Artificial Blood Development Implications for Military Medicine.

Massive hemorrhaging remains the most common cause of preventable battlefield deaths. Blood used for trauma care requires a robust donation network, capacity for long-term storage, and extensive and accurate testing. Bioengineering technologies could offer a remedy to these constraints in the form of blood substitutes-fluids that could be transfused into patients to provide oxygen, carry away waste, and aid in coagulation-that would be used in prolonged casualty care and in far-forward settings, overcoming the obstacles of distance and time. The different molecular properties of red blood cells (RBCs), blood substitutes, and platelet replacements contribute to their respective utilities, and each type is currently represented in ongoing clinical trials. Hemoglobin oxygen carriers (HBOCs) are the most advanced RBC replacements, many of which are currently being evaluated in clinical trials in the United States and other countries. Despite recent advancements, challenges remaining in the development of blood alternatives include stability, oxygen capacity, and compatibility. The continued research and investment in new technologies has the potential to significantly benefit the treatment of life-threatening emergency injuries, both on the battlefield and in the civilian sector. In this review, we discuss military blood-management practices and military-specific uses of individual blood components, as well as describe and analyze several artificial blood products that could be options for future battlefield use.

The Epistemic Fallacy: Unintended Consequences of Empirically Treating (Clinically Diagnosed) Chronic Lyme Disease in a Soldier.

OBJECTIVE: We document a military patient presenting with a diffuse set of symptoms suggestive of chronic Lyme disease (CLD) and the subsequent empiric treatment and health complications arising therein. The lay medical community, spurred by the internet, has ascribed these diffuse symptoms to various illnesses including CLD without confirmatory serological evidence of any underlying disease. With a growing community of patient advocates, CLD has become an illness with broad and highly generalized list of clinical symptoms and an absence of agreed-upon confirmatory laboratory tests. Further complicating matters, diagnostic criteria and treatment protocols differ between the Infectious Diseases Society of America and the International Lyme and Associated Diseases Society guidelines. Clinicians also face serious challenges in diagnosing and treating patients who present with generalized symptoms and close to 50 diagnostic tests for Lyme disease available in North America. Further complicating the picture for military patients seeking medical confirmation of a disease and resolution of their symptoms, medical fitness boards use putative diagnoses as prima faciae evidence in disability. Here a military patient with a long list of complaints that defy any clear or easy diagnosis and treatment is discussed. However, these symptoms taken together with selectively summed notes in the medical record in the absence of convincing and clear laboratory confirmation are suggestive of CLD and its complications, but no resolution was ultimately reached. With the presumptive determination of a medical disability due to CLD by the medical board, the medical dismissal of this service member from active duty occurred.

Prevalence of Lyme Disease Attributable to Military Service at the USMA, West Point NY: FY2016-2018.

INTRODUCTION: Lyme disease incidence rates have steadily increased since its official recognition in 1975. Since exposure to Lyme is associated with activities conducted in and around tick-habitats including tall grass, shrubs, deciduous forest, and leaf litter, it has been suggested that service members, who are thought to spend higher amounts of time in these habitats due to training requirements, may have higher risk for exposure. Specifically, this study looks at service member and family member exposure to Ixodes scapularis, the vector for Lyme in the Northeastern and Midwestern United States. While literature pertaining to occupational and military specific exposures to Lyme vectors have attempted to quantify the possible elevated risk of Lyme disease for service members, thus far, studies have not consistently confirmed that service members are at a greater risk than family members. MATERIALS AND METHODS: This cross-sectional study looks at cases of Lyme disease at Keller Army Community Hospital (KACH) on the West Point Military Reservation in New York during Fiscal Year (FY) 2016 through FY2018. Lyme cases were pulled from Military Health System Mart using current ICD-10-CM codes for Lyme related conditions (A69.20 and A69.29). In total, 144 cases were considered for the analysis. Totaling all service members and family members enrolled at KACH over the three-year period account for 35,526 person-years. Period prevalence, attributable risk percentage, population attributable risk percentage, and corresponding 95% confidence intervals were calculated for service members and family member categories. This study was conducted on human subject research according to 32CFR219 and meets the requirements of exempt status under 32CFR219.101(b)(4) because it is a cross-sectional study on existing de-identified patient data. RESULTS: During FY2016-2018, service members accounted for 63 cases of Lyme totaling 21,595 person-years with a period prevalence of 292 cases per 100,000 (219.8, 363.7). Family members accounted for 81 cases with a total of 13,931 person-years with a period prevalence of 581 cases per 100,000 (455.2, 707.7). The percentage of attributable risk during the three-year period credited to military status is -99.30% (-145.69%, -52.91%). The population attributable risk percentage is -43.4%. CONCLUSIONS: While this study was unable to capture the military specific occupational exposure to I. scapularis, it does show a difference in period prevalence between service members and family members with the family members being at higher risk to contract Lyme instead of service members as is commonly suggested in the literature. Additional studies may be conducted to see if this holds true across service member Military Occupational Specialties as a proxy for occupational exposure. Similar studies should be conducted at military installations situated in Lyme endemic areas to determine if these results are comparable across the military or specific to West Point. Future research should attempt to identify all the service member protective factors against Lyme with attribution to permethrin-treated uniforms and other military interventions designed to defend soldiers against vector-borne diseases.

Modeling Lyme disease host animal habitat suitability, West Point, New York.

As the most frequently reported vector-borne disease among active component U.S. service members, with an incidence rate of 16 cases per 100,000 person-years in 2011, Lyme disease poses both a challenge to healthcare providers in the Military Health System and a threat to military readiness. Spread through the bite of an infected blacklegged tick, infection with the bacterial cause of Lyme disease can have lasting effects that may lead to medical discharge from the military. The U.S. Military Academy at West Point is situated in a highly endemic area in New York State. To identify probable areas where West Point cadets as well as active duty service members stationed at West Point and their families might contract Lyme disease, this study used Geographic Information System mapping methods and remote sensing data to replicate an established spatial model to identify the likely habitat of a key host animal--the white-tailed deer.

Improving vector-borne pathogen surveillance: A laboratory-based study exploring the potential to detect dengue virus and malaria parasites in mosquito saliva.

BACKGROUND & OBJECTIVES: Vector-borne pathogen surveillance programmes typically rely on the collection of large numbers of potential vectors followed by screening protocols focused on detecting pathogens in the arthropods. These processes are laborious, time consuming, expensive, and require screening of large numbers of samples. To streamline the surveillance process, increase sample throughput, and improve cost-effectiveness, a method to detect dengue virus and malaria parasites (Plasmodium falciparum) by leveraging the sugar-feeding behaviour of mosquitoes and their habit of expectorating infectious agents in their saliva during feeding was investigated in this study. METHODS: Dengue virus 2 (DENV-2) infected female Aedes aegypti mosquitoes and P. falciparum infected female Anopheles stephensi mosquitoes were allowed to feed on honey coated Flinders Technical Associates -FTA® cards dyed with blue food colouring. The feeding resulted in deposition of saliva containing either DENV-2 particles or P. falciparum sporozoites onto the FTA card. Nucleic acid was extracted from each card and the appropriate real-time PCR (qPCR) assay was run to detect the pathogen of interest. RESULTS: As little as one plaque forming unit (PFU) of DENV-2 and as few as 60 P. falciparum parasites deposited on FTA cards from infected mosquitoes were detected via qPCR. Hence, their use to collect mosquito saliva for pathogen detection is a relevant technique for vector surveillance. INTERPRETATION & CONCLUSION: This study provides laboratory confirmation that FTA cards can be used to capture and stabilize expectorated DENV-2 particles and P. falciparum sporozoites from infectious, sugar-feeding mosquitoes in very low numbers. Thus, the FTA card-based mosquito saliva capture method offers promise to overcome current limitations and revolutionize traditional mosquito-based pathogen surveillance programmes. Field testing and further method development are required to optimize this strategy.

Development and validation of an arthropod maceration protocol for zoonotic pathogen detection in mosquitoes and fleas.

Arthropod-borne diseases remain a pressing international public health concern. While progress has been made in the rapid detection of arthropod-borne pathogens via quantitative real-time (qPCR), or even hand-held detection devices, a simple and robust maceration and nucleic acid extraction method is necessary to implement biosurveillance capabilities. In this study, a comparison of maceration techniques using five types of beads followed by nucleic acid extraction and detection were tested using two morphologically disparate arthropods, the Aedes aegypti mosquito and Xenopsylla spp. flea, to detect the zoonotic diseases dengue virus serotype-1 and Yersinia pestis. Post-maceration nucleic acid extraction was carried out using the 1-2-3 Platinum-Path-Sample-Purification (PPSP) kit followed by qPCR detection using the Joint Biological Agent Identification and Diagnostic System (JBAIDS). We found that the 5mm stainless steel beads added to the beads provided in the PPSP kit were successful in macerating the exoskeleton for both Ae. aegypti and Xenopsylla spp. Replicates in the maceration/extraction/detection protocol were increased in a stepwise fashion until a final 128 replicates were obtained. For dengue virus detection there was a 99% positivity rate and for Y. pestis detection there was a 95% positive detection rate. In the examination of both pathogens, there were no significant differences between qPCR instruments, days ran, time of day ran, or operators.

Ebola virus infection induces irregular dendritic cell gene expression.

Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

Leveraging arthropod-borne disease surveillance assays for clinical diagnostic use.

Researchers at the Walter Reed Army Institute of Research have taken a joint service approach to filling an identified diagnostic capability gap by leveraging a vector surveillance assay. Specifically, the Army took a field-stable real-time polymerase chain reaction assay, developed by the Air Force, for dengue virus surveillance in arthropod vectors and collaborated with Navy researchers for utility in human diagnostics. As current Department of Defense diagnostic PCR assays employ the Joint Biological Agent Identification and Diagnostic System, the dengue assay was tested for use on this platform. The low rates of false negative and false positive dengue samples in clinical matrices demonstrate excellent utility as a human diagnostic assay. Overall, converting an arboviral vector surveillance assay to human diagnostic assay and potentially vice versa is both cost effective and labor reducing. Codevelopment with harmonization of vector surveillance and diagnostics offers monetary and resource advantages to the Department of Defense and should be considered as a path forward in times when downsizing threatens assay development and pathogen discovery.

Real-time Leishmania genus master mix: a platform compatibility and stability study.

Performing diagnostics and vector-pathogen surveillance in austere environments is challenging. On-site diagnostic/detection mitigates vector-borne disease complications during military or humanitarian deployments to disease endemic locals. The mobile molecular diagnostic platform, Joint Biological Agent Identification and Diagnostic System (JBAIDS; BioFire Diagnostics Inc., Salt Lake City, UT, USA), rapidly identifies biothreat pathogens. Although ideal for remote diagnostics, the platform was validated for specific pathogens of insignificant epidemiological consequence. Recognizing the JBAIDS's remote diagnostic/detection versatility, we tested a Leishmania genus real-time PCR master mix validated for use on the SmartCycler® (Cepheid, Sunnyvale, CA, USA) for concomitant use on the JBAIDS. We evaluated assay sensitivity, precision, and specificity of one or more Leishmania spp. on the JBAIDS and found that the JBAIDS produces superior detection sensitivity and specificity compared to the SmartCycler®. We also examined the storage stability of a bulk lot preparation of the Leishmania genus real-time PCR master mix on the SmartCycler® to ensure that long periods of frozen storage that would translate to a field environment with the JBAIDS were not detrimental to the reagent. We found that the bulk master mix maintains its stability over a 13-month time period. Overall, these studies confirm JBAIDS's versatility and demonstrate a streamlined assay development approach where reagents are compatible with both platforms.

Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes.

Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens.

Plasmodium-specific molecular assays produce uninterpretable results and non-Plasmodium spp. sequences in field-collected Anopheles vectors.

The Malaria Research and Reference Reagent Resource-recommended PLF/UNR/VIR polymerase chain reaction (PCR) was used to detect Plasmodium vivax in Anopheles spp. mosquitoes collected in South Korea. Samples that were amplified were sequenced and compared with known Plasmodium spp. by using the PlasmoDB.org Basic Local Alignment Search Tool/n and the National Center for Biotechnology Information Basic Local Alignment Search Tool/n tools. Results show that the primers PLF/UNR/VIR used in this PCR can produce uninterpretable results and non-specific sequences in field-collected mosquitoes. Three additional PCRs (PLU/VIV, specific for 18S small subunit ribosomal DNA; Pvr47, specific for a nuclear repeat; and GDCW/PLAS, specific for the mitochondrial marker, cytB) were then used to find a more accurate and interpretable assay. Samples that were amplified were again sequenced. The PLU/VIV and Pvr47 assays showed cross-reactivity with non-Plasmodium spp. and an arthropod fungus (Zoophthora lanceolata). The GDCW/PLAS assay amplified only Plasmodium spp. but also amplified the non-human specific parasite P. berghei from an Anopheles belenrae mosquito. Detection of P. berghei in South Korea is a new finding.

Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

Antibody to the E3 glycoprotein protects mice against lethal venezuelan equine encephalitis virus infection.

Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.

Glycoprotein interactions in paramyxovirus fusion.

The Paramyxoviridae are enveloped, negative-stranded RNA viruses, some of which recognize sialic acid-containing receptors, while others recognize specific proteinaceous receptors. The major cytopathic effect of paramyxovirus infection is membrane fusion-induced syncytium formation. Paramyxoviruses are unusual in that the receptor-binding and fusion-promoting activities reside on two different spike structures, the attachment and fusion glycoproteins, respectively. For most paramyxoviruses, this distribution of functions requires a mechanism by which the two processes can be linked for the promotion of fusion. This is accomplished by a virus-specific interaction between the two proteins. An increasing body of evidence supports the notion that members of this family of viruses utilize this glycoprotein interaction in different ways in order to mediate the regulation of the fusion protein activation, depending on the type of receptor utilized by the virus.

Addition of N-glycans in the stalk of the Newcastle disease virus HN protein blocks its interaction with the F protein and prevents fusion.

Most paramyxovirus fusion (F) proteins require the coexpression of the homologous attachment (HN) protein to promote membrane fusion, consistent with the existence of a virus-specific interaction between the two proteins. Analysis of the fusion activities of chimeric HN proteins indicates that the stalk region of the HN spike determines its F protein specificity, and analysis of a panel of site-directed mutants indicates that the F-interactive site resides in this region. Here, we use the addition of oligosaccharides to further explore the role of the HN stalk in the interaction with F. N-glycans were individually added at several positions in the stalk to determine their effects on the activities of HN, as well as its structure. N-glycan addition at positions 69 and 77 in the stalk specifically blocks fusion and the HN-F interaction without affecting either HN structure or its other activities. N-glycans added at other positions in the stalk modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein, as indicated by sucrose gradient sedimentation analyses. Finally, N-glycan addition in another region of HN (residues 124 to 152), predicted by a peptide-based analysis to mediate the interaction with F, does not significantly reduce the level of fusion, arguing strongly against this site being part of the F-interactive domain in HN. Our data support the idea that the F-interactive site on HN is defined by the stalk region of the protein.

Decreased dependence on receptor recognition for the fusion promotion activity of L289A-mutated newcastle disease virus fusion protein correlates with a monoclonal antibody-detected conformational change.

It has been shown that the L289A-mutated Newcastle disease virus (NDV) fusion (F) protein gains the ability to promote fusion of Cos-7 cells independent of the viral hemagglutinin-neuraminidase (HN) protein and exhibits a 50% enhancement in HN-dependent fusion over wild-type (wt) F protein. Here, we show that HN-independent fusion by L289A-F is not exhibited in BHK cells or in several other cell lines. However, similar to the results in Cos-7 cells, the mutated protein plus HN does promote 50 to 70% more fusion above wt levels in all of the cell lines tested. L289A-F protein exhibits the same specificity as the wt F protein for the homologous HN protein, as well as NDV-human parainfluenza virus 3 HN chimeras. The mutated F protein promotes fusion more effectively than the wt when it is coexpressed with either the chimeras or HN proteins deficient in receptor recognition activity. In addition, its fusogenic activity is significantly more resistant to removal of sialic acid on target cells. These findings are consistent with the demonstration that L289A-F interacts more efficiently with wt and mutated HN proteins than does wt F by a cell surface coimmunoprecipitation assay. Taken together, these findings indicate that L289A-F promotes fusion by a mechanism analogous to that of the wt protein with respect to the HN-F interaction but is less dependent on the attachment activity of HN. The phenotype of the mutated F protein correlates with a conformational change in the protein detectable by two different monoclonal antibodies. This conformational change may reflect a destabilization of F structure induced by the L289A substitution, which may in turn indicate a lower energy requirement for fusion activation.

Amino acid substitutions in the F-specific domain in the stalk of the newcastle disease virus HN protein modulate fusion and interfere with its interaction with the F protein.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus mediates attachment to sialic acid receptors, as well as cleavage of the same moiety. HN also interacts with the other viral glycoprotein, the fusion (F) protein, to promote membrane fusion. The ectodomain of the HN spike consists of a stalk and a terminal globular head. The most conserved part of the stalk consists of two heptad repeats separated by a nonhelical intervening region (residues 89 to 95). Several amino acid substitutions for a completely conserved proline residue in this region not only impair fusion and the HN-F interaction but also decrease neuraminidase activity in the globular domain, suggesting that the substitutions may alter HN structure. Substitutions for L94 also interfere with fusion and the HN-F interaction but have no significant effect on any other HN function. Amino acid substitutions at other positions in the intervening region also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the intervening region is critical to the role of HN in the promotion of fusion and may be directly involved in its interaction with the homologous F protein.