The East African Community (EAC) mobile laboratory networks in Kenya, Burundi, Tanzania, Rwanda, Uganda, and South Sudan-from project implementation to outbreak response against Dengue, Ebola, COVID-19, and epidemic-prone diseases.

BACKGROUND: East Africa is home to 170 million people and prone to frequent outbreaks of viral haemorrhagic fevers and various bacterial diseases. A major challenge is that epidemics mostly happen in remote areas, where infrastructure for Biosecurity Level (BSL) 3/4 laboratory capacity is not available. As samples have to be transported from the outbreak area to the National Public Health Laboratories (NPHL) in the capitals or even flown to international reference centres, diagnosis is significantly delayed and epidemics emerge. MAIN TEXT: The East African Community (EAC), an intergovernmental body of Burundi, Rwanda, Tanzania, Kenya, Uganda, and South Sudan, received 10 million € funding from the German Development Bank (KfW) to establish BSL3/4 capacity in the region. Between 2017 and 2020, the EAC in collaboration with the Bernhard-Nocht-Institute for Tropical Medicine (Germany) and the Partner Countries' Ministries of Health and their respective NPHLs, established a regional network of nine mobile BSL3/4 laboratories. These rapidly deployable laboratories allowed the region to reduce sample turn-around-time (from days to an average of 8h) at the centre of the outbreak and rapidly respond to epidemics. In the present article, the approach for implementing such a regional project is outlined and five major aspects (including recommendations) are described: (i) the overall project coordination activities through the EAC Secretariat and the Partner States, (ii) procurement of equipment, (iii) the established laboratory setup and diagnostic panels, (iv) regional training activities and capacity building of various stakeholders and (v) completed and ongoing field missions. The latter includes an EAC/WHO field simulation exercise that was conducted on the border between Tanzania and Kenya in June 2019, the support in molecular diagnosis during the Tanzanian Dengue outbreak in 2019, the participation in the Ugandan National Ebola response activities in Kisoro district along the Uganda/DRC border in Oct/Nov 2019 and the deployments of the laboratories to assist in SARS-CoV-2 diagnostics throughout the region since early 2020. CONCLUSIONS: The established EAC mobile laboratory network allows accurate and timely diagnosis of BSL3/4 pathogens in all East African countries, important for individual patient management and to effectively contain the spread of epidemic-prone diseases.

Commentary: mobile laboratories for SARS-CoV-2 diagnostics: what Europe could learn from the East African Community to assure trade in times of border closures.

BACKGROUND: The emergence of SARS-CoV-2 mutants might lead to European border closures, which impact on trade and result in serious economic losses. In April 2020, similar border closures were observed during the first SARS-CoV-2 wave in East Africa. MAIN BODY: Since 2017 the East African Community EAC together with the Bernhard-Nocht-Institute for Tropical Medicine BNITM established a mobile laboratory network integrated into the National Public Health Laboratories of the six Partner States for molecular diagnosis of viral haemorrhagic fevers and SARS-CoV-2. Since May 2020, the National Public Health Laboratories of Kenya, Rwanda, Burundi, Uganda and South Sudan deployed these mobile laboratories to their respective borders, issuing a newly developed "Electronic EAC COVID-19 Digital Certificate" to SARS-CoV-2 PCR-negative truck drivers, thus assuring regional trade. CONCLUSION: Considering the large financial damages of border closures, such a mobile laboratory network as demonstrated in East Africa is cost-effective, easy to implement and feasible. The East African Community mobile laboratory network could serve as a blueprint for Europe and other countries around the globe.

Elucidation of cellular targets and exploitation of the receptor-binding domain of SARS-CoV-2 for vaccine and monoclonal antibody synthesis.

The pandemic caused by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has resulted in over 452 822 deaths in the first 20 days of June 2020 due to the coronavirus virus disease 2019 (COVID-19). The SARS-CoV-2 uses the host angiotensin-converting enzyme 2 (ACE2) receptor to gain entry inside the human cells where it replicates by using the cell protein synthesis mechanisms. The knowledge of the tissue distribution of ACE2 in human organs is therefore important to predict the clinical course of the COVID-19. Also important is the understanding of the viral receptor-binding domain (RBD), a region within the spike (S) proteins, that enables the entry of the virus into the host cells to synthesize vaccine and monoclonal antibodies (mAbs). We performed an exhaustive search of human protein databases to establish the tissues that express ACE2 and performed an in-depth analysis like sequence alignments and homology modeling of the spike protein (S) of the SARS-CoV-2 to identify antigenic regions in the RBD that can be exploited to synthesize vaccine and mAbs. Our results show that ACE2 is widely expressed in human organs that may explain the pulmonary, systemic, and neurological deficits seen in COVID-19 patients. We show that though the S protein of the SARS-CoV-2 is a homolog of S protein of SARS-CoV-1, it has regions of dissimilarities in the RBD and transmembrane segments. We show peptide sequences in the RBD of SARS-CoV-2 that can bind to the major histocompatibility complex alleles and serve as effective epitopes for vaccine and mAbs synthesis.

Persistence of Lassa Virus Associated With Severe Systemic Arteritis in Convalescing Guinea Pigs (Cavia porcellus).

Lassa fever (LF) survivors develop various clinical manifestations including polyserositis, myalgia, epididymitis, and hearing loss weeks to months after recovery from acute infection. We demonstrate a systemic lymphoplasmacytic and histiocytic arteritis and periarteritis in guinea pigs more than 2 months after recovery from acute Lassa virus (LASV) infection. LASV was detected in the arterial tunica media smooth muscle cells by immunohistochemistry, in situ hybridization, and transmission electron microscopy. Our results suggest that the sequelae of LASV infection may be due to virus persistence resulting in systemic vascular damage. These findings shed light on the pathogenesis of LASV sequelae in convalescent human survivors.

Biosafety Level 4 Laboratory User Training Program, China.

Experienced, qualified personnel certified to work in high-level biocontainment laboratories contribute to the safe operation of these facilities. China began a training program for laboratory users after establishing its first Biosafety Level 4 laboratory, the Wuhan National Biosafety Laboratory (Level 4) of the Chinese Academy of Sciences. We provide an overview of the content of this training program, which can serve as a reference for developing national norms for high-containment laboratory training.

Detection of Ebola Virus RNA Through Aerosol Sampling of Animal Biosafety Level 4 Rooms Housing Challenged Nonhuman Primates.

Ebola virus disease is a serious illness of humans and nonhuman primates (NHPs). Direct contact has been shown to be the primary source of Ebola (EBOV) transmission. We used a high-volume air sampler to determine whether EBOV could be detected during 3 independent studies with EBOV-challenged NHPs. Viral RNA was recovered during days 9 and 10 of Study I and days 7 and 8 of Study III. Viral RNA levels were below limits of detection during all other collections. The results demonstrate that the biosafety level 4 (BSL-4) suit protects workers from aerosols in a BSL-4 environment using proper engineering and administrative controls.

Comparison of respiratory inductive plethysmography versus head-out plethysmography for anesthetized nonhuman primates in an animal biosafety level 4 facility.

For inhalational studies and aerosol exposures to viruses, head-out plethysmography acquisition has been traditionally used for the determination of estimated inhaled dose in anesthetized nonhuman primates prior to or during an aerosol exposure. A pressure drop across a pneumotachograph is measured within a sealed chamber during inspiration/exhalation of the nonhuman primate, generating respiratory values and breathing frequencies. Due to the fluctuation of depth of anesthesia, pre-exposure respiratory values can be variable, leading to less precise and accurate dosing calculations downstream. Although an anesthesia infusion pump may help stabilize the depth of sedation, pumps are difficult to use within a sealed head-out plethysmography chamber. Real-time, head-out plethysmography acquisition could increase precision and accuracy of the measurements, but the bulky equipment needed for head-out plethysmography precludes real-time use inside a Class III biological safety cabinet, where most aerosol exposures occur. However, the respiratory inductive plethysmography (RIP) acquisition method measures the same respiratory parameters by detecting movement of the chest and abdomen during breathing using two elastic bands within the Class III biological safety cabinet. As respiratory values are relayed to a computer for software integration and analysis real-time, adjustment of aerosol exposure duration is based on the depth of sedation of the animal. The objective of this study was to compare values obtained using two methodologies (pre-exposure head-out plethysmography and real-time RIP). Transitioning to RIP technology with real-time acquisition provides more consistent, precise, and accurate aerosol dosing by reducing reported errors in respiratory values from anesthesia variability when using pre-exposure head-out plethysmography acquisition.

Generation and characterization of large-particle aerosols using a center flow tangential aerosol generator with a non-human-primate, head-only aerosol chamber.

Aerosol droplets or particles produced from infected respiratory secretions have the potential to infect another host through inhalation. These respiratory particles can be polydisperse and range from 0.05 to 500 µm in diameter. Animal models of infection are generally established to facilitate the potential licensure of candidate prophylactics and/or therapeutics. Consequently, aerosol-based animal infection models are needed to properly study and counter airborne infections. Ideally, experimental aerosol exposure should reliably result in animal disease that faithfully reproduces the modeled human disease. Few studies have been performed to explore the relationship between exposure particle size and induced disease course for infectious aerosol particles. The center flow tangential aerosol generator (CenTAG™) produces large-particle aerosols capable of safely delivering a variety of infectious aerosols to non-human primates (NHPs) within a Class III Biological Safety Cabinet (BSC) for establishment or refinement of NHP infectious disease models. Here, we report the adaptation of this technology to the Animal Biosafety Level 4 (ABSL-4) environment for the future study of high-consequence viral pathogens and the characterization of CenTAG™-created sham (no animal, no virus) aerosols using a variety of viral growth media and media supplements.

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Development of a Novel Positive Pressure Protective Suit for a Biosafety Level 4 Laboratory in Japan.

Biosafety level 4 (BSL-4) laboratories are necessary to study microorganisms that are highly pathogenic to humans and have no prevention or therapeutic measures. Currently, most BSL-4 facilities have suit-type laboratories to conduct experiments on highly pathogenic microorganisms. In 2021, the first Japanese suit-type BSL-4 laboratory was constructed at Nagasaki University. Positive pressure protection suit (PPPS) is a primary barrier that protects and isolates laboratory workers from pathogens and the laboratory environment. Here, we developed a novel PPPS originally designed to be used in the Nagasaki BSL-4 laboratory. We modified several parts of a domestic chemical protective suit, including its front face shield, cuff, and air supply hose, for safe handling of microbiological agents. The improved suit, PS-790BSL4-AL, showed resistance to several chemicals, including quaternary ammonium disinfectant, and did not show any permeation against blood and phages. To validate the suit's integrity, we also established an airtight test that eliminated individual differences for quantitative testing. In conclusion, our developed suit performs sufficiently as a primary barrier and allows for the safe handling of pathogens in our new BSL-4 laboratory.

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses.

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories.

Mouse Models of Henipavirus Infection.

The Nipah and Hendra viruses, belonging to henipavirus genus, are recently emerged zoonotic pathogens that cause severe and often fatal, neurologic, and/or respiratory diseases in both humans and various animals. As mice represent a small animal model convenient to study viral infections and provide a well-developed experimental toolbox for analysis in immunovirology, we describe in this chapter a few basic methods used in biosafety 4 level (BSL4) conditions to study henipavirus infection in mice.

Transcriptome profiling highlights regulated biological processes and type III interferon antiviral responses upon Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a biosafety level-4 (BSL-4) pathogen that causes Crimean-Congo hemorrhagic fever (CCHF) characterized by hemorrhagic manifestation, multiple organ failure and high mortality rate, posing great threat to public health. Despite the recently increasing research efforts on CCHFV, host cell responses associated with CCHFV infection remain to be further characterized. Here, to better understand the cellular response to CCHFV infection, we performed a transcriptomic analysis in human kidney HEK293 â€‹cells by high-throughput RNA sequencing (RNA-seq) technology. In total, 496 differentially expressed genes (DEGs), including 361 up-regulated and 135 down-regulated genes, were identified in CCHFV-infected cells. These regulated genes were mainly involved in host processes including defense response to virus, response to stress, regulation of viral process, immune response, metabolism, stimulus, apoptosis and protein catabolic process. Therein, a significant up-regulation of type III interferon (IFN) signaling pathway as well as endoplasmic reticulum (ER) stress response was especially remarkable. Subsequently, representative DEGs from these processes were well validated by RT-qPCR, confirming the RNA-seq results and the typical regulation of IFN responses and ER stress by CCHFV. Furthermore, we demonstrate that not only type I but also type III IFNs (even at low dosages) have substantial anti-CCHFV activities. Collectively, the data may provide new and comprehensive insights into the virus-host interactions and particularly highlights the potential role of type III IFNs in restricting CCHFV, which may help inform further mechanistic delineation of the viral infection and development of anti-CCHFV strategies.

Dangerous Risk Group-4 (RG-4) emergent viruses.

World Health Organization (WHO) Risk Group-4 (RG-4) pathogens are among the most dangerous of the emergent and re-emergent viruses. International health agencies, working in concert, bridge the gaps in health care for populations at risk for RG-4 viral pathogen exposure. RG-4 virus research incorporates Biodefense Program and Biosafety Laboratory (BSL)-4 technologies. RG-4 viruses include Arena-viridae, Filo-viridae, Flavi-viridae, Herpes-viridae, Nairo-viridae, Paramyxo-viridae, and Pox-viridae.

Emergent Risk Group-4 (RG-4) Filoviruses: A paradox in progress.

Filoviruses, categorized as World Health Organization (WHO) Risk Group 4 (RG-4) pathogens, represent significant global health risks due to their extraordinary virulence. The Filoviridae family encompasses Ebola strains such as Sudan, Zaire, Bundibugyo, Tai Forest (formerly known as Ivory Coast), Reston, and Bombali, in addition to the closely related Marburg and Ravn virus strains. Filoviruses originated from a common ancestor about 10,000 years ago and displayed remarkable consistency in genetic heterogeneity until the 20th century. However, they overcame a genetic bottleneck by mid-century. Paradoxically, this resulted in the emergence of boosted virulent strains from the 1970's onward. Filovirus research is included in the NIAID Biodefense Program and utilizes the highest level specialized protective laboratories, Biosafety Laboratory (BSL)-4. The spread of Filoviruses as well as other RG-4 pathogens within Africa poses a significant health threat increasingly both in Africa and out of Africa.

Nipah and Hendra Viruses: Deadly Zoonotic Paramyxoviruses with the Potential to Cause the Next Pandemic.

Nipah and Hendra viruses are deadly zoonotic paramyxoviruses with a case fatality rate of upto 75%. The viruses belong to the genus henipavirus in the family Paramyxoviridae, a family of negative-sense single-stranded RNA viruses. The natural reservoirs of NiV and HeV are bats (flying foxes) in which the virus infection is asymptomatic. The intermediate hosts for NiV and HeV are swine and equine, respectively. In humans, NiV infections result in severe and often fatal respiratory and neurological manifestations. The Nipah virus was first identified in Malaysia and Singapore following an outbreak of encephalitis in pig farmers and subsequent outbreaks have been reported in Bangladesh and India almost every year. Due to its extreme pathogenicity, pandemic potential, and lack of established antiviral therapeutics and vaccines, research on henipaviruses is highly warranted so as to develop antivirals or vaccines that could aid in the prevention and control of future outbreaks.

Inactivation Methods for Experimental Nipah Virus Infection.

Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe disease in humans and livestock. Due to its high pathogenicity in humans and the lack of available vaccines and therapeutics, NiV needs to be handled in biosafety level 4 (BSL-4) laboratories. Safe inactivation of samples containing NiV is thus necessary to allow further processing in lower containment areas. To date, there is only limited information available on NiV inactivation methods validated by BSL-4 facilities that can be used as a reference. Here, we compare some of the most common inactivation methods in order to evaluate their efficacy at inactivating NiV in infected cells, supernatants and organs. Thus, several physical and chemical inactivation methods, and combinations thereof, were assessed. Viral replication was monitored for 3 weeks and NiV presence was assessed by RT-qPCR, plaque assay and indirect immunofluorescence. A total of nineteen methods were shown to reduce NiV infectious particles in cells, supernatants and organs to undetectable levels. Therefore, we provide a list of methods for the safe and efficient inactivation of NiV.

Maintaining differential pressure gradients does not increase safety inside modern BSL-4 laboratories.

This article discusses a previously unrecognized contradiction in the design of biosafety level-4 (BSL-4) suit laboratories, also known as maximum or high containment laboratories. For decades, it is suggested that both directional airflow and pressure differentials are essential safety measures to prevent the release of pathogens into the environment and to avoid cross-contamination between laboratory rooms. Despite the absence of an existing evidence-based risk analyses demonstrating increased safety by directional airflow and pressure differentials in BSL-4 laboratories, they were anchored in various national regulations. Currently, the construction and operation of BSL-4 laboratories are subject to rigorous quality and technical requirements including airtight containment. Over time, BSL-4 laboratories evolved to enormously complex technical infrastructures. With the aim to counterbalance this development towards technical simplification while still maintaining maximum safety, we provide a detailed risk analysis by calculating pathogen mitigation in maximum contamination scenarios. The results presented and discussed herein, indicate that both directional airflow or a differential pressure gradient in airtight rooms within a secondary BSL-4 containment do not increase biosafety, and are not necessary. Likewise, reduction of pressure zones from the outside into the secondary containment may also provide sufficient environmental protection. We encourage laboratory design professionals to consider technical simplification and policymakers to adapt corresponding legislation and regulations surrounding directional airflow and pressure differentials for technically airtight BSL-4 laboratories.

Livestock and Risk Group 4 Pathogens: Researching Zoonotic Threats to Public Health and Agriculture in Maximum Containment.

Maximum-containment laboratories are a unique and essential component of the bioeconomy of the United States. These facilities play a critical role in the national infrastructure, supporting research on a select set of especially dangerous pathogens, as well as novel, emerging diseases. Understanding the ecology, biology, and pathology at the human-animal interface of zoonotic spillover events is fundamental to efficient control and elimination of disease. The use of animals as human surrogate models or as target-host models in research is an integral part of unraveling the interrelated components involved in these dynamic systems. These models can prove vitally important in determining both viral- and host-factors associated with virus transmission, providing invaluable information that can be developed into better risk mitigation strategies. In this article, we focus on the use of livestock in maximum-containment, biosafety level-4 agriculture (BSL-4Ag) research involving zoonotic, risk group 4 pathogens and we provide an overview of historical associated research and contributions. Livestock are most commonly used as target-host models in high-consequence, maximum-containment research and are routinely used to establish data to assist in risk assessments. This article highlights the importance of animal use, insights gained, and how this type of research is essential for protecting animal health, food security, and the agriculture economy, as well as human public health in the face of emerging zoonotic pathogens. The utilization of animal models in high-consequence pathogen research and continued expansion to include available species of agricultural importance is essential to deciphering the ecology of emerging and re-emerging infectious diseases, as well as for emergency response and mitigation preparedness.

Significance of High-Containment Biological Laboratories Performing Work During the COVID-19 Pandemic: Biosafety Level-3 and -4 Labs.

High containment biological laboratories (HCBL) are required for work on Risk Group 3 and 4 agents across the spectrum of basic, applied, and translational research. These laboratories include biosafety level (BSL)-3, BSL-4, animal BSL (ABSL)-3, BSL-3-Ag (agriculture livestock), and ABSL-4 laboratories. While SARS-CoV-2 is classified as a Risk Group 3 biological agent, routine diagnostic can be handled at BSL-2. Scenarios involving virus culture, potential exposure to aerosols, divergent high transmissible variants, and zoonosis from laboratory animals require higher BSL-3 measures. Establishing HCBLs especially those at BSL-4 is costly and needs continual investments of resources and funding to sustain labor, equipment, infrastructure, certifications, and operational needs. There are now over 50 BSL-4 laboratories and numerous BSL-3 laboratories worldwide. Besides technical and funding challenges, there are biosecurity and dual-use risks, and local community issues to contend with in order to sustain operations. Here, we describe case histories for distinct HCBLs: representative national centers for diagnostic and reference, nonprofit organizations. Case histories describe capabilities and assess activities during COVID-19 and include capacities, gaps, successes, and summary of lessons learned for future practice.