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At least 127 records · Page 7

North American Wintering Mallards Infected With Highly Pathogenic Avian Influenza Show Few Signs of Altered Local or Migratory Movements

Avian influenza viruses pose a threat to wildlife and livestock health. The emergence of highly pathogenic avian influenza (HPAI) in wild birds and poultry in North America in late 2021 was the first such outbreak since 2015 and the largest outbreak in North America to date. Despite its prominence and economic impacts, we know relatively little about how HPAI spreads in wild bird populations. In January 2022, we captured 43 mallards (Anas platyrhynchos) in Tennessee, USA, 11 of which were actively infected with HPAI. These were the first confirmed detections of HPAI H5N1 clade 2.3.4.4b in the Mississippi Flyway. We compared movement patterns of infected and uninfected birds and found no clear differences; infected birds moved just as much during winter, migrated slightly earlier, and migrated similar distances as uninfected birds. Infected mallards also contacted and shared space with uninfected birds while on their wintering grounds, suggesting ongoing transmission of the virus. We found no differences in body condition or survival rates between infected and uninfected birds. Together, these results show that HPAI H5N1 clade 2.3.4.4b infection was unrelated to body condition or movement behavior in mallards infected at this location during winter; if these results are confirmed in other seasons and as HPAI H5N1 continues to evolve, they suggest that these birds could contribute to the maintenance and dispersal of HPAI in North America. Further research on more species across larger geographic areas and multiple seasons would help clarify potential impacts of HPAI on waterfowl and how this emerging disease spreads at continental scales, across species, and potentially between wildlife and domestic animals.

animal migration↗

Bring'em Back Alive or at Least Carefully

Within the next decade, the world's space agencies plan to launch a variety of robotic spacecraft that will return samples from the surface of Mars, the tail of a comet, the nucleus of a comet, the surface of an asteroid, and the solar wind. Most of these places are not considered likely spots for life, but any mission returning from a location with the potential for harboring life will require special containment and handling because of the possible inclusion of living entities within returned samples. In its 1997 report on sample return from Mars, the Space Studies Board of the National Research Council (NRC) noted that the only risk of significant adverse effects would be from returning a replicating organism. Furthermore, the report noted: 'While the probability of returning a replicating biological entity in a sample from Mars' is judged to be low and the risk of pathogenic or ecological effects is lower still, the risk is not zero. Therefore, it is reasonable that NASA adopt a prudent approach, erring on the side of caution and safety when dealing with returned samples. More recently, a 1998 NRC report on small solar system bodies (asteroids, comets, planetary satellites, and interplanetary dust) recommended a similarly cautious approach for samples returned from anywhere else within the solar system that could have environmental conditions conducive for harboring life. We have not detected life elsewhere in the solar system, at least not yet. Nonetheless, the rationale behind the conservative approach to sample handling is similar to the environmental, health, and safety measures taken on Earth when transporting or handling infectious agents or importing non-native organisms to a new area. Better safe than sorry.

Race, Margaret S.↗

The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157:H7

A suite of fluorescent intracellular stains and probes was used, in conjunction with viable plate counts, to assess the effect of chlorine disinfection on membrane potential (rhodamine 123; Rh123 and bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC4(3)), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride; CTC) and substrate responsiveness (direct viable counts; DVC) in the commensal pathogen Escherichia coli O157:H7. After a 5 min exposure to the disinfectant, physiological indices were affected in the following order: viable plate counts > substrate responsiveness > membrane potential > respiratory activity > membrane integrity. In situ assessment of physiological activity by examining multiple targets, as demonstrated in this study, permits a more comprehensive determination of the site and extent of injury in bacterial cells following sublethal disinfection with chlorine. This approach to assessing altered bacterial physiology has application in various fields where detection of stressed bacteria is of interest.

NASA Discipline Environmental Health↗

Adenosine Monophosphate-Based Detection of Bacterial Spores

A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

Kern, Roger G.↗

In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment

ABSTRACT Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998–2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infectionin vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancementin vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operatingin vivomay modulate ZIKV infection outcomes. IMPORTANCE We investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998–2020) to neutralize or enhance Zika virus infectionin vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, bothin vitroandin vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Microbiology↗

The mechanosensory calcium-selective ion channel: key component of a plasmalemmal control centre?

Mechanosensory calcium-selective ion channels probably serve to detect not only mechanical stress but also electrical, thermal, and diverse chemical stimuli. Because all stimuli result in a common output, most notably a shift in second messenger calcium concentration, the channels are presumed to serve as signal integrators. Further, insofar as second messenger calcium in turn gives rise to mechanical, electrical, and diverse chemical changes, the channels are postulated to initiate regulatory feedbacks. It is proposed that the channels and the feedback loops play a wide range of roles in regulating normal plant function, as well as in mediating disturbance of normal function by environmental stressors and various pathogens. In developing evidence for the physiological performance of the channel, a model for a cluster of regulatory plasmalemmal proteins and cytoskeletal elements grouped around a set of wall-to-membrane and transmembrane linkers has proved useful. An illustration of how the model might operate is presented. It is founded on the demonstration that several xenobiotics interfere both with normal channel behaviour and with gravitropic reception. Accordingly, the first part of the illustration deals with how the channels and the control system within which they putatively operate might initiate gravitropism. Assuming that gravitropism is an asymmetric expression of growth, the activities of the channels and the plasmalemmal control system are extrapolated to account for regulation of both rate and allometry of cell expansion. Finally, it is discussed how light, hormones, redox agents and herbicides could in principle affect growth via the putative plasmalemmal control cluster or centre.

NASA Program Space Biology↗

Efficacy of commercial recombinant HVT vaccines against a North American clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus in chickens

The outbreak of clade 2.3.4.4b H5 highly pathogenic avian influenza (HPAI) in North America that started in 2021 has increased interest in applying vaccination as a strategy to help control and prevent the disease in poultry. Two commercially available vaccines based on the recombinant herpes virus of turkeys (rHVT) vector were tested against a recent North American clade 2.3.4.4b H5 HPAI virus isolate: A/turkey/Indiana/22-003707-003/2022 H5N1 in specific pathogen free white leghorn (WL) chickens and commercial broiler chickens. One rHVT-H5 vaccine encodes a hemagglutinin (HA) gene designed by the computationally optimized broadly reactive antigen method (COBRA-HVT vaccine). The other encodes an HA gene of a clade 2.2 virus (2.2-HVT vaccine). There was 100% survival of both chicken types COBRA-HVT vaccinated groups and in the 2.2-HVT vaccinated groups there was 94.8% and 90% survival of the WL and broilers respectively. Compared to the 2.2-HVT vaccinated groups, WL in the COBRA-HVT vaccinated group shed significantly lower mean viral titers by the cloacal route and broilers shed significantly lower titers by the oropharyngeal route than broilers. Virus titers detected in oral and cloacal swabs were otherwise similar among both vaccine groups and chicken types. To assess antibody-based tests to identify birds that have been infected after vaccination (DIVA-VI), sera collected after the challenge were tested with enzyme-linked lectin assay-neuraminidase inhibition (ELLA-NI) for N1 neuraminidase antibody detection and by commercial ELISA for detection of antibodies to the NP protein. As early as 7 days post challenge (DPC) 100% of the chickens were positive by ELLA-NI. ELISA was less sensitive with a maximum of 75% positive at 10DPC in broilers vaccinated with 2.2-HVT. Both vaccines provided protection from challenge to both types of chickens and ELLA-NI was sensitive at identifying antibodies to the challenge virus therefore should be evaluated further for DIVA-VI.

60 APPLIED LIFE SCIENCES↗

Remote Sensing, GIS, and Vector-Borne Disease

The concept of global climate change encompasses more than merely an alteration in temperature; it also includes spatial and temporal covariations in precipitation and humidity, and more frequent occurrence of extreme weather events. The impact of these variations, which can occur at a variety of temporal and spatial scales, could have a direct impact on disease transmission through their environmental consequences for pathogen, vector, and host survival, as well as indirectly through human demographic and behavioral responses. New and future sensor systems will allow scientists to investigate the relationships between climate change and environmental risk factors at multiple spatial, temporal and spectral scales. Higher spatial resolution will provide better opportunities for mapping urban features previously only possible with high resolution aerial photography. These opportunities include housing quality (e.g., Chagas'disease, leishmaniasis) and urban mosquito habitats (e.g., dengue fever, filariasis, LaCrosse encephalitis). There are or will be many new sensors that have higher spectral resolution, enabling scientists to acquire more information about parameters such as soil moisture, soil type, better vegetation discrimination, and ocean color, to name a few. Although soil moisture content is now detectable using Landsat, the new thermal, shortwave infrared, and radar sensors will be able to provide this information at a variety of scales not achievable using Landsat. Soil moisture could become a key component in transmission risk models for Lyme disease (tick survival), helminthiases (worm habitat), malaria (vector-breeding habitat), and schistosomiasis (snail habitat).

Beck, Louisa R.↗

Conducting polymer-based electrochemical sensors: Progress, challenges, and future perspectives

Conducting polymers are promising due to their unique properties, such as excellent electrical and optical properties, physical and chemical stability, high conductivity, and effective redox properties with high-temperature stability and biocompatibility. Due to these properties, conducting polymers are useful in diverse applications like sensors, batteries, oil industries, biosensors, biomedicines, catalysis, cancer treatment, etc. This review article aims to discuss the recent trends and analysis of conducting polymer-based electrochemical sensors in diverse areas with all required sensor characteristics, such as the derived limit of detection, utilized techniques for the sensing analysis and derived linear dynamic range with the stability of the sensors. Conducting polymers and their nanocomposites-based electrochemical sensors have demonstrated exceptional capabilities towards detecting various biomolecules, heavy metals, pesticides, and viruses like SARS-COV-2. Incorporation of redox mediators, use of conducting hydrogels, and molecular imprinting are promising strategies for better performance of the derived sensor. The article has demonstrated the existing challenges and limitations and provided solutions in the field. In the future, conducting polymers-based electrochemical sensors can be utilized in wearable sensors and integrated with IoT devices for better reach in real-time applications. They can also be made more accessible with precise control and data output by following specific methodologies. Utilizing green and sustainable conducting polymers can be crucial in advancing eco-friendly practices in the future. Conducting polymer-based electrochemical sensors has affectivity in neurochemical and pathogen sensing, which is essential for brain function and mental health.

42 ENGINEERING↗

3D Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent VZV Infection

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

Goodwin, Thomas J.↗

405 nm light microbicidal efficacy on Treponema pallidum spiked in ex vivo human platelets

Abstract Pathogen reduction technologies using chemicals and or UV light have been demonstrated to improve the safety of ex vivo platelets from infectious diseases. However, UV light exposure also may affect the treated products, depending on wavelength and exposure. Alternatively, visible spectra 405 nm violet-blue light has broad-spectrum microbicidal activity. Here we tested the effect of 405 nm light onTreponema pallidum, the bacterium that causes syphilis. We contaminated platelets stored in plasma with two treponemal concentrations (low and high titers) and treated an aliquot with 270 J/cm 2 dose (irradiance = 15 mW/cm 2 ) of 405 nm light while another aliquot remained untreated. Next, we inoculated intradermally an aliquot of both samples into rabbits. Rabbits inoculated with untreated samples developed syphilis while animals inoculated with light-treated samples did not. Thus, inactivation was demonstrated to the limit of detection of the bioassay. We estimated > 2 log 10 and > 4 log 10 reduction in the low and high dose studies, respectively. These results provide proof-of-concept that 405 nm light is effective in reducing syphilis risk in ex vivo platelets.

Science & Technology - Other Topics↗

Evaluating an Automated Approach for Monitoring Forest Disturbances in the Pacific Northwest from Logging, Fire and Insect Outbreaks with Landsat Time Series Data

Forests are the largest aboveground sink for atmospheric carbon (C), and understanding how they change through time is critical to reduce our C-cycle uncertainties. We investigated a strong decline in Normalized Difference Vegetation Index (NDVI) from 1982 to 1991 in Pacific Northwest forests, observed with the National Ocean and Atmospheric Administration's (NOAA) series of Advanced Very High Resolution Radiometers (AVHRRs). To understand the causal factors of this decline, we evaluated an automated classification method developed for Landsat time series stacks (LTSS) to map forest change. This method included: (1) multiple disturbance index thresholds; and (2) a spectral trajectory-based image analysis with multiple confidence thresholds. We produced 48 maps and verified their accuracy with air photos, monitoring trends in burn severity data and insect aerial detection survey data. Area-based accuracy estimates for change in forest cover resulted in producer's and user's accuracies of 0.21 +/- 0.06 to 0.38 +/- 0.05 for insect disturbance, 0.23 +/- 0.07 to 1 +/- 0 for burned area and 0.74 +/- 0.03 to 0.76 +/- 0.03 for logging. We believe that accuracy was low for insect disturbance because air photo reference data were temporally sparse, hence missing some outbreaks, and the annual anniversary time step is not dense enough to track defoliation and progressive stand mortality. Producer's and user's accuracy for burned area was low due to the temporally abrupt nature of fire and harvest with a similar response of spectral indices between the disturbance index and normalized burn ratio. We conclude that the spectral trajectory approach also captures multi-year stress that could be caused by climate, acid deposition, pathogens, partial harvest, thinning, etc. Our study focused on understanding the transferability of previously successful methods to new ecosystems and found that this automated method does not perform with the same accuracy in Pacific Northwest forests. Using a robust accuracy assessment, we demonstrate the difficulty of transferring change attribution methods to other ecosystems, which has implications for the development of automated detection/attribution approaches. Widespread disturbance was found within AVHRR-negative anomalies, but identifying causal factors in LTSS with adequate mapping accuracy for fire and insects proved to be elusive. Our results provide a background framework for future studies to improve methods for the accuracy assessment of automated LTSS classifications.

Landsat↗

Study to define and verify the personal oral hygiene requirements for extended manned space flight: Oral physiology and microbiology in Skylab manned space missions

Methods for metabolic fingerprinting of pathogenic oral bacteria were developed and the effects of Skylab missions on salivary electrolyte levels were studied. High resolution gas liquid chromatographic (GLC) and pyrolysis-GLC procedures were used to obtain metabolic profiles of closely related bacteria associated with dental caries and periodontal disease. It was found that the GLC procedures provide a practical and reproducible means of obtaining metabolic markers for identifying closely related strains of these organisms. Fractions of stimulated whole saliva samples from the prime and back-up crews of the three Skylab missions were used to measure salivary electrolyte concentrations. All the electrolytes previously reported as having increased in urine and feces during the missions were assessed. Sodium, potassium, calcium, magnesium, phosphorous and chloride were studied. A decrease in sodium and an increase in magnesium were observed, but the mineral imbalances attributable to the mission-related increases in urinary electrolytes were not detected.

Brown, L. R.↗

A non-canonical fungal peroxisome PTS-1 signal, SYM, and its evolutionary aspects

Abstract Proteins localized to peroxisomes, particularly those expressed under specific conditions or in low abundance, are often undetected by routine proteomics methods due to detection sensitivity limits. In silico identification and experimental validation of peroxisomal targeting signals (PTSs) offer a reliable alternative. We demonstrate that SYM, a non-canonical plant PTS-1 signal, functions similarly inAspergillus nidulans, as GFP tagged with a SYM C-terminal tripeptide localizes to peroxisomes. One of two nativeA. nidulansproteins with C-terminal SYM tripeptide shows weak peroxisomal localization alongside cytoplasmic presence, indicating that only a subset of proteins with non-canonical signals access peroxisomes.In silicoanalysis of 1,010 fungal genomes identified diverse SYM-proteins with variable functions, suggesting that non-canonical PTS-1 signals may evolve spontaneously. Two-thirds of SYM-proteins are predicted to localize to specific intracellular compartments other than the peroxisome. We propose that despite their predicted localization, these proteins possessing SYM as a non-canonical peroxisomal signal might also have peroxisomal presence. Among SYM-proteins, pectinesterases, known plant pathogen virulence factors, were frequent. Notably, 25% of fungal pectinesterases harbor non-canonical PTS-1 signals, suggesting that partial peroxisomal localization of pectinesterases has evolved convergently. This suggests that partial peroxisomal localization may enhance protein functional flexibility, contributing to the organism’s adaptability.

Science & Technology - Other Topics↗

On-orbit RNA Purification and qRT-PCR Capabilities of the WetLab-2 System

NASA Ames Research Center’s WetLab-2 system brings new capabilities to the ISS for researchers. The system can lyse cells and extract RNA on-orbit from different sample types ranging from microbial cultures to animal tissues. Our purification method has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. qRT-PCR reactions are performed by dispensing the RNA into reaction tubes that contain all lyophilized reagents needed to perform the analysis. The system uses a Cepheid® SmartCycler that allows for multiplexing of assays, this can be used to normalize for RNA concentration and integrity and to study multiple genes of interest in each tube. There are a total of 16 independent PCR modules each capable of detecting up to four fluorescent channels. The WetLab- 2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to purify and stabilize RNA on-orbit can eliminate the confounding effects of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of some samples. It also has the benefit of minimizing the needed downmass. Conducting qRT-PCR and generating results on-orbit is also an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also provide benchmarking prior to sample return. The system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver is scheduled to launch on SpaceX-8 this spring. Pending operations, the preliminary results from the validation flight will be presented. To support the needs of future researchers, we are adapting our system to purify RNA from two additional sample types: fibrous tissue such as muscle and mammalian adherent cells grown on alginate beads. Progress of this work will also be presented. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

Parra, Macarena↗

Latent Viruses: A Space Travel Hazard??

A major issue associated with long-duration space flight is the possibility of infectious disease causing an unacceptable medical risk to crew members. Our proposal is designed to gain information that addresses several issues outlined in the Immunology/Infectious disease critical path. The major hypothesis addressed is that space flight causes alterations in the immune system that may allow latent viruses which are endogenous in the human population to reactivate and shed to higher levels than normal which can affect the health of crew members during a long term space-flight mission. We will initially focus our studies on the human herpesviruses and human polyomaviruses which are important pathogens known to establish latent infections in the human population. Both primary infection and reactivation from latent infection with this group of viruses can cause a variety of illnesses that result in morbidity and occasionally mortality of infected individuals. Effective vaccines exist for only one of the eight known human herpesviruses and the vaccine itself can still reactivate from latent infection. Available antivirals are of limited use and are effective against only a few of the human herpesviruses. Although most individuals display little if any clinical consequences from latent infection, events which alter immune function such as immunosuppressive therapy following solid organ transplantation are known to increase the risk of developing complications as a result of latent virus reactivation. This proposal will measure both the frequency and magnitude of viral shedding and genome loads in the blood from humans participating in activities that serve as ground based models of space flight conditions. Our initial goal is to develop sensitive quantitative competitive PCR- based assays (QC-PCR) to detect the herpesvirus Epstein-Barr virus (EBV), and the polyomaviruses SV40, BKV, and JCV. Using these assays we will establish baseline patterns of viral genome load in the blood and viral shedding from normal volunteers in a longitudinal study over I year in length. As a comparison, we will measure patterns of viral genome loads and shedding from individuals who are severely immunosuppressed, in whom herpesvirus reactivation or primary infection with a herpesvirus is known to cause complications. In addition, we will proceed to testing ground based analogs in collaboration with Dr. Duane Pierson (Lyndon B. Johnson Space Center). This will include measuring samples obtained from individuals living and working in the extreme environment of Antarctica. We expect to detect viral shedding or reactivation from most of the test groups, although the magnitude of shedding or reactivation cannot be predicted. The data accumulated from studies in this proposal should allow us to evaluate whether events that simulate certain aspects of space flight reactivate viral infections severe enough in nature that they may compromise the success of long-term space flight missions. These studies will also provide a foundation to monitor viral reactivation and shedding from crew members participating in actual space flight missions. We will present data showing the establishment of our QC-PCR assay for detection of EBV.

Ling, P. D.↗

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.

ISS tools↗

A Comprehensive Assessment of Biologicals Contained Within Commercial Airliner Cabin Air

Both culture-based and culture-independent, biomarker-targeted microbial enumeration and identification technologies were employed to estimate total microbial and viral burden and diversity within the cabin air of commercial airliners. Samples from each of twenty flights spanning three commercial carriers were collected via air-impingement. When the total viable microbial population was estimated by assaying relative concentrations of the universal energy carrier ATP, values ranged from below detection limits (BDL) to 4.1 x 106 cells/cubic m of air. The total viable microbial population was extremely low in both of Airline A (approximately 10% samples) and C (approximately 18% samples) compared to the samples collected aboard flights on Airline A and B (approximately 70% samples). When samples were collected as a function of time over the course of flights, a gradual accumulation of microbes was observed from the time of passenger boarding through mid-flight, followed by a sharp decline in microbial abundance and viability from the initiation of descent through landing. It is concluded in this study that only 10% of the viable microbes of the cabin air were cultivable and suggested a need to employ state-of-the art molecular assay that measures both cultivable and viable-but-non-cultivable microbes. Among the cultivable bacteria, colonies of Acinetobacter sp. were by far the most profuse in Phase I, and Gram-positive bacteria of the genera Staphylococcus and Bacillus were the most abundant during Phase II. The isolation of the human pathogens Acinetobacter johnsonii, A. calcoaceticus, Janibacter melonis, Microbacterium trichotecenolyticum, Massilia timonae, Staphylococcus saprophyticus, Corynebacterium lipophiloflavum is concerning, as these bacteria can cause meningitis, septicemia, and a handful of sometimes fatal diseases and infections. Molecular microbial community analyses exhibited presence of the alpha-, beta-, gamma-, and delta- proteobacteria, as well as Gram-positive bacteria, Fusobacteria, Cyanobacteria, Deinococci, Bacterioidetes, Spirochetes, and Planctomyces in varying abundance. Neisseria meningitidis rDNA sequences were retrieved in great abundance from Airline A followed by Streptococcus oralis/mitis sequences. Pseudomonas synxantha sequences dominated Airline B clone libraries, followed by those of N. meningitidis and S. oralis/mitis. In Phase II, Airline C, sequences representative of more than 113 species, enveloping 12 classes of bacteria, were retrieved. Proteobacterial sequences were retrieved in greatest frequency (58% of all clone sequences), followed in short order by those stemming from Gram-positives bacteria (31% of all clone sequences). As for overall phylogenetic breadth, Gram-positive and alpha-proteobacteria seem to have a higher affinity for international flights, whereas beta-and gamma-proteobacteria are far more common about domestic cabin air parcels in Airline C samples. Ultimately, the majority of microbial species circulating throughout the cabin airs of commercial airliners are commensal, infrequently pathogenic normal flora of the human nasopharynx and respiratory system. Many of these microbes likely originate from the oral and nasal cavities, and lungs of passengers and flight crew and are disseminated unknowingly via routine conversation, coughing, sneezing, and stochastic passing of fomites. The data documented in this study will be useful to generate a baseline microbial population database and can be utilized to develop biosensor instrumentation for monitoring microbial quality of cabin or urban air.

microbial diversity↗