Strategies to Improve Multi-enzyme Compatibility and Coordination in One-Pot SHERLOCK.

While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60-69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.

Omicron Spike Protein is Vulnerable to Reduction.

SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.

Validation and Establishment of the SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a Prescreening Tool for the Plaque Reduction Neutralization Test.

Neutralization assays are important for understanding and quantifying neutralizing antibody responses toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and prescreening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity assays produced acceptable results, with 100% (95% confidence interval [CI], 94% to 100%) specificity and 100% (95% CI, 94% to 100%) sensitivity against ancestral Wuhan spike-pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike-pseudotyped lentivirus were 88.3% (95% CI, 77.8% to 94.2%) and 100% (95% CI, 94% to 100%), respectively. Assay precision measuring intra-assay variability produced acceptable results for high (50% PRNT [PRNT50], 1:≥640), mid (PRNT50, 1:160), and low (PRNT50, 1:40) antibody titer concentration ranges based on the PRNT50, with coefficients of variation (CVs) of 14.21%, 12.47%, and 13.28%, respectively. Intermediate precision indicated acceptable ranges for the high and mid concentrations, with CVs of 15.52% and 16.09%, respectively. However, the low concentration did not meet the acceptance criteria, with a CV of 26.42%. Acceptable ranges were found in the robustness evaluation for both intra-assay and interassay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT. IMPORTANCE Neutralization studies play an important role in providing guidance and justification for vaccine administration and helping prevent the spread of diseases. The neutralization data generated in our laboratory have been included in the decision-making process of the National Advisory Committee on Immunization (NACI) in Canada. During the coronavirus 2019 (COVID-19) pandemic, the plaque reduction neutralization test (PRNT) has been the gold standard for determining neutralization of SARS-CoV-2. We validated a SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) as an alternative method to help support the PRNT. The advantages of using the SCLSNA is that it can process more samples, is less tedious to perform, and can be used in laboratories with a lower biosafety level. The use of the SCLSNA can further expand our capabilities to help fulfill the requirements for NACI and other important collaborations.

Salivary testing for SARS-CoV-2 in the pediatric population: a diagnostic accuracy study.

BACKGROUND: Accurate and timely testing for SARS-CoV-2 in the pediatric population is crucial to control the COVID-19 pandemic; saliva testing has been proposed as a less invasive alternative to nasopharyngeal swabs. We sought to compare the detection of SARS-CoV-2 using saliva versus nasopharyngeal swab in the pediatric population, and to determine the optimum time of testing for SARS-CoV-2 using saliva. METHODS: We conducted a longitudinal diagnostic study in Ottawa, Canada, from Jan. 19 to Mar. 26, 2021. Children aged 3-17 years were eligible if they exhibited symptoms of COVID-19, had been identified as a high-risk or close contact to someone confirmed positive for SARS-CoV-2 or had travelled outside Canada in the previous 14 days. Participants provided both nasopharyngeal swab and saliva samples. Saliva was collected using a self-collection kit (DNA Genotek, OM-505) or a sponge-based kit (DNA Genotek, ORE-100) if they could not provide a saliva sample into a tube. RESULTS: Among 1580 paired nasopharyngeal and saliva tests, 60 paired samples were positive for SARS-CoV-2. Forty-four (73.3%) were concordant-positive results and 16 (26.6%) were discordant, among which 8 were positive only on nasopharyngeal swab and 8 were positive only on saliva testing. The sensitivity of saliva was 84.6% (95% confidence interval 71.9%-93.1%). INTERPRETATION: Salivary testing for SARS-CoV-2 in the pediatric population is less invasive and shows similar detection of SARS-CoV-2 to nasopharyngeal swabs. It may therefore provide a feasible alternative for diagnosis of SARS-CoV-2 infection in children.

Pandemic 1918 Influenza Virus Does Not Cause Lethal Infection in Rhesus or Cynomolgus Macaques.

The 1918 H1N1 influenza pandemic was among the most severe in history, taking the lives of approximately 50 million people worldwide, and novel prophylactic vaccines are urgently needed to prevent another pandemic. Given that macaques are physiologically relevant preclinical models of human immunology that have advanced the clinical treatment of infectious diseases, a lethal pandemic influenza challenge model would provide a stringent platform for testing new influenza vaccine concepts. To this end, we infected rhesus macaques and Mauritian cynomolgus macaques with highly pathogenic 1918 H1N1 influenza virus and assessed pathogenesis and disease severity. Despite infection with a high dose of 1918 influenza delivered via multiple routes, rhesus macaques demonstrated minimal signs of disease, with only intermittent viral shedding. Cynomolgus macaques infected via intrabronchial instillation demonstrated mild symptoms, with disease severity depending on the infection dose. Cynomolgus macaques infected with a high dose of 1918 influenza delivered via multiple routes experienced moderate disease characterized by consistent viral shedding, pulmonary infiltrates, and elevated inflammatory cytokine levels. However, 1918 influenza was uniformly nonlethal in these two species, demonstrating that this isolate is insufficiently pathogenic in rhesus and Mauritian cynomolgus macaques to support testing novel prophylactic influenza approaches where protection from severe disease combined with a lethal outcome is desired as a highly stringent indication of vaccine efficacy. IMPORTANCE The world remains at risk of an influenza pandemic, and the development of new therapeutic and preventative modalities is critically important for minimizing human death and suffering during the next influenza pandemic. Animal models are central to the development of new therapies and vaccine approaches. In particular, nonhuman primates like rhesus and cynomolgus macaques are highly relevant preclinical models given their physiological and immunological similarities to humans. Unfortunately, there remains a scarcity of macaque models of pandemic influenza with which to test novel antiviral modalities. Here, we demonstrate that even at the highest doses tested, 1918 influenza was not lethal in these two macaque species, suggesting that they are not ideal for the development and testing of novel pandemic influenza-specific vaccines and therapies. Therefore, other physiologically relevant nonhuman primate models of pandemic influenza are needed.

Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus.

CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/µl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens.

Role of Key Infectivity Parameters in the Transmission of Ebola Virus Makona in Macaques.

Many characteristics associated with Ebola virus disease remain to be fully understood. It is known that direct contact with infected bodily fluids is an associated risk factor, but few studies have investigated parameters associated with transmission between individuals, such as the dose of virus required to facilitate spread and route of infection. Therefore, we sought to characterize the impact by route of infection, viremia, and viral shedding through various mucosae, with regards to intraspecies transmission of Ebola virus in a nonhuman primate model. Here, challenge via the esophagus or aerosol to the face did not result in clinical disease, although seroconversion of both challenged and contact animals was observed in the latter. Subsequent intramuscular or intratracheal challenges suggest that viral loads determine transmission likelihood to naive animals in an intramuscular-challenge model, which is greatly facilitated in an intratracheal-challenge model where transmission from challenged to direct contact animal was observed consistently.

Host parameters and mode of infection influence outcome in SARS-CoV-2-infected hamsters.

The golden hamster model of SARS-CoV-2 infection recapitulates key characteristics of COVID-19. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in hamsters. We report that delivery of SARS-CoV-2 by a low- versus high-volume intranasal or intragastric route results in comparable viral titers in the lung and viral shedding. However, low-volume intranasal exposure results in milder weight loss, whereas intragastric exposure leads to a diminished capacity to regain body weight. Male hamsters, and particularly older male hamsters, display an impaired capacity to recover from illness and delayed viral clearance. These factors were found to influence the nature of the host inflammatory cytokine response but had a minimal effect on the quality and durability of the humoral immune response and susceptibility to re-infection. These data further elucidate key factors that impact pre-clinical challenge studies carried out in the hamster model of COVID-19.

SARS-CoV-2 infection and transmission in the North American deer mouse.

Widespread circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive nondomesticated animals. Here we report that North American deer mice (Peromyscus maniculatus) are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naïve deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 remains unknown.

Characterization of Ebola Virus Risk to Bedside Providers in an Intensive Care Environment.

BACKGROUND: The 2014-2016 Ebola outbreak in West Africa recapitulated that nosocomial spread of Ebola virus could occur and that health care workers were at particular risk including notable cases in Europe and North America. These instances highlighted the need for centers to better prepare for potential Ebola virus cases; including understanding how the virus spreads and which interventions pose the greatest risk. METHODS: We created a fully equipped intensive care unit (ICU), within a Biosafety Level 4 (BSL4) laboratory, and infected multiple sedated non-human primates (NHPs) with Ebola virus. While providing bedside care, we sampled blood, urine, and gastric residuals; as well as buccal, ocular, nasal, rectal, and skin swabs, to assess the risks associated with routine care. We also assessed the physical environment at end-point. RESULTS: Although viral RNA was detectable in blood as early as three days post-infection, it was not detectable in the urine, gastric fluid, or swabs until late-stage disease. While droplet spread and fomite contamination were present on a few of the surfaces that were routinely touched while providing care in the ICU for the infected animal, these may have been abrogated through good routine hygiene practices. CONCLUSIONS: Overall this study has helped further our understanding of which procedures may pose the highest risk to healthcare providers and provides temporal evidence of this over the clinical course of disease.

Comparison analysis of different swabs and transport mediums suitable for SARS-CoV-2 testing following shortages.

On March 11, 2020, the World Health Organization (WHO) assessed COVID-19, caused by SARS-CoV-2, as a pandemic. As of June 1, 2020, SARS-CoV-2 has had a documented effect of over 6 million cases world-wide, amounting to over 370,000 deaths (World Health Organization, 2020. Novel Coronavirus (COVID-19) Situation. http://https://covid19.who.int/). Consequently, the high demand for testing has resulted in a depletion of commercially available consumables, including the recommended swabs and viral transport media (VTM) required for nasopharyngeal sampling. Therefore, the potential use of unvalidated alternatives must be explored to address the global shortage of testing supplies. To tackle this issue, we evaluated the utility of different swabs and transport mediums for the molecular detection of SARS-CoV-2. This study compared the performance of six swabs commonly found in primary and tertiary health care settings (PurFlock Ultra, FLOQSwab, Puritan Pur-Wraps cotton tipped applicators, Puritan polyester tipped applicators, MedPro 6" cotton tipped applicators, and HOLOGIC Aptima) for their efficacy in testing for SARS-CoV-2. Separately, the molecular detection of SARS-CoV-2 was completed from different transport mediums (DMEM, PBS, 100 % ethanol, 0.9 % normal saline and VTM), which were kept up to three days at room temperature (RT). The results indicate that there is no meaningful difference in viral yield from different swabs and most transport mediums for the collection and detection of SARS-CoV-2, indicating swab and medium alternatives could be used if supplies run out.

Predicting Infectious Severe Acute Respiratory Syndrome Coronavirus 2 From Diagnostic Samples.

BACKGROUND: Reverse-transcription polymerase chain reaction (RT-PCR) has become the primary method to diagnose viral diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RT-PCR detects RNA, not infectious virus; thus, its ability to determine duration of infectivity of patients is limited. Infectivity is a critical determinant in informing public health guidelines/interventions. Our goal was to determine the relationship between E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples, symptom onset to test (STT), and infectivity in cell culture. METHODS: In this retrospective cross-sectional study, we took SARS-CoV-2 RT-PCR-confirmed positive samples and determined their ability to infect Vero cell lines. RESULTS: Ninety RT-PCR SARS-CoV-2-positive samples were incubated on Vero cells. Twenty-six samples (28.9%) demonstrated viral growth. Median tissue culture infectious dose/mL was 1780 (interquartile range, 282-8511). There was no growth in samples with a Ct > 24 or STT > 8 days. Multivariate logistic regression using positive viral culture as a binary predictor variable, STT, and Ct demonstrated an odds ratio (OR) for positive viral culture of 0.64 (95% confidence interval [CI], .49-.84; P < .001) for every 1-unit increase in Ct. Area under the receiver operating characteristic curve for Ct vs positive culture was OR, 0.91 (95% CI, .85-.97; P < .001), with 97% specificity obtained at a Ct of > 24. CONCLUSIONS: SARS-CoV-2 Vero cell infectivity was only observed for RT-PCR Ct < 24 and STT < 8 days. Infectivity of patients with Ct > 24 and duration of symptoms > 8 days may be low. This information can inform public health policy and guide clinical, infection control, and occupational health decisions. Further studies of larger size are needed.

Correction to: Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

Please note that four authors (Logan Banadyga, Alixandra Albietz, Brad Pickering, and Gary Wong) have been erroneously omitted from the author list in the published original article [1].

Impact of intensive care unit supportive care on the physiology of Ebola virus disease in a universally lethal non-human primate model.

BACKGROUND: There are currently limited data for the use of specific antiviral therapies for the treatment of Ebola virus disease (EVD). While there is anecdotal evidence that supportive care may be effective, there is a paucity of direct experimental data to demonstrate a role for supportive care in EVD. We studied the impact of ICU-level supportive care interventions including fluid resuscitation, vasoactive medications, blood transfusion, hydrocortisone, and ventilator support on the pathophysiology of EVD in rhesus macaques infected with a universally lethal dose of Ebola virus strain Makona C07. METHODS: Four NHPs were infected with a universally lethal dose Ebola virus strain Makona, in accordance with the gold standard lethal Ebola NHP challenge model. Following infection, the following therapeutic interventions were employed: continuous bedside supportive care, ventilator support, judicious fluid resuscitation, vasoactive medications, blood transfusion, and hydrocortisone as needed to treat cardiovascular compromise. A range of physiological parameters were continuously monitored to gage any response to the interventions. RESULTS: All four NHPs developed EVD and demonstrated a similar clinical course. All animals reached a terminal endpoint, which occurred at an average time of 166.5 ± 14.8 h post-infection. Fluid administration may have temporarily blunted a rise in lactate, but the effect was short lived. Vasoactive medications resulted in short-lived improvements in mean arterial pressure. Blood transfusion and hydrocortisone did not appear to have a significant positive impact on the course of the disease. CONCLUSIONS: The model employed for this study is reflective of an intramuscular infection in humans (e.g., needle stick) and is highly lethal to NHPs. Using this model, we found that the animals developed progressive severe organ dysfunction and profound shock preceding death. While the overall impact of supportive care on the observed pathophysiology was limited, we did observe some time-dependent positive responses. Since this model is highly lethal, it does not reflect the full spectrum of human EVD. Our findings support the need for continued development of animal models that replicate the spectrum of human disease as well as ongoing development of anti-Ebola therapies to complement supportive care.

Protective Efficacy and Long-Term Immunogenicity in Cynomolgus Macaques by Ebola Virus Glycoprotein Synthetic DNA Vaccines.

Background: There remains an important need for prophylactic anti-Ebola virus vaccine candidates that elicit long-lasting immune responses and can be delivered to vulnerable populations that are unable to receive live-attenuated or viral vector vaccines. Methods: We designed novel synthetic anti-Ebola virus glycoprotein (EBOV-GP) DNA vaccines as a strategy to expand protective breadth against diverse EBOV strains and evaluated the impact of vaccine dosing and route of administration on protection against lethal EBOV-Makona challenge in cynomolgus macaques. Long-term immunogenicity was monitored in nonhuman primates for >1 year, followed by a 12-month boost. Results: Multiple-injection regimens of the EBOV-GP DNA vaccine, delivered by intramuscular administration followed by electroporation, were 100% protective against lethal EBOV-Makona challenge. Impressively, 2 injections of a simple, more tolerable, and dose-sparing intradermal administration followed by electroporation generated strong immunogenicity and was 100% protective against lethal challenge. In parallel, we observed that EBOV-GP DNA vaccination induced long-term immune responses in macaques that were detectable for at least 1 year after final vaccination and generated a strong recall response after the final boost. Conclusions: These data support that this simple intradermal-administered, serology-independent approach is likely important for additional study towards the goal of induction of anti-EBOV immunity in multiple at-risk populations.

DNA vaccination protects mice against Zika virus-induced damage to the testes.

Zika virus (ZIKV) is an emerging pathogen causally associated with serious sequelae in fetuses, inducing fetal microcephaly and other neurodevelopment defects. ZIKV is primarily transmitted by mosquitoes, but can persist in human semen and sperm, and sexual transmission has been documented. Moreover, exposure of type-I interferon knockout mice to ZIKV results in severe damage to the testes, epididymis and sperm. Candidate ZIKV vaccines have shown protective efficacy in preclinical studies carried out in animal models, and several vaccines have entered clinical trials. Here, we report that administration of a synthetic DNA vaccine encoding ZIKV pre-membrane and envelope (prME) completely protects mice against ZIKV-associated damage to the testes and sperm and prevents viral persistence in the testes following challenge with a contemporary strain of ZIKV. These data suggest that DNA vaccination merits further investigation as a potential means to reduce ZIKV persistence in the male reproductive tract.

Delivering Prolonged Intensive Care to a Non-human Primate: A High Fidelity Animal Model of Critical Illness.

Critical care needs have been rising in recent decades as populations age and comorbidities increase. Sepsis-related admissions to critical care contribute up to 50% of volume and septic shock carries a 35-54% fatality rate. Improvements in sepsis-related care and mortality would have a significant impact of a resource-intensive area of health care delivery. Unfortunately, research has been hampered by the lack of an animal model that replicates the complex care provided to humans in an intensive care unit (ICU). We developed a protocol to provide full ICU type supportive care to Rhesus macaques. This included mechanical ventilation, continuous sedation, fluid and electrolyte management and vasopressor support in response to Ebolavirus-induced septic shock. The animals accurately recapitulated human responses to a full range of ICU interventions (e.g. fluid resuscitation). This model can overcome current animal model limitations by accurately emulating the complexity of ICU care and thereby provide a platform for testing new interventions in critical care and sepsis without placing patients at risk.

Pathogenicity Comparison Between the Kikwit and Makona Ebola Virus Variants in Rhesus Macaques.

Enhanced virulence and/or transmission of West African Ebola virus (EBOV) variants, which are divergent from their Central African counterparts, are suspected to have contributed to the sizable toll of the recent Ebola virus disease (EVD) outbreak. This study evaluated the pathogenicity and shedding in rhesus macaques infected with 1 of 2 West African isolates (EBOV-C05 or EBOV-C07) or a Central African isolate (EBOV-K). All animals infected with EBOV-C05 or EBOV-C07 died of EVD, whereas 2 of 3 EBOV-K-infected animals died. The viremia level was elevated 10-fold in EBOV-C05-infected animals, compared with EBOV-C07- or EBOV-K-infected animals. More-severe lung pathology was observed in 2 of 6 EBOV-C05/C07-infected macaques. This is the first detailed analysis of the recently circulating EBOV-C05/C07 in direct comparison to EBOV-K with 6 animals per group, and it showed that EBOV-C05 but not EBOV-C07 can replicate at higher levels and cause more tissue damage in some animals. Increased virus shedding from individuals who are especially susceptible to EBOV replication is possibly one of the many challenges facing the community of healthcare and policy-making responders since the beginning of the outbreak.

Diagnosis and management of Ebola samples in the laboratory.

The magnitude of the 2014-2016 West African Ebola virus outbreak has highlighted the importance of immediate and rapid deployment of control measures in affected areas. While many prophylactic and therapeutic options entered clinical trials in the past two years, larger use to impact on Ebola spread will not be possible until at least one product meets final approval by regulatory agencies. Control of the West African outbreak was achieved almost entirely by breaking chain of transmissions through case identification and specialized treatment, communication, safe burials and other proven methods. To achieve this in a timely manner, epidemiologists and medical teams are working in concert with laboratories to identify infected individuals and provide care within Ebola treatment units. Herein, we review an outbreak response workflow from the point of view of mobile laboratories and summarize methods that have been used by them during the West African Ebola virus outbreak of 2014-2016.

Two-mAb cocktail protects macaques against the Makona variant of Ebola virus.

The 2014-2015 Ebola virus (EBOV) outbreak in West Africa highlighted the urgent need for specific therapeutic interventions for infected patients. The human-mouse chimeric monoclonal antibody (mAb) cocktail ZMapp, previously shown to be efficacious in EBOV (variant Kikwit) lethally infected nonhuman primates (NHPs) when administration was initiated up to 5 days, was used in some patients during the outbreak. We show that a two-antibody cocktail, MIL77E, is fully protective in NHPs when administered at 50 mg/kg 3 days after challenge with a lethal dose of EBOV variant Makona, the virus responsible for the ongoing 2014-2015 outbreak, whereas a similar formulation of ZMapp protected two of three NHPs. The chimeric MIL77E mAb cocktail is produced in engineered Chinese hamster ovary cells and is based on mAbs c13C6 and c2G4 from ZMapp. The use of only two antibodies in MIL77E opens the door to a pan-ebolavirus cocktail.