Cytokine Profile Distinguishes Children With Plasmodium falciparum Malaria From Those With Bacterial Blood Stream Infections.

BACKGROUND: Malaria presents with unspecific clinical symptoms that frequently overlap with other infectious diseases and is also a risk factor for coinfections, such as non-Typhi Salmonella. Malaria rapid diagnostic tests are sensitive but unable to distinguish between an acute infection requiring treatment and asymptomatic malaria with a concomitant infection. We set out to test whether cytokine profiles could predict disease status and allow the differentiation between malaria and a bacterial bloodstream infection. METHODS: We created a classification model based on cytokine concentration levels of pediatric inpatients with either Plasmodium falciparum malaria or a bacterial bloodstream infection using the Luminex platform. Candidate markers were preselected using classification and regression trees, and the predictive strength was calculated through random forest modeling. RESULTS: Analyses revealed that a combination of 7-15 cytokines exhibited a median disease prediction accuracy of 88% (95th percentile interval, 73%-100%). Haptoglobin, soluble Fas-Ligand, and complement component C2 were the strongest single markers with median prediction accuracies of 82% (with 95th percentile intervals of 71%-94%, 62%-94%, and 62%-94%, respectively). CONCLUSIONS: Cytokine profiles possess good median disease prediction accuracy and offer new possibilities for the development of innovative point-of-care tests to guide treatment decisions in malaria-endemic regions.

Phylogeography of Lassa Virus in Nigeria.

Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatiotemporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian states. The final data set, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 states. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while a few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi Within lineages II and III, seven and five sublineages, respectively, were distinguishable. Phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal toward the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast toward Taraba and Adamawa and south toward Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sublineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at a higher resolution. In addition, we estimated the direction and time frame of virus dispersal in the country.IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sublineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows us to predict into which geographical areas the virus might spread in the future and prioritize areas for Lassa fever surveillance. Our study not only aimed to generate Lassa virus sequences from across Nigeria but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.

Kinetics of Soluble Mediators of the Host Response in Ebola Virus Disease.

Background: The pathophysiology of Ebola virus disease (EVD) is still poorly understood. This study aimed at identifying soluble biomarkers that inform on disease mechanisms. Methods: Fifty-four soluble mediators of the immune, coagulation, and endothelial system were measured in baseline and follow-up samples from hospitalized patients with EVD, using Luminex technology. Cross-sectional expression levels and changes over time were correlated with outcome. Results: Levels of circulating proinflammatory cytokines and chemokines, as well as markers of endothelial dysfunction and coagulopathy, were elevated on admission to hospital in patients who died from EVD as compared to survivors. These markers further increased in patients who died and/or decreased over time in survivors. In contrast, markers of gut integrity and T-cell response were higher in survivors and increased until discharge. Conclusions: Inflammatory response, endothelial integrity, gastric tissue protection, and T cell immunity play a role in EVD pathophysiology.

New Lineage of Lassa Virus, Togo, 2016.

We describe a strain of Lassa virus representing a putative new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo. This finding extends the known range of Lassa virus to Togo.

Chimeric Mice with Competent Hematopoietic Immunity Reproduce Key Features of Severe Lassa Fever.

Lassa fever (LASF) is a highly severe viral syndrome endemic to West African countries. Despite the annual high morbidity and mortality caused by LASF, very little is known about the pathophysiology of the disease. Basic research on LASF has been precluded due to the lack of relevant small animal models that reproduce the human disease. Immunocompetent laboratory mice are resistant to infection with Lassa virus (LASV) and, to date, only immunodeficient mice, or mice expressing human HLA, have shown some degree of susceptibility to experimental infection. Here, transplantation of wild-type bone marrow cells into irradiated type I interferon receptor knockout mice (IFNAR-/-) was used to generate chimeric mice that reproduced important features of severe LASF in humans. This included high lethality, liver damage, vascular leakage and systemic virus dissemination. In addition, this model indicated that T cell-mediated immunopathology was an important component of LASF pathogenesis that was directly correlated with vascular leakage. Our strategy allows easy generation of a suitable small animal model to test new vaccines and antivirals and to dissect the basic components of LASF pathophysiology.

Emergence of Zaire Ebola virus disease in Guinea.

In March 2014, the World Health Organization was notified of an outbreak of a communicable disease characterized by fever, severe diarrhea, vomiting, and a high fatality rate in Guinea. Virologic investigation identified Zaire ebolavirus (EBOV) as the causative agent. Full-length genome sequencing and phylogenetic analysis showed that EBOV from Guinea forms a separate clade in relationship to the known EBOV strains from the Democratic Republic of Congo and Gabon. Epidemiologic investigation linked the laboratory-confirmed cases with the presumed first fatality of the outbreak in December 2013. This study demonstrates the emergence of a new EBOV strain in Guinea.

Control measures following a case of imported Lassa fever from Togo, North Rhine Westphalia, Germany, 2016.

In a patient transferred from Togo to Cologne, Germany, Lassa fever was diagnosed 12 days post mortem. Sixty-two contacts in Cologne were categorised according to the level of exposure, and gradual infection control measures were applied. No clinical signs of Lassa virus infection or Lassa specific antibodies were observed in the 62 contacts. Thirty-three individuals had direct contact to blood, other body fluids or tissue of the patients. Notably, with standard precautions, no transmission occurred between the index patient and healthcare workers. However, one secondary infection occurred in an undertaker exposed to the corpse in Rhineland-Palatinate, who was treated on the isolation unit at the University Hospital of Frankfurt. After German authorities raised an alert regarding the imported Lassa fever case, an American healthcare worker who had cared for the index patient in Togo, and who presented with diarrhoea, vomiting and fever, was placed in isolation and medevacked to the United States. The event and the transmission of Lassa virus infection outside of Africa underlines the need for early diagnosis and use of adequate personal protection equipment (PPE), when highly contagious infections cannot be excluded. It also demonstrates that larger outbreaks can be prevented by infection control measures, including standard PPE.

Evaluation of RealStar Reverse Transcription-Polymerase Chain Reaction Kits for Filovirus Detection in the Laboratory and Field.

BACKGROUND: Diagnosis of Ebola virus (EBOV) disease (EVD) requires laboratory testing. METHODS: The RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and the derived RealStar Zaire Ebolavirus RT-PCR kit were validated using in vitro transcripts, supernatant of infected cell cultures, and clinical specimens from patients with EVD. RESULTS: The Filovirus Screen kit detected EBOV, Sudan virus, Taï Forest virus, Bundibugyo virus, Reston virus, and Marburg virus and differentiated between the genera Ebolavirus and Marburgvirus The amount of filovirus RNA that could be detected with a probability of 95% ranged from 11 to 67 RNA copies/reaction on a LightCycler 480 II. The Zaire Ebolavirus kit is based on the Filovirus Screen kit but was optimized for detection of EBOV. It has an improved signal-to-noise ratio at low EBOV RNA concentrations and is somewhat more sensitive than the Filovirus kit. Both kits show significantly lower analytical sensitivity on a SmartCycler II. Clinical evaluation revealed that the SmartCycler II, compared with other real-time PCR platforms, decreases the clinical sensitivity of the Filovirus Screen kit to diagnose EVD at an early stage. CONCLUSIONS: The Filovirus Screen kit detects all human-pathogenic filoviruses with good analytical sensitivity if performed on an appropriate real-time PCR platform. High analytical sensitivity is important for early diagnosis of EVD.

Efficacy of Favipiravir Alone and in Combination With Ribavirin in a Lethal, Immunocompetent Mouse Model of Lassa Fever.

We studied the therapeutic potential of favipiravir (T-705) for Lassa fever, both alone and in combination with ribavirin. Favipiravir suppressed Lassa virus replication in cell culture by 5 log10 units. In a novel lethal mouse model, it lowered the viremia level and the virus load in organs and normalized levels of cell-damage markers. Treatment with 300 mg/kg per day, commenced 4 days after infection, when the viremia level had reached 4 log10 virus particles/mL, rescued 100% of Lassa virus-infected mice. We found a synergistic interaction between favipiravir and ribavirin in vitro and an increased survival rate and extended survival time when combining suboptimal doses in vivo.

Ebola virus disease in mice with transplanted human hematopoietic stem cells.

The development of treatments for Ebola virus disease (EVD) has been hampered by the lack of small-animal models that mimick human disease. Here we show that mice with transplanted human hematopoetic stem cells reproduce features typical of EVD. Infection with Ebola virus was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Our study provides a small-animal model with human components for the development of EVD therapies.

Health care response to CCHF in US soldier and nosocomial transmission to health care providers, Germany, 2009.

In 2009, a lethal case of Crimean-Congo hemorrhagic fever (CCHF), acquired by a US soldier in Afghanistan, was treated at a medical center in Germany and resulted in nosocomial transmission to 2 health care providers (HCPs). After his arrival at the medical center (day 6 of illness) by aeromedical evacuation, the patient required repetitive bronchoscopies to control severe pulmonary hemorrhage and renal and hepatic dialysis for hepatorenal failure. After showing clinical improvement, the patient died suddenly on day 11 of illness from cerebellar tonsil herniation caused by cerebral/cerebellar edema. The 2 infected HCPs were among 16 HCPs who received ribavirin postexposure prophylaxis. The infected HCPs had mild or no CCHF symptoms. Transmission may have occurred during bag-valve-mask ventilation, breaches in personal protective equipment during resuscitations, or bronchoscopies generating infectious aerosols. This case highlights the critical care and infection control challenges presented by severe CCHF cases, including the need for experience with ribavirin treatment and postexposure prophylaxis.

Rodent control strategies and Lassa virus: some unexpected effects in Guinea, West Africa.

The Natal multimammate mouse (Mastomys natalensis) is the host of Lassa mammarenavirus, causing Lassa haemorrhagic fever in West Africa. As there is currently no operational vaccine and therapeutic drugs are limited, we explored rodent control as an alternative to prevent Lassa virus spillover in Upper Guinea, where the disease is highly endemic in rural areas. In a seven-year experiment, we distributed rodenticides for 10-30 days once a year and, in the last year, added intensive snap trapping for three months in all the houses of one village. We also captured rodents both before and after the intervention period to assess their effectiveness by examining alterations in trapping success and infection rates (Lassa virus RNA and IgG antibodies). We found that both interventions reduced the rodent population by 74-92% but swiftly rebounded to pre-treatment levels, even already six months after the last snap-trapping control. Furthermore, while we observed that chemical control modestly decreased Lassa virus infection rates annually (a reduction of 5% in seroprevalence per year), the intensive trapping unexpectedly led to a significantly higher infection rate (from a seroprevalence of 28% before to 67% after snap trapping control). After seven years, we conclude that annual chemical control, alone or with intensive trapping, is ineffective and sometimes counterproductive in preventing Lassa virus spillover in rural villages. These unexpected findings may result from density-dependent breeding compensation following culling and the survival of a small percentage of chronically infected rodents that may spread the virus to a new susceptible generation of mice.

Spatio-temporal spread of Lassa virus and a new rodent host in the Mano River Union area, West Africa.

The spread of Lassa virus (LASV) in Guinea, Liberia and Sierra Leone, which together are named the Mano River Union (MRU) area, was examined phylogeographically. To provide a reliable evolutionary scenario, new rodent-derived, whole LASV sequences were included. These were generated by metatranscriptomic next-generation sequencing from rodents sampled between 2003 and 2020 in 21 localities of Guinea and Sierra Leone. An analysis was performed using BEAST to perform continuous phylogeographic inference and EvoLaps v36 to visualize spatio-temporal spread. LASV was identified as expected in its primary host reservoir, the Natal multimammate mouse (Mastomys natalensis), and also in two Guinean multimammate mice (Mastomys erythroleucus) in northern Sierra Leone and two rusty-bellied brush-furred mice (Lophuromys sikapusi) in southern Sierra Leone. This finding is consistent with the latter two species being secondary host reservoirs. The strains in these three species were very closely related in LASV lineage IV. Phylogenetic analysis indicated that the most recent common ancestor of lineage IV existed 316-374 years ago and revealed distinct, well-supported clades from Sierra Leone (Bo, Kabala and Kenema), Guinea (Faranah, Kissidougou-Guekedou and Macenta) and Liberia (Phebe-Ganta). The phylogeographic scenario suggests southern Guinea as the point of origin of LASV in the MRU area, with subsequent spread to towards Mali, Liberia and Sierra Leone at a mean speed of 1.6 to 1.1 km/year.

A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells.

Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells.

Cross-species analysis of the replication complex of Old World arenaviruses reveals two nucleoprotein sites involved in L protein function.

Lassa virus (LASV) causing hemorrhagic Lassa fever in West Africa, Mopeia virus (MOPV) from East Africa, and lymphocytic choriomeningitis virus (LCMV) are the main representatives of the Old World arenaviruses. Little is known about how the components of the arenavirus replication machinery, i.e., the genome, nucleoprotein (NP), and L protein, interact. In addition, it is unknown whether these components can function across species boundaries. We established minireplicon systems for MOPV and LCMV in analogy to the existing LASV system and exchanged the components among the three systems. The functional and physical integrity of the resulting complexes was tested by reporter gene assay, Northern blotting, and coimmunoprecipitation studies. The minigenomes, NPs, and L proteins of LASV and MOPV could be exchanged without loss of function. LASV and MOPV L protein was also active in conjunction with LCMV NP, while the LCMV L protein required homologous NP for activity. Analysis of LASV/LCMV NP chimeras identified a single LCMV-specific NP residue (Ile-53) and the C terminus of NP (residues 340 to 558) as being essential for LCMV L protein function. The defect of LASV and MOPV NP in supporting transcriptional activity of LCMV L protein was not caused by a defect in physical NP-L protein interaction. In conclusion, components of the replication complex of Old World arenaviruses have the potential to functionally and physically interact across species boundaries. Residue 53 and the C-terminal domain of NP are important for function of L protein during genome replication and transcription.

T cell-dependence of Lassa fever pathogenesis.

Lassa virus (LASV), the causative agent of Lassa fever (LF), is endemic in West Africa, accounting for substantial morbidity and mortality. In spite of ongoing research efforts, LF pathogenesis and mechanisms of LASV immune control remain poorly understood. While normal laboratory mice are resistant to LASV, we report that mice expressing humanized instead of murine MHC class I (MHC-I) failed to control LASV infection and develop severe LF. Infection of MHC-I knockout mice confirmed a key role for MHC-I-restricted T cell responses in controlling LASV. Intriguingly we found that T cell depletion in LASV-infected HHD mice prevented disease, irrespective of high-level viremia. Widespread activation of monocyte/macrophage lineage cells, manifest through inducible NO synthase expression, and elevated IL-12p40 serum levels indicated a systemic inflammatory condition. The absence of extensive monocyte/macrophage activation in T cell-depleted mice suggested that T cell responses contribute to deleterious innate inflammatory reactions and LF pathogenesis. Our observations in mice indicate a dual role for T cells, not only protecting from LASV, but also enhancing LF pathogenesis. The possibility of T cell-driven enhancement and immunopathogenesis should be given consideration in future LF vaccine development.

Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections.

Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-alpha, IFN-beta, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-lambda) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-lambda plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-lambda receptor defect. Careful analysis revealed that expression of functional IFN-lambda receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-lambda contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.

Experimental Morogoro Virus Infection in Its Natural Host, Mastomys natalensis.

Natural hosts of most arenaviruses are rodents. The human-pathogenic Lassa virus and several non-pathogenic arenaviruses such as Morogoro virus (MORV) share the same host species, namely Mastomys natalensis (M. natalensis). In this study, we investigated the history of infection and virus transmission within the natural host population. To this end, we infected M. natalensis at different ages with MORV and measured the health status of the animals, virus load in blood and organs, the development of virus-specific antibodies, and the ability of the infected individuals to transmit the virus. To explore the impact of the lack of evolutionary virus-host adaptation, experiments were also conducted with Mobala virus (MOBV), which does not share M. natalensis as a natural host. Animals infected with MORV up to two weeks after birth developed persistent infection, seroconverted and were able to transmit the virus horizontally. Animals older than two weeks at the time of infection rapidly cleared the virus. In contrast, MOBV-infected neonates neither developed persistent infection nor were able to transmit the virus. In conclusion, we demonstrate that MORV is able to develop persistent infection in its natural host, but only after inoculation shortly after birth. A related arenavirus that is not evolutionarily adapted to M. natalensis is not able to establish persistent infection. Persistently infected animals appear to be important to maintain virus transmission within the host population.

Validation of Inactivation Methods for Arenaviruses.

Several of the human-pathogenic arenaviruses cause hemorrhagic fever and have to be handled under biosafety level 4 conditions, including Lassa virus. Rapid and safe inactivation of specimens containing these viruses is fundamental to enable downstream processing for diagnostics or research under lower biosafety conditions. We established a protocol to test the efficacy of inactivation methods using the low-pathogenic Morogoro arenavirus as surrogate for the related highly pathogenic viruses. As the validation of chemical inactivation methods in cell culture systems is difficult due to cell toxicity of commonly used chemicals, we employed filter devices to remove the chemical and concentrate the virus after inactivation and before inoculation into cell culture. Viral replication in the cells was monitored over 4 weeks by using indirect immunofluorescence and immunofocus assay. The performance of the protocol was verified using published inactivation methods including chemicals and heat. Ten additional methods to inactivate virus in infected cells or cell culture supernatant were validated and shown to reduce virus titers to undetectable levels. In summary, we provide a robust protocol for the validation of chemical and physical inactivation of arenaviruses in cell culture, which can be readily adapted to different inactivation methods and specimen matrices.

Discovery and genetic characterization of a novel orthonairovirus in Ixodes ricinus ticks from Danube Delta.

Different arthropod species are vectors of a wide array of arboviruses (arthropod-borne viruses) and have likely been central to viral evolution. To better understand the extent of arthropod-borne pathogens, as well as their origin and evolutionary history, it is crucial to uncover the full range of microbial agents, including viruses associated with arthropods. In this study, a collection of ticks obtained in 2016 directly from mammal and bird hosts from several rural and natural sites of Danube Delta was subjected to transcriptome sequencing and amplification assays. Vector surveillance revealed the presence of a novel orthonairovirus species, designated Sulina virus, in Ixodes ricinus ticks. Phylogenetic clustering of each viral protein consistently placed the new virus in the Orthonairovirus genus as a new genogroup closely related to Tamdy orthonairovirus, a genogroup comprising both pathogenic and tick-associated orthonairoviruses. The serological testing of engorged ticks and blood of infected hosts, along with the inoculation of vertebrate cells and mice found no specific antibodies or viral replication, suggesting that Sulina virus is an orthonairovirus associated with the virome of Ixodes ricinus. Finally, the characterization of a novel orthonairovirus identified using high throughput sequencing will advance our knowledge of interactions between viruses and tick vectors, expanding our perspective on fundamental questions regarding orthonairovirus evolution, diversity, ecology and potential of emergence as pathogens.