Rapid establishment of a frontline field laboratory in response to an imported outbreak of Ebola virus disease in western Uganda, June 2019.

The Democratic Republic of the Congo (DRC) declared an Ebola virus disease (EVD) outbreak in North Kivu in August 2018. By June 2019, the outbreak had spread to 26 health zones in northeastern DRC, causing >2,000 reported cases and >1,000 deaths. On June 10, 2019, three members of a Congolese family with EVD-like symptoms traveled to western Uganda's Kasese District to seek medical care. Shortly thereafter, the Viral Hemorrhagic Fever Surveillance and Laboratory Program (VHF program) at the Uganda Virus Research Institute (UVRI) confirmed that all three patients had EVD. The Ugandan Ministry of Health declared an outbreak of EVD in Uganda's Kasese District, notified the World Health Organization, and initiated a rapid response to contain the outbreak. As part of this response, UVRI and the United States Centers for Disease Control and Prevention, with the support of Uganda's Public Health Emergency Operations Center, the Kasese District Health Team, the Superintendent of Bwera General Hospital, the United States Department of Defense's Makerere University Walter Reed Project, and the United States Mission to Kampala's Global Health Security Technical Working Group, jointly established an Ebola Field Laboratory in Kasese District at Bwera General Hospital, proximal to an Ebola Treatment Unit (ETU). The laboratory consisted of a rapid containment kit for viral inactivation of patient specimens and a GeneXpert Instrument for performing Xpert Ebola assays. Laboratory staff tested 76 specimens from alert and suspect cases of EVD; the majority were admitted to the ETU (89.3%) and reported recent travel to the DRC (58.9%). Although no EVD cases were detected by the field laboratory, it played an important role in patient management and epidemiological surveillance by providing diagnostic results in <3 hours. The integration of the field laboratory into Uganda's National VHF Program also enabled patient specimens to be referred to Entebbe for confirmatory EBOV testing and testing for other hemorrhagic fever viruses that circulate in Uganda.

Isolation of Angola-like Marburg virus from Egyptian rousette bats from West Africa.

Marburg virus (MARV) causes sporadic outbreaks of severe Marburg virus disease (MVD). Most MVD outbreaks originated in East Africa and field studies in East Africa, South Africa, Zambia, and Gabon identified the Egyptian rousette bat (ERB; Rousettus aegyptiacus) as a natural reservoir. However, the largest recorded MVD outbreak with the highest case-fatality ratio happened in 2005 in Angola, where direct spillover from bats was not  shown. Here, collaborative studies by the Centers for Disease Control and Prevention, Njala University, University of California, Davis USAID-PREDICT, and the University of Makeni identify MARV circulating in ERBs in Sierra Leone. PCR, antibody and virus isolation data from 1755 bats of 42 species shows active MARV infection in approximately 2.5% of ERBs. Phylogenetic analysis identifies MARVs that are similar to the Angola strain. These results provide evidence of MARV circulation in West Africa and demonstrate the value of pathogen surveillance to identify previously undetected threats.

Laboratory Response to Ebola - West Africa and United States.

The 2014-2016 Ebola virus disease (Ebola) epidemic in West Africa highlighted the need to maintain organized laboratory systems or networks that can be effectively reorganized to implement new diagnostic strategies and laboratory services in response to large-scale events. Although previous Ebola outbreaks enabled establishment of critical laboratory practice safeguards and diagnostic procedures, this Ebola outbreak in West Africa highlighted the need for planning and preparedness activities that are better adapted to emerging pathogens or to pathogens that have attracted little commercial interest. The crisis underscored the need for better mechanisms to streamline development and evaluation of new diagnostic assays, transfer of material and specimens between countries and organizations, and improved processes for rapidly deploying health workers with specific laboratory expertise. The challenges and events of the outbreak forced laboratorians to examine not only the comprehensive capacities of existing national laboratory systems to recognize and respond to events, but also their sustainability over time and the mechanisms that need to be pre-established to ensure effective response. Critical to this assessment was the recognition of how response activities (i.e., infrastructure support, logistics, and workforce supplementation) can be used or repurposed to support the strengthening of national laboratory systems during the postevent transition to capacity building and recovery. This report compares CDC's domestic and international laboratory response engagements and lessons learned that can improve future responses in support of the International Health Regulations and Global Health Security Agenda initiatives.The activities summarized in this report would not have been possible without collaboration with many U.S. and international partners (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/partners.html).

Integrated Disease Investigations and Surveillance planning: a systems approach to strengthening national surveillance and detection of events of public health importance in support of the International Health Regulations.

The international community continues to define common strategic themes of actions to improve global partnership and international collaborations in order to protect our populations. The International Health Regulations (IHR[2005]) offer one of these strategic themes whereby World Health Organization (WHO) Member States and global partners engaged in biosecurity, biosurveillance and public health can define commonalities and leverage their respective missions and resources to optimize interventions. The U.S. Defense Threat Reduction Agency's Cooperative Biological Engagement Program (CBEP) works with partner countries across clinical, veterinary, epidemiological, and laboratory communities to enhance national disease surveillance, detection, diagnostic, and reporting capabilities. CBEP, like many other capacity building programs, has wrestled with ways to improve partner country buy-in and ownership and to develop sustainable solutions that impact integrated disease surveillance outcomes. Designing successful implementation strategies represents a complex and challenging exercise and requires robust and transparent collaboration at the country level. To address this challenge, the Laboratory Systems Development Branch of the U.S. Centers for Disease Control and Prevention (CDC) and CBEP have partnered to create a set of tools that brings together key leadership of the surveillance system into a deliberate system design process. This process takes into account strengths and limitations of the existing system, how the components inter-connect and relate to one another, and how they can be systematically refined within the local context. The planning tools encourage cross-disciplinary thinking, critical evaluation and analysis of existing capabilities, and discussions across organizational and departmental lines toward a shared course of action and purpose. The underlying concepts and methodology of these tools are presented here.

Genetic diversity of coastal bottlenose dolphins revealed by structurally and functionally diverse hemoglobins.

Studies of structure-function relationships in the respiratory proteins of marine mammals revealed unexpected variations in the number and types of hemoglobins (Hbs) present in coastal bottlenose dolphins, Tursiops truncatus. We obtained blood samples from free-ranging coastal bottlenose dolphins as a component of capture-release studies. We found that the oxygen-binding functions of bottlenose dolphin blood are poised between effector-saturated and unsaturated levels, enabling exercise-dependent shifts in oxygen transfer functions. Isolated bottlenose dolphin Hbs showed elevated pH sensitivities (Bohr effects) and appreciably lower oxygen affinities than adult human Hb in the absence of allosteric effectors. These properties may be an adaptive modification that enhances oxygen delivery during diving episodes when oxygen tensions and effector levels are low. The Hbs of individual dolphins showed similar oxygen affinities, responses to effectors, and expression of heme-heme interaction in oxygen binding, but differed in their redox potentials and rates of autoxidation. The heterogeneity suggested by these functional variations in Hbs of individual dolphins was born out by variations in the molecular weights and numbers of their alpha and beta globin chains. Although coastal bottlenose dolphins were expected to have a single type of Hb, the mass differences observed revealed considerable genetic diversity. There were multiple Hb forms in some individuals and differences in Hb patterns among individuals within the same community.

Redox properties of human transferrin bound to its receptor.

Virtually all organisms require iron, and iron-dependent cells of vertebrates (and some more ancient species) depend on the Fe(3+)-binding protein of the circulation, transferrin, to meet their needs. In its iron-donating cycle, transferrin is first captured by the transferrin receptor on the cell membrane, and then internalized to a proton-pumping endosome where iron is released. Iron exits the endosome to enter the cytoplasm via the ferrous iron transporter DMT1, a molecule that accepts only Fe(2+), but the reduction potential of ferric iron in free transferrin at endosomal pH (approximately 5.6) is below -500 mV, too low for reduction by physiological agents such as the reduced pyridine nucleotides with reduction potentials of -284 mV. We now show that in its complex with the transferrin receptor, which persists throughout the transferrin-to-cell cycle of iron uptake, the potential is raised by more than 200 mV. Reductive release of iron from transferrin, which binds Fe(2+) very weakly, is therefore physiologically feasible, a further indication that the transferrin receptor is more than a passive conveyor of transferrin and its iron.

The influence of the synergistic anion on iron chelation by ferric binding protein, a bacterial transferrin.

Although the presence of an exogenous anion is a requirement for tight Fe(3+) binding by the bacterial (Neisseria) transferrin nFbp, the identity of the exogenous anion is not specific in vitro. nFbp was reconstituted as a stable iron containing protein by using a number of different exogenous anions [arsenate, citrate, nitrilotriacetate, pyrophosphate, and oxalate (symbolized by X)] in addition to phosphate, predominantly present in the recombinant form of the protein. Spectroscopic characterization of the Fe(3+)anion interaction in the reconstituted protein was accomplished by UV-visible and EPR spectroscopies. The affinity of the protein for Fe(3+) is anion dependent, as evidenced by the effective Fe(3+) binding constants (K'(eff)) observed, which range from 1 x 10(17) M(-1) to 4 x 10(18) M(-1) at pH 6.5 and 20 degrees C. The redox potentials for Fe(3+)nFbpXFe(2+)nFbpX reduction are also found to depend on the identity of the synergistic anion required for Fe(3+) sequestration. Facile exchange of exogenous anions (Fe(3+)nFbpX + X' --> Fe(3+)nFbpX' + X) is established and provides a pathway for environmental modulation of the iron chelation and redox characteristics of nFbp. The affinity of the iron loaded protein for exogenous anion binding at pH 6.5 was found to decrease in the order phosphate > arsenate approximately pyrophosphate > nitrilotriacetate > citrate approximately oxalate carbonate. Anion influence on the iron primary coordination sphere through iron binding and redox potential modulation may have in vivo application as a mechanism for periplasmic control of iron delivery to the cytosol.

Heme redox properties of S-nitrosated hemoglobin A0 and hemoglobin S: implications for interactions of nitric oxide with normal and sickle red blood cells.

S-Nitrosated hemoglobin is remarkably stable and can be cycled between deoxy, oxygenated, or oxidized forms without significant loss of NO. Here we show that S-nitrosation of adult human hemoglobin (Hb A(0)) or sickle cell Hb (Hb S) results in an increased ease of anaerobic heme oxidation, while anions cause redox shifts in the opposite direction. The negatively charged groups of the cytoplasmic domain of Band 3 protein also produce an allosteric effect on S-nitrosated Hb. Formation and deoxygenation of a SNO-Hb/Band 3 protein assembly does not in itself cause NO release, even in the presence of glutathione; however, this assembly may play a role in the migration of NO from the red blood cells to other targets and may be linked to Heinz body formation. Studies of the anaerobic oxidation of Hb S revealed an altered redox potential relative to Hb A(0) that favors met-Hb formation and may therefore underlie the increased rate of autoxidation of Hb S under aerobic conditions, the increased formation of Heinz bodies in sickle cells, and the decreased lifetime of red cells containing Hb S. A model for the interrelationships between the deoxy, oxy, and met forms of Hb A(0) and Hb S, and their S-nitrosated counterparts, is presented.