Viral haemorrhagic fevers are characterized by fever and bleeding disorders and can progress to high fever, shock and death in many cases. Filoviruses constitute a family of RNA Viruses, causing severe haemorrhagic fever disease in human an non-human primates, represented by the the Genus Marburgvirus (MARV), with one species, and Ebolavirus (EBOV), which encompasses at least four species of viruses, named after the locations were the first Filovirus outbreaks were registered, including experimental facilities out of the natural geographical range: Zaire Ebolavirus, Sudan Ebolavirus, Bundibugyo Ebolavirus and Reston Ebolavirus (named after the experimental facility were macaques carrying the virus were imported in Reston, Virginia, USA). Other viruses causing haemorrhagic fever syndromes, at least in humans, include the families of Arenaviruses (including Old World Arenaviruses like Lassa haemorrhagic fever virus (LHFV), and New World Arenaviruses (like Guaranito (GHFV), Junín (JHFV) or Machupo (MHFV) viruses); Bunyaviruses like the Crimean-Congo haemorrhagic fever virus (CCHFV); Flaviviruses like the Dengue virus (DENV), which causes severe haemorrhagic fever only in some cases, under not yet determined circumstances; and Hantaviruses (HANV), like the viruses causing the haemorrhagic fever with renal syndrome (HFRS).

In the present opinion article, it will be disclosed that the haemorrhagic features of viral haemorrhagic fevers in general, and those caused by Lassa virus, dengue virus and especially Ebola virus, in particular, are caused by common patterns of metabolic disturbances of the glucose and ascorbate homeostasis. According to the experimental data available and some clues pointed out in literature, this work or opinion article will focus on the possible pathophysiological process that governs haemorrhagic fevers caused by Filoviruses, and from that point, expand on the hypothesis to other viruses for which experimental data suggest some commonalty in factors driving the possible pathophysiology of haemorrhagic fevers caused by filoviruses, as is the case of Lassa virus and Dengue virus. Other haemorrhagic fever-causing viruses, as the New World Arenaviruses (GHFV, JHFV, MHFV, for example), Bunyaviruses like the CCHFV, or Hantaviruses, could be classified as being governed by other pathophysiological metabolic mechanisms, although interactions of these viruses with the same host factors considered for Filoviruses, Lassa and Dengue viruses (as is supported by literature), cannot yet be excluded, although theses interactions are not yet supported by experimental research.

Disruption of glucose and ascorbate homeostasis could be the reason of both enhanced inflammatory cytokine storming and haemorrhagic manifestations at the level of endothelial cells and vasculature injury. ^{1 – 5} Endothelial damage during EBOV infection with no evidence of direct endothelial cytolysis has previously been described, ⁶ reinforcing the idea that other indirect mechanisms governing vasculature injury are present. Intracellular ascorbic acid has been considered as an enhancer of nitric oxide (NO) synthesis ⁷ and the macromolecular permeability of cultured human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid and L-ascorbic acid 2-phosphate. ⁸ Apoptosis in endothelial cells can be induced by hyperglycemia and ascorbate helps to prevent endothelial dysfunction, stimulates type-IV collagen synthesis and enhances cell proliferation. Haemorrhages and vasculature disfunctions are a clinical feature not only of viral haemorrhagic fevers, but also in scurvy, diabetes and thrombotic microangiopathic haemolytic anaemia. Inflammatory cytokine concentrations were found to be acutely increased by hyperglycaemia in humans, ^{4 , 5} which has been suggested as the causative role in the vasculature disorders and immune activation of diabetes. Hypoascorbinaemia and diabetes mellitus share several clinical symptoms including microangiopathy, capillary hyperperfusion and haemorrhages.

An early description by Symmers in 1952 of the thrombotic microangiopathic haemolytic anaemia, ⁹ which extraordinarily fits the haemorrhagic and vascular manifestations of filovirus haemorrhagic fever (FHF), included haemorrhagic manifestations in the form of widespread purpura. As described by Symmers, “ there might be haemorrhage from any of the orifices of the body”. A prodromal period of fever, muscle and joint pains and vague abdominal pain existed and included also mental confusion and neurological symptoms. The parallel of the thrombotic microangiopathic haemolytic anaemia with the filovirus pathological manifestations is important and would open-up the consideration of the haemorrhagic fever disease as a pathophysiological process driven by a mechanism different from the viral lysis that shares commonalties with metabolic and haematological diseases pivoting on the role of blood cells as central players of the homeostasis.

Glucose and ascorbate homeostasis, the presence of Glut-1 on erythrocytes and the erythrocyte physiology ⁵² might play an important role in Filovirus disease, not excluding viral interactions with the erythrocyte membrane. The blood glucose concentration is maintained within narrow limits by an inter-play between tissue glucose uptake, hepatic glucose production and insulin production. Erythrocytes, because of their number in blood, perform an important buffering function of glucose and ascorbate/dehydroascorbate levels in plasma. Erythrocyte counts of frugivorous species have been shown lower than those reported for insectivorous bats. ⁵³ In those species not able to synthesize ascorbate and expressing Glut-1 on erythrocytes virus interactions would lead to severe disturbance of the glucose and ascorbate levels in plasma, activation of hypoxia-inducible factors and haemolysis. Coagulation abnormalities and haemorrhages are a clinical feature of Ebola virus haemorrhagic fever. It should also be tested the possible importance of haemolysis and erythrocyte-virus interactions. As it is shown in an important paper ⁵⁴ erythrocytes can regulate platelet reactivity directly through chemical signalling and adhesive erythrocyte-platelet interactions. Cell free haemoglobin induces platelet aggregation contributing to high risk of thrombotic complications. Adhesion of abnormal and/or stimulated erythrocytes to vascular endothelium can contribute to vasculature occlusions associated with thrombosis. Efficient blood coagulation requires sufficient pro-thrombotic surfaces, which are provided by cells that expose phosphatidylserine, which is normally in the cytoplasmic side of the membrane to separate this pro-coagulant surface from plasma coagulation factors. The questions are: Could virus-erythrocytes interactions induce the exposure of phosphatidylserine on the cell surface? Is the phosphatidylserine-enriched envelope of some haemorrhagic fever-causing virions involved in this pro-thrombotic process mediated by exposed phosphatidylserine?

As the filovirus glycoproteins could interact with Glut-1 or other functionally related protein, the influx of glucose into the cells is severely impaired. As a consequence, transient hyperglycemia and a marked oxidative stress coupled with the high levels of glucose in plasma would be established, and then promote the activation of NF–κB transcription, exacerbating a pro-inflammatory response mediated by cytokines and chemokines, as described by Katherine Esposito et al. ⁴ On the other hand, the inability to synthesize ascorbate and the unavailability of ascorbate to entry into the cells through Glut-1 is an Achilles Heel when trying to counteract the oxidative stress. Additionally, there is another pathophysiological process to be mentioned: interactions with Glut-1 or other functionally related protein lead to an imbalance in the glucose homeostasis and the availability of glucose, not only for the erythrocytes, but also for CD8+ and CD4+ cells. These phenomena result in the bystander apoptosis of lymphocytes mediated by the lack of glucose, as described by Maclver et al. ⁵⁵ Activated T cells have dramatically increased metabolic requirements to support their demands. ⁵⁶ Therefore, activation of T cells causes a large increase in Glut-1 expression and surface localization, also promoting the Rab11b recycling of Glut-1 intracellular pools. ⁵⁵ A work on the mechanisms and consequences of Ebolavirus-induced lymphocyte apoptosis concluded undefined that Ebola virus induces multiple pro-apoptotic stimuli. ⁵⁷ As identified by Zhao and colleagues ⁵⁸ in 2007 there is a glucose-initiated signaling pathway that leads to inhibition of GSK-3 and prevents cell death through stabilization of the anti-apoptotic BcI2 family protein McI. Normally targeted for proteasomal degradation by the ubiquitin E3 ligases, in highly glycolytic cells as are cancer cells and activated T cells, McI remains unphosphorylated and is not degraded, increasing the threshold for cell-death and maintaining cell survival. Conversely, when glucose uptake is limited, glycolytic flux decreases to a level that no longer sustains viability and pro-apoptotic signals promote cell death. ⁵⁵

At a strictly cellular level, cell–adhesion dependent membrane trafficking of a binding partner for the EBOV glycoprotein has been shown to be a determinant of viral entry ⁵⁹ and attachment to adhesion substratum has been shown to induce the accumulation of glucose transporters and stimulate glucose metabolism in PC12 cells. ⁶⁰ According to Dube and colleagues a membrane trafficking event translocates the unknown binding partner of the receptor binding region (RBR) of EBOV glycoprotein (GP) to the cell surface and they identified two adherent primate lymphocytic cell lines that bind viral glycoprotein RBR at their surface and supported GP-mediated entry and infection. Lymphocytes are normally described in literature as non-permissive for EBOV-GP mediated entry and infection, ⁶¹ and activation of lymphocytes could be the trigger that renders them susceptible. According to the present hypothesis, this binding partner related to lymphocyte activation may possibly be or be related to Glut-1.

This first clue about a link between EBOV species range, expression of Glut-1 on erythrocytes and inability to synthesize ascorbate in humans and non-human primates should be further analyzed. In contrast to primates, and according to a study by Cui et al., bats are perhaps in the process of large-scale loss of ascorbate biosynthesis ability, ⁶² and show varying degrees of lack of gluconolactone oxidase function. This evolutionary recent loss of the ability to synthesize ascorbate could account for differences in Glut-1 expression and ascorbate metabolism resulting in differences in filovirus pathophysiology between species. In another study the same authors showed that the fruit bat Rousettus leschenaultii has retained the ability to synthesize ascorbate although at low levels compared with the mouse. ⁶³ Olival et al. found serological evidence to both species Reston and Zaire of the genus Ebolavirus in Rousettus leschenaultii, one of the fruit bats species considered to be reservoir of EBOV, ⁶⁴ and Marburgvirus has even been isolated from Rousettus aegyptiacus, ⁶⁵ another Pteropodidae member. It is known that frugivorous and nectarivorous bats are able to ingest large quantities of sugar in a short time span while avoiding the potentially adverse side-effects of elevated blood glucose, which could be an important fact supporting the hypothesis that glucose and ascorbate metabolism account for the severity of the haemorrhagic fevers in primates and for the role of fruit bats as reservoir species maintaining low levels of filovirus replication. This ability to ingest large quantities of sugar in a short time span while avoiding the potentially adverse side-effects of elevated blood glucose has been related to the adaptive evolution in the glucose transporter 4 (Glut-4) gene in skeletal muscle ⁶⁶ and also to high passive paracellular absorption of glucose in the gut of Rousettus aegyptiacus. ⁶⁷ The importance of Glut-4 and the active sodium-ascorbate co-transporters (SVCTs) and active sodium-dependent glucose cotransporters (SGLTs) in regard to Filovirus pathophysiology should be further investigated.

Regarding LHFV, the differences in glucose and ascorbate physiology could be an explanation of the absence of haemorrhagic manifestations of the illness in mice while primates and humans develop haemorrhagic fevers. If direct interactions of filoviral glycoproteins with Glut-1 could be demonstrated, it could partialy explain the differences in severity of the disease manifestations between Ebola and Lassa infections and others. It has been shown that the EBOV glycoprotein is the main determinant of cell cytotoxicity and injury. ⁶⁸ It should be further researched which role plays the viral glycoprotein in cell cytotoxicity for LHFV and DENV. The differences in haemorrhagic fever prevalence and severity between EBOV, LHFV and DENV could be explained by the differences in the pools of cellular factors and receptors involved in virus activity. Direct interactions with Glut-1 could explain the direct incidence of the EBOV on the glucose and ascorbate transport and homeostasis, causing the transient hyperglycemia, cytokine storming and haemorrhagic features. On the other hand, TIM-1 and Axl are entry factors for LHFV only under specific circumstances: in other words, are only a dystroglycan-independent entry route. The concentration of phosphatidylserine displayed in the viral envelope influences virus binding via Axl, as is described in another important paper. ⁶⁹ According to these authors, since functional α-dystroglycan is recognized by the viral envelope protein GP1 of LHFV, the exact ratio of LHFV GP1 to phosphatidylserine in the viral envelope appears critical for receptor use. When the presence of phosphatidylserine on the virion surface is qualitatively important in relation to the expression of GP1 in Lassa virus, the cellular entry of the virus through the interaction phosphatidylserine-Gas6-Axl or phosphatidylserine-TIM-1 would lead to the hypothesized metabolic disturbances, mediated by TIM-1 and/or Axl, probably via Glut-1. When Axl or TIM-1 are not an important receptor for LHFV entry and entry occurs via interaction of the viral GP1 with α-dystroglycan, the incidence of metabolic disorders and haemorrhagic manifestations would be lower.

For Dengue virus, when virus-host interactions mainly take place via viral glycoprotein-DC-SIGN interaction, the metabolic glucose and ascorbate disorders and haemorrhagic manifestations would be less important than in those cases in which viral interactions with host factors involve the interaction of the viral envelope with Axl and/or TIM-1. These different host factors that can interact with the viral envelope would explain the differences in pathophysiology: normal Dengue fever when DC-SIGN-viral glycoprotein interactions are predominant, or Dengue severe haemorrhagic fever when Axl and/or TIM-1 interact predominantly with the dengue virus envelope. The presence and importance of phosphatidylserine in the virus envelope could vary from virus to virus and from one virus type or strain (DENV-1, DENV-2, DENV-3, DENV-4, LV-1, LV-2, LFV-3, L V-4) to another. This should be matter of further research. It would be interesting to know which factors are governing the significant presence of phosphatidylserine on the virion envelope, and this considering the four haemorrhagic viruses: Ebola virus, Marburg virus, Lassa virus and Dengue virus.

Pathogenesis of viral haemorrhagic fevers is not yet well understood. The interaction of viruses with metabolism is a matter that will gain more attention in the next years. If direct interactions of filoviral glycoproteins with Glut-1 could be experimentally confirmed, it would even open up new opportunities to challenge Glut-1 transporters in cancer cells.

If some assumptions hypothesized in this opinion article were correct, regardless of other means of challenging the viruses, supportive care of haemorrhagic fevers almost in general could be significantly improved. It encompasses the use of insulin, ascorbate, glutathione (which participates, along with NADH, in the reduction of dehydroascorbate to ascorbate), maintaining constant and normal levels of glucose in plasma and general measures to counteract oxidative stress. Filoviruses originate from Africa, where almost all outbreaks have taken place, and a low-cost approach of the treatment would be advantageous for practical reasons.