microbial community samples. When tested against serum, tissue, and fecal samples from pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, the microarray correctly detected these two viruses and other common viral and bacterial microbiome species. This cost-effective and high-throughput microarray is an efficient tool to rapidly analyze large numbers of clinical and environmental samples for the presence of multiple viral and bacterial pathogens.

Double-stranded viral RNA persists in vitro and in vivo during prolonged infection of porcine reproductive and respiratory syndrome virus.

In order to study the mechanism of PRRSV persistence, an in vitro model of persistence was developed by serially passaging PRRSV-infected MARC-145 cells 109 times. Viral persistence was detected to be associated with increased double-stranded (dsRNA) in the infected cells. In PRRSV infected pigs, reduced ratio of plus to minus strands of viral RNA was observed in lymphoid tissues from PRRSV persistent pigs at 52 days post infection. Viral dsRNA was mostly detected in the germinal center during persistent infection compared to the localization of dsRNA in the inter-follicular zones during acute infection. RNA array analysis of antiviral cytokines in persistently infected lymph nodes showed that the presence of dsRNA did not stimulate antiviral immunity. These results suggest that PRRSV dsRNA functions as a mediator for viral persistence. The localization of PRRSV dsRNA in the germinal center of lymphoid tissues reveals a novel mechanism for PRRSV persistence.

Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses.

Recently, we identified a unique -2/-1 ribosomal frameshift mechanism in PRRSV, which yields two truncated forms of nonstructural protein (nsp) 2 variants, nsp2TF and nsp2N. Here, in vitro expression of individual PRRSV nsp2TF and nsp2N demonstrated their ability to suppress cellular innate immune responses in transfected cells. Two recombinant viruses were further analyzed, in which either nsp2TF was C-terminally truncated (vKO1) or expression of both nsp2TF and nsp2N was knocked out (vKO2). Host cellular mRNA profiling showed that a panel of cellular immune genes, in particular those involved in innate immunity, was upregulated in cells infected with vKO1 and vKO2. Compared to the wild-type virus, vKO1 and vKO2 expedited the IFN-α response and increased NK cell cytotoxicity, and subsequently enhanced T cell immune responses in infected pigs. Our data strongly implicate nsp2TF/nsp2N in arteriviral immune evasion and demonstrate that nsp2TF/nsp2N-deficient PRRSV is less capable of counteracting host innate immune responses.

Increased microbiome diversity at the time of infection is associated with improved growth rates of pigs after co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2).

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most important pathogens affecting the swine industry worldwide. Co-infections are common on a global scale, resulting in pork production losses through reducing weight gain and causing respiratory disease in growing pigs. Our initial work demonstrated that the fecal microbiome was associated with clinical outcome of pigs 70days post-infection (dpi) with PRRSV and PCV2. However, it remained uncertain if microbiome characteristics could predispose response to viral infection. The purpose of this study was to determine if microbiome characteristics present at the time of virus exposure were associated with outcome after co-infection. Using the Lawrence Livermore Microbial Detection Array, we profiled the microbiome in feces prior to infection from pigs identified retrospectively as having high or low growth rates after co-infection. High growth rate pigs had less severe interstitial pneumonia, reduced virus replication, and a significant increase in average daily weight gain throughout the study. At the level of the fecal microbiome, high growth rate pigs had increased microbial diversity on both a family and species level. Shifts in the microbiome composition of high growth rate pigs included reduced Methanobacteriaceae species, increased Ruminococcaceae species, and increased Streptococcaceae species when compared to low growth rate pigs. The results indicate that both microbiome diversity and composition at the time of virus exposure may play a role in the subsequent response of pigs to PRRSV/PCV2 co-infection.