The deployment of mobile diagnostic laboratories for Ebola virus disease diagnostics in Sierra Leone and Guinea.

BACKGROUND: Ebola virus emerged in West Africa in December 2013. The ease of mobility, porous borders, and lack of public health infrastructure led to the largest Ebola virus disease (EVD) outbreak to date. INTERVENTION: The 2013 EVD outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. As part of the United States' Department of Defense response, MRIGlobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (MDLs). The first laboratory analysed blood samples from patients in an adjacent Ebola Treatment Centre (ETC) and buccal swabs from the deceased in the community in Moyamba, Sierra Leone. The second laboratory was deployed to support an ETC in Conakry, Guinea. The Department of Defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site. LESSONS LEARNT: Prompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. Experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for MDL setup and operation. As the Ebola response slowed, the sustainment of the MDLs' operations was prioritised, including staff training and the transition of the MDLs to local governments. Training programmes for local staff were prepared in Sierra Leone and Guinea. RECOMMENDATIONS: The MRIGlobal MDL team significantly contributed to establishing increased laboratory capacity during the EVD outbreak in West Africa. Using the MDLs for molecular diagnosis is highly recommended until more sustainable solutions can be provided.

Sustainable Laboratory Capacity Building After the 2014 Ebola Outbreak in the Republic of Guinea.

Background: The 2014-2016 West Africa Ebola virus disease outbreak heavily impacted the Republics of Guinea, Sierra Leone, and Liberia. The outbreak uncovered the weaknesses of the public health systems, including inadequately trained and insufficient health personnel as well as limited and poorly equipped health infrastructures. These weaknesses represent significant threats to global health security. In the wake of the outbreak, affected countries made urgent requests for international engagement to help strengthening the public health systems. Methods: This work describes the successful multi-year implementation of a laboratory capacity building program in the Republic of Guinea. The program integrated biorisk and quality management systems training, infectious diseases diagnostic training, facility engineering and maintenance training, and mentorship to strengthen Guinea's bio-surveillance capacity. Results: The major outcome of these efforts was an established and local staff-operated public health laboratory that performs disease surveillance and reporting and diagnostic of priority diseases and pathogens of security concerns. Conclusions: This work has improved the Guinea country's capabilities to address country public health issues and preparedness to respond to future infectious disease threats.

Antiviral activity of the EB peptide against zoonotic poxviruses.

BACKGROUND: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox. FINDINGS: In yield reduction assays, EB had an EC50 of 26.7 µM against cowpox and 4.4 µM against monkeypox. The EC50 for plaque reduction was 26.3 µM against cowpox and 48.6 µM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice. CONCLUSIONS: While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.

Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone.

Mitoxantrone, an FDA-approved therapeutic for the treatment of cancer and multiple sclerosis, was previously reported to exhibit antiviral activity against vaccinia virus. To determine whether this activity extends to other orthopoxviruses, mitoxantrone was tested against cowpox and monkeypox. Mitoxantrone demonstrated an EC(50) of 0.25 µM against cowpox and 0.8 µM against monkeypox. Intraperitoneal treatment of cowpox virus-challenged C57Bl/6 mice with 0.5 mg/kg mitoxantrone resulted in 25% survival and a significant increase in survival time. In an effort to improve its efficacy, mitoxantrone was tested for synergistic activity with cidofovir. In vitro tests demonstrated significant synergy between the two drugs against cowpox; however, no synergistic effect on animal survival or median time-to-death was seen in intranasally-infected BALB/c mice. Significantly fewer animals survived when treated with a combination of 0.5 mg/kg mitoxantrone and 100 mg/kg cidofovir than with 100 mg/kg cidofovir alone. This is, to our knowledge, the first report of limited anti-orthopoxvirus activity by mitoxantrone in an animal model.

Evaluation of therapeutic interventions for vaccinia virus keratitis.

BACKGROUND: Vaccinia virus keratitis (VACVK) is a complication of smallpox vaccination that can result in blindness. There are no Food and Drug Administration-approved treatments for VACVK, and vaccinia immunoglobulin (VIG) is contraindicated in isolated VACVK. We used a rabbit model of infection to compare several therapeutic options for VACVK. METHODS: Rabbit eyes were infected with 10(5) plaque-forming units of the Dryvax strain of vaccinia virus and scored daily for 28 days using a modified MacDonald-Shadduck scoring system. Animals were treated for 10 days after the onset of keratitis with albumin, VIG, prednisolone acetate, trifluridine, or combinations thereof. Ocular viral titers and vaccinia-specific antibody titers were determined by plaque assay and enzyme-linked immunosorbent assay, respectively. RESULTS: Treatment with intravenous VIG neither exacerbated nor ameliorated VACVK. Topical prednisolone acetate interfered with viral clearance, and ocular disease rebounded in prednisolone-treated groups. The most effective treatment was topical trifluridine alone. CONCLUSIONS: We conclude that (1) VIG did not negatively affect the treatment of isolated keratitis, (2) topical corticosteroids should not be used for treating VACVK, and (3) treatment with topical trifluridine, with or without intravenous VIG, is the preferred therapeutic regimen for treating VACVK.

Kinetics of immune cell infiltration in vaccinia virus keratitis.

PURPOSE: Vaccinia virus keratitis leading to blindness is a severe complication of smallpox vaccination. The clinical manifestations of vaccinia virus keratitis are similar to those of herpes simplex virus keratitis, a well-studied immunopathologic disease. Vaccinia virus keratitis is likely to involve an immunopathologic component, but little is known about the pathogenesis of the disease. The goal of this study was to determine type and kinetics of immune cell infiltration in the cornea during vaccinia virus keratitis. METHODS: Rabbit eyes were trephined and inoculated with 1x10(5) pfu of the Dryvax strain of the vaccinia virus. On days 2, 4, 7, 10, 14, and 28 after infection, the animals were scored for clinical disease and eye sections were stained for B cells, CD4+ cells, CD8+ cells, and neutrophils. The eyelid, ciliary body, cornea, iris, iridocorneal angle, and choroid were examined. RESULTS: Corneal vaccinia virus challenge resulted in the infiltration of B cells, CD4+ cells, CD8+ cells, and neutrophils into the cornea and eyelids. Neutrophils were the predominant cell type on days 2 and 3 after infection, whereas CD4+ cells were the predominant cell type detected in corneas on days 4 through 10. CD8+ cells and B cells peaked on day 10, but at lower levels than CD4+ cells and neutrophils. CONCLUSIONS: These results suggest that sequential migration of neutrophils, then CD4+ cells, plays an important role in vaccinia virus keratitis.

A quantitative rabbit model of vaccinia keratitis.

PURPOSE: The goal of this study was to use multiple quantitative disease measures to evaluate the effect of various viral inocula on the development of vaccinia keratitis in rabbits. METHODS: Trephined eyes of female rabbits were infected with 10(4), 10(5), 10(6), or 10(7) plaque-forming units (pfu) of the Dryvax strain of the vaccinia virus and scored daily for disease for 14 days according to a modification of the MacDonald-Shadduck scoring system. Ocular viral titers and vaccinia-specific antibody titers were determined by plaque assay and ELISA, respectively. RESULTS: The amount of virus used for infection affected the severity of disease, with 10(4) pfu eliciting milder keratitis after delayed onset compared with higher amounts of virus. At inocula above 10(5) pfu the course and severity of corneal disease was not significantly different. The time to reach peak titers was delayed in the 10(4) group but peak titers were similar in all groups. Severe conjunctival chemosis interfered with scoring in animals infected with 10(6) or 10(7) pfu. Virus-specific antibody titers were similar in all groups at day 14. Body weights decreased less than 10% in all groups. CONCLUSIONS: The course of vaccinia keratitis in rabbits paralleled that in humans. A viral inoculum of 10(5) pfu/eye was determined to be optimal for use in further studies of vaccinia keratitis.

Evaluation of a theta-defensin in a Murine model of herpes simplex virus type 1 keratitis.

PURPOSE: To test the activity of a synthetic theta-defensin, retrocyclin (RC)-2, in a murine herpes simplex virus (HSV)-1 keratitis model. METHODS: The in vitro antiviral activity of RC-2 against HSV-1 KOS was determined by yield reduction and viral inactivation assays. Efficacy in an experimental murine HSV-1 keratitis model was tested using pre- or postinfection treatment with 0.1% peptide in PBS with or without 2% methylcellulose. Viral titers in the tear film were determined by plaque assay. RESULTS: RC-2 inhibited HSV-1 KOS in vitro with an EC(50) of 10 microM (~20 microg/mL) in yield-reduction assays, but was not directly virucidal. RC-106 (a less active analogue) did not inhibit HSV-1 KOS in culture. Incubating the virus with RC-2 or applying the peptide in 2% methylcellulose to the cornea before viral infection significantly reduced the severity of ocular disease, but postinfection treatment with 0.1% RC-2 in PBS with or without 2% methylcellulose did not. Viral titers were significantly reduced on some days after infection in the preincubation and prophylaxis groups. CONCLUSIONS: RC-2 was active against HSV-1 KOS in cultures and showed protective activity in vivo when used in a prophylactic mode, but the peptide showed limited activity in a postinfection herpes keratitis model. These findings support data obtained from experiments with HIV-1, HSV-2, and influenza A, indicating that RCs inhibit the entry of viruses rather than their replication.

A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumor cell hybrid vaccines.

Fusion of tumor cells with antigen-presenting cells (APCs) has been proposed for the preparation of cancer vaccines. However, generation of these hybrids, using physical or chemical methods such as electrofusion or polyethylene glycol (PEG), has been difficult to standardize. Characterization of cell fusion has also been problematic because of difficulties in differentiating fusion from cell aggregation, leakage of cellular dyes and dendritic-cell (DC) phagocytosis of tumor material. In this report, we describe a new method to generate hybrid cell vaccines, based on gene transfer of a viral fusogenic membrane glycoprotein (FMG) into tumor cells, and incorporate a genetic method by which true hybrid formation can be unambiguously detected. We describe a new class of tumor cell-DC hybrid that can be rapidly isolated after cell fusion. These hybrids are highly potent in in vitro antigen presentation assays, target lymph nodes in vivo and are powerful immunogens against established metastatic disease.