please remove the (#) after the volume in each reference.

Asked the authors to and they said they couldn't

Invited Review

[10015] Biomarkers; [10194] Thrombosis; [10195] Vascular Disease; [10030] Inflammation; [10054] Vascular

Biology

Running title: Mechanisms of bleeding in viral hemorrhagic feverMechanisms of bleeding in viral hemorrhagic

fever

Perkins and Mackman

Potential mechanisms underlying bleeding during infection with

hemorrhagic fever viruses

Megan V. Perkins 1,2 & , Nigel Mackman 2,3,*

1
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill.

2
UNC Blood Research Center, Department of Medicine, University of North Carolina at Chapel Hill.

3
Division of Hematology, Department of Medicine, University of North Carolina at Chapel Hill.

For Sources of Funding and Disclosures, see page XXX.

*
Corresponding Author: Nigel Mackman, Ph.D., Department of Medicine, 116 Manning Drive 8004B Mary

Ellen Jones Building, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA. Email:

nmackman@[Link]. Tel: (919) 843-3961. Fax: (919) 966-6012

ABSTRACT: Viral hemorrhagic fever (VHF) describes different diseases caused by several viruses from 6

virus families: Filoviridae, Nairoviridae, Phenuiviridae, Hanaviridae, Arenaviridae, and Flaviviridae. VHF

was once considered a geographically localized problem, but due to expanding vector ranges and increased

human contact with animal reservoirs and hosts the number of VHF cases is increasing. As the name indicates,

VHF is associated with bleeding. Both direct effects from viral infection of host cells and indirect effects caused

by the host response to the virus contribute to the dysregulation of the hemostatic system. Many studies have

measured different parameters and various biomarkers in samples from infected humans and non-human
primate models. For example, Ebola virus infection in a non-human primate model leads to increased tissue

factor expression in peripheral blood mononuclear cells and extracellular vesicles. In dengue virus infection,

thrombocytopenia and platelet dysfunction occur. There are likely both common and distinct mechanisms

underlying bleeding in different VHFs, as sites of bleeding differ between the viruses. Herein, we discuss the

potential mechanisms leading to bleeding during VHF, which include a consumptive coagulopathy, decreased

coagulation factor production, thrombocytopenia and platelet dysfunction, and endothelial cell activation and

damage resulting in increased vascular permeability. While a significant body of work exists examining

different aspects of the various viral infections that may lead to bleeding, there are still many open questions

and areas for investigation. Therefore, more studies are needed to better understand the mechanisms underlying

bleeding in VHF caused by different viruses.

Non-standard Abbreviations and AcronymsNonstandard Abbreviations and Acronyms

CCHFV – Crimean Congo hemorrhagic fever virus

CFR – Case fatality rate

DIC – Disseminated intravascular coagulation

EBOV – Ebola virus

EC – Endothelial cell

EV – Extracellular vesicle

EVD – Ebola virus disease

F – Factor

FDP – Fibrin degradation product

HCPS – Hantavirus cardiopulmonary syndrome

HFRS – Hemorrhagic fever with renal syndrome

HFV – hemorrhagic fever virus

HTNV – Hantaan virus

LASV – Lassa fever virus

MARV – Marburg virus

MVD – Marburg virus disease

NHP – Non-human primate

PBMC – Peripheral blood mononuclear cell

PS – Phosphatidylserine

RVFV – Rift Valley fever virus

SNV – Sin nombre virus

TF – Tissue factor

tPA – Tissue plasminogen activator

uPA – Urokinase plasminogen activator

VHF – viral hemorrhagic fever

vWF – von Willebrand factor

YF – Yellow fever

YFV – Yellow fever virus

Section 1: Hemorrhagic fever viruses

Viral hemorrhagic fever (VHF) is a catch-all term for the disease induced by viruses designated as hemorrhagic

fever viruses (HFVs). VHF generally begins with non-specific, febrile symptoms that may progress to more

severe disease, including hemorrhagic symptoms, with a variable clinical course. While all HFVs can induce

hemorrhagic symptoms, including coagulopathy, thrombocytopenia, and bleeding, they do so to differing

frequencies, severity, and possibly by different mechanisms. HFVs are from 6 virus families: Filoviridae,

Nairoviridae, Phenuiviridae, Hanaviridae, Arenaviridae, and Flaviviridae (Figure 1). This review will discuss

Ebola virus (EBOV), Marburg virus (MARV), Lassa fever virus (LASV), Rift Valley fever virus (RVFV),

Crimean Congo hemorrhagic fever virus (CCHFV), Hantaan virus (HTNV), sin nombre virus (SNV), yellow

fever virus (YFV), and dengue virus (DENV). All of these viruses have small (<20 kilobases) RNA genomes. A

brief overview of the virological information for each virus is presented in Table 1. The case presentation

information is presented in Table 2. Importantly, the case presentation information for many of the viruses (e.g.,

number of infections, number of cases presenting with severe and/or hemorrhagic manifestations, and case
fatality rate) is derived primarily from data on individuals presenting to a clinical setting or from

epidemiological estimates. Notably, many individuals infected with some of these viruses experience sub-

clinical disease or do not present to a clinic. Thus, these numbers should be considered as estimates based off of

available data. The geographic distribution of the different HFVs is shown in Figure 2.

Filoviridae: Ebola virus and Marburg virus

EBOV belongs to the genus Ebolavirus that has 5 other species. Not all members of the Ebolavirus genus cause

disease in humans. For those that do, the disease is collectively called Ebola virus disease (EVD) and has a

pooled case fatality rate (CFR) ~60 percent (Table 2). (24) Most individuals with EVD are symptomatic and

experience severe disease (Table 2). (32) EVD can be separated into 3 phases: early, peak, and resolution. The

early phase begins with febrile symptoms (e.g., arthralgia, headache, fatigue, rash) that progress to severe

gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea). During the early phase, hemorrhagic symptoms

(e.g., ecchymoses, bloody vomit or stool, bloody gums, conjunctival injection, nosebleeds, and bruising) may

begin (Table 2). Hemorrhagic symptoms may occur in up to 50 percent of cases, but the incidence is highly

variable (Table 2). (37) During the peak phase, about 4-12 days after disease onset, viral load begins to peak.

With an increase in viral load, symptoms, such as gastrointestinal and hemorrhagic symptoms, continue and

may worsen. Additionally, multi-organ dysfunction may be observed, including renal failure, respiratory failure,

neurologic symptoms (e.g., meningoencephalitis and stroke), and cardiac problems (e.g., myocarditis and

pericarditis). (37) Individuals surviving EVD enter the resolution or convalescent phase and are more

susceptible to secondary infections leading to sepsis. A significant proportion of surviving individuals also

report long-term sequalae, including arthralgias, myalgias, visual and auditory changes, extreme fatigue, rashes,

and gastrointestinal issues. The cause of such symptoms is unknown, but these secondary complications are

debilitating for affected individuals. (44)

MARV is 1 of 2 species of the genus Marburgvirus. MARV causes Marburg virus disease (MVD),

which is separated into early, peak, and resolution phases and has a similar clinical course to EVD in terms of

symptoms and overall timing. (3) There is no evidence of asymptomatic MARV disease currently. Similar to
EBOV, MVD has a high proportion of cases with hemorrhagic manifestations (34-83 percent) and an average

CFR of 50 percent (Table 2). (25, 28, 38)

Arenaviridae: Lassa fever virus

LASV belongs to the genus Mammarenavirus. There are 41 members of this genus, and only 8 of the members

cause disease in humans, including LASV. (4) LASV is transmitted to humans primarily through contact with

secretions from infected rodents (Table 1)(Figure 2). (17) The primary rodent reservoir is the multimammate

rat, which is found in Western Africa (Table 1). (17) The majority (~80 percent) of individuals infected with

LASV are asymptomatic and only ~20 percent of infected individuals progress to severe disease (Table 2). (17)

Lassa fever progresses through 3 stages. The first is the viral prodrome, characterized by non-specific febrile

symptoms lasting 4 to 7 days. The peak phase occurs next and is characterized by sore throat, chest pain,

conjunctival injection, vomiting, diarrhea, and abdominal pain. These symptoms may then progress further to

hypotension, shock, neurological symptoms (e.g., altered consciousness, seizures, tremors, hearing loss), head

and neck edema, and hemorrhage from the mouth, nose, rectum, or vagina (Table 2). Overt bleeding can occur

in up to 40% of cases and has a strong association with mortality (Table 2). (17) The final phase is the

convalescent phase. Survivors may suffer from long-term complications, such as hearing loss, seizures,

cognitive impairment, and ophthalmic complications. (45)

Phenuiviridae: Rift Valley fever virus

RVFV belongs to the genus Phlebovirus, which has 67 species, but only 11 of those species, including RVFV,

cause disease in humans. (46) RVFV geographic distribution follows that of its primary reservoir and vector,

Aedes mosquitoes, and other mosquito and arthropod species that serve as secondary vectors (Table 1)(Figure

2). (19) While mosquitos can infect humans after taking a blood meal, most people become infected through

contact with tissues and fluids from infected domesticated livestock. (19) RVFV induces severe and lethal

disease in domesticated ruminates, causing large economic impacts in affected communities. Most humans

infected with RVFV experience mild or subclinical symptoms. (19, 21) Due to the mild nature of many RVFV

infections in humans and differences between outbreaks, the exact incidence of RVFV is unclear. However,
estimates suggest that half a million people were infected with RVFV between 1997 and 2010. (18) Less than 1

percent of infected individuals develop hemorrhagic manifestations (Table 2). (19, 21) Initial symptoms begin

in a manner similar to other VHFs, with non-specific febrile symptoms for 3-4 days. Hemorrhagic disease may

begin about 1 week after symptom onset with the development of a macular rash, extended ecchymoses, gum or

gastrointestinal bleeding, and bloody stools or vomit. (21) Hemorrhagic symptoms frequently portend a fatal

outcome. While the overall CFR for RVFV is low (less than 1 percent), it can be up to 50 percent in cases with

hemorrhagic symptoms (Table 2). Other severe disease manifestations for RVFV infection include ocular (up to

10 percent of cases) and encephalitic (less than 1 percent of cases) symptoms. (19, 21)

Nairoviridae: Crimean-Congo hemorrhagic fever virus

The Hyalomma tick is the primary reservoir and vector of CCHFV (Table 1)(Figure 2). Humans, livestock, and

wild animals can become infected with CCHFV through the bite of an infected tick. Humans may also become

infected upon handling parts or fluids of infected animals. Most infections (up to 88 percent) with CCHFV are

thought to be asymptomatic. (33) Of the approximately 12 percent of infections that are severe enough to cause

individuals to seek medical help, hemorrhagic symptoms (e.g., nosebleeds, ecchymoses, and blood vomit) can

occur in up to 50 percent of individuals (<6 percent of all infections)(Table 2). (33) As with other VHFs, initial

symptoms of CCHFV infection are non-specific and febrile. (6) A week or less after symptom onset, the

hemorrhagic phase begins. Hemorrhagic disease is characterized by mucous membrane and skin petechia and

extended ecchymoses, nosebleeds, coughing up blood (hemoptysis), bleeding from injection sites, and bloody

stools, vomit, or urine (Table 2). These symptoms usually persist for 2-3 days. Severe cases may develop

disseminated intravascular coagulation (DIC), hypovolemic shock, or multi-organ failure, including that of the

liver and lungs. Survivors can suffer from a variety of complications, including hypotension, arrhythmias,

difficulty breathing, and issues with their vision, hearing, or memory. Long-term complications remain

undefined. (6)

Hantaviridae: Hantaan virus (HTNV) and sin nombre virus (SNV)

Orthohantaviruses are divided into Old World (e.g., HTNV) and New World (e.g., SNV) based on their

geographic distributions. Old World viruses are primarily found throughout Europe and Asia, while New World

viruses are found primarily in the Americas (Table 1)(Figure 2). (20) Of the 60 different viruses in the genus

Orthohantavirus, 28 viruses, including HTNV and SNV, are clinically important. (7, 20)

HTNV and other Old World Orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS).

Moderate (50 percent) and severe disease (20 percent) account for up to 70 percent of clinical HTNV cases and

are differentiated by severity of symptoms such as bleeding (Table 2). (34) The overall CFR for HTNV

infection is about 1 percent. (7, 20) HFRS can be divided into 5 phases. (7, 34) The first is a non-specific

febrile phase lasting a week or less that progresses into a hypotensive phase. Hemorrhagic manifestations occur

in less than 30 percent of cases and may begin near the end of the febrile phase. (34, 39) Hemorrhagic

symptoms include conjunctival hemorrhages and petechiae in the mouth (Table 2). (7) The hypotensive phase

is variable in duration (hours to a couple of days) and is characterized by hypotension that may lead to

hypovolemic shock. Hemorrhagic signs may increase during this phase, including bruising, petechiae on the

skin and mucosal surfaces, reddening of the conjunctiva, nose bleeds, intracranial hemorrhage, and bloody

vomit, urine, or stools (Table 2). (7) Following this phase is the oliguric phase, which lasts less than a week and

is characterized by temporarily decreased kidney function, which leads to low urine output (oliguria) or other

abnormal characteristics (e.g., no urine output, increased protein or blood in the urine). Up to a third of

individuals with severe HFRS die during the hypotensive phase and about 50 percent die during the oliguric

phase. (7) The next phase is the polyuric phase, where the body begins to return to normal, including a recovery

of kidney function, including urinary output. This phase can last up to several weeks. The final phase is

convalescence, which can last up to half a year. Chronic renal failure and hypotension are rare complications of

HFRS. (7)

SNV and other New World Orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS).

Like HFRS and other VHFs, HCPS begins with a non-specific febrile phase that is usually short (< 1 week). (7)

The cardiopulmonary phase onsets suddenly in more than 50 percent of cases with the rapid development of a
cough, shortness of breath, non-cardiac pulmonary edema, pleural effusions, hypotension, and tachycardia.

Cardiogenic shock, lactic acidosis, respiratory failure, and decreased plasma volume may also occur in severe

cases. (7) HCPS has a high CFR relative to HFRS (35 percent vs 1 percent)(Table 2). Individuals surviving the

cardiopulmonary phase enter the polyuric phase, where pulmonary edema resolves and the body begins to

return to normal. Convalescence is the final stage. While recovery is slow, survivors do not typically have any

long-term complications that are directly attributed to viral infection rather than intense hospitalization.

Notably, while SNV is considered a HFV, severe bleeding is not a hallmark symptom of the pathology ( Table

2). (7, 20)

Flaviviridae: Yellow fever virus and dengue virus

YFV is an Orthoflavivirus. There are 53 total Orthoflaviviruses, with over half causing disease in humans.

Yellow fever (YF) begins with non-specific febrile symptoms. Approximately 20-60 percent of individuals

enter a remission phase for up to 2 days before progressing to the toxic phase, which is marked by yellowing of

the skin or eyes (leading to the name YF) caused by liver and renal dysfunction or failure, thrombocytopenia,

and hemorrhagic symptoms (Table 2). (9, 10) Hemorrhagic manifestations occur in approximately 12 percent

of cases and include petechiae, bruising, bloody gums, and bloody vomit. Prior to death, individuals may also

experience encephalitic manifestations, such as confusion, seizures, and coma. For individuals surviving YF,

long-term complications are rarely reported. (9, 10)

DENV is also an Orthoflavivirus. The disease caused by DENV has historically been broadly classified

as an acute febrile illness [Dengue fever(DF)], a severe illness with fever, hemorrhagic symptoms,

thrombocytopenia, and evidence of plasma leakage, such as increased/progressively increasing hematocrit or

decreased albumin in the plasma or blood (Dengue hemorrhagic fever), or dengue hemorrhagic fever with

additional circulatory failure (Dengue shock syndrome). In 2008, the World Health Organization reclassified

the DENV presentations as DF, DF with warning signs, and severe DF. The primary difference between DF

with warning signs and severe DF is the severity of the symptoms, where individuals with severe DF have

severe plasma leakage, which can lead to Dengue shock syndrome, severe bleeding, and severe spleen and liver
enlargement. (47) All types of DF can be broadly divided into the febrile, toxic, and recovery phases. Signs of

severe disease occur during the toxic phase, including hemorrhagic symptoms. Hemorrhagic manifestations

occur in 0.5-5 percent of clinically presenting DENV cases and include petechiae, bruising, and bloody vomit

and stool (Table 2). (11, 16) There are 4 distinct DENV serotypes, which can be thought of as distinct viruses.

Importantly, most severe DENV infections occur upon reinfection with a different DENV serotype. This is due

to antibody-dependent enhancement, in which antibodies from the first infection with one DENV serotype bind

and enhance infection of a different DENV serotype by facilitating entry into target cells expressing Fcγ

receptors. This aspect of DENV infection has also complicated vaccine development (Table 1). (43)

Section 2: The hemostatic system

Hemostasis is defined as the processes that stop bleeding following blood vessel injury. The hemostatic system

has several different components, including platelets, the coagulation system, the fibrinolytic system, the

endothelium, and the blood vessel wall. Under normal conditions, these components are in balance, leading to

the formation of a hemostatic plug comprised of platelets and cross-linked fibrin that stops bleeding. However,

during disease, the hemostatic system can become dysregulated, leading to bleeding and thrombosis.

The coagulation system

The coagulation system contains pro-coagulant factors that are required for the formation of fibrin and anti-

coagulant factors that regulate different points of the coagulation cascade. The coagulation cascade can be

divided into the extrinsic, intrinsic, and common pathways. The extrinsic pathway [tissue factor (TF)/Factor (F)

VIIa complex] primarily initiates blood coagulation. Under normal conditions, TF is expressed by cells

underlying the vascular endothelium. (48) Damage to a blood vessel leads to the activation of the coagulation

protease cascade by the TF/FVIIa complex. (48) The intrinsic pathway is initiated by the activation of FXII.

(49) FXIIa activates FXI to FXIa, which then activates FIX to FIXa. FIXa in association with its cofactor

FVIIIa forms the intrinsic Xase complex. The common pathway includes the prothrombinase complex

(FXa/FVa) that converts prothrombin to thrombin. Thrombin is the central protease of the coagulation cascade

and cleaves fibrinogen to form fibrin monomers and activates the transglutaminase FXIII, the cofactors FV and
FVIII, as well as platelets. (49) It should be noted that FXIIa can also activate pre-kallikrein to kallikrein, which

cleaves high molecular weight kininogen resulting in the liberation of bradykinin, which increases vascular

permeability. (49)

Fibrinogen is comprised of 2 copies each of 3 chains: Aα, Bβ, and γ. (50) Fibrinogen is assembled in

hepatocytes and secreted as a soluble protein. (50) Upon cleavage by thrombin, fibrinogen is converted to fibrin

monomers. These monomers oligomerize and eventually form protofibrils. These protofibrils aggregate to form

an insoluble fibrin mesh. The stability of the fibrin mesh is further enhanced through cross-linking by FXIIIa.

(50)

Platelets

Platelets are small, anucleate blood cells produced from megakaryocytes in the bone marrow. (51) They are

rapidly recruited to sites of vessel injury. Initial platelet tethering to the injured vessel is mainly mediated by the

platelet receptor GPIb-V-IX binding von Willebrand Factor (vWF) that is deposited from the plasma or released

from activated endothelial cells (ECs). Firm adhesion requires the interaction of various platelet integrins with

components in the extracellular matrix. Next, binding of soluble agonists, such as thrombin, ADP, or

thromboxane A2, to platelet G protein-coupled receptors activates the platelets. Finally, binding of fibrinogen to

activated αIIbβ3 on platelets mediates platelet aggregation. (51)

The endothelium

The vascular endothelium is an anti-thrombotic surface that inhibits the activation of platelets and coagulation.

(52) For example, ECs inhibit platelet activation by expressing CD39, which degrades ATP, and by releasing

nitric oxide and prostacyclin. The endothelium also expresses anti-coagulant proteins, such as tissue factor

pathway inhibitor and components of the protein C system (e.g., thrombomodulin, endothelial protein C

receptor), as well as molecules (e.g., heparin sulfates) that bind antithrombin. In addition, ECs release tissue

plasminogen activator (tPA), which promotes fibrinolysis. (52) The integrity of the EC barrier is maintained by

tight junctions and adherens junctions between adjacent ECs and the EC glycocalyx. (53, 54) Under

pathological conditions, however, the endothelium is converted into a prothrombotic surface by the release of
vWF and P-selectin from Weibel-Palade bodies, down regulation or shed of anticoagulant proteins (e.g.,

thrombomodulin), and induction of TF expression. (52)

The fibrinolytic system

The fibrinolytic system generates plasmin that degrades cross-linked fibrin. tPA and urokinase plasminogen

activator (uPA) activate plasminogen to plasmin. (55) tPA and plasminogen bind to fibrin, which enhances tPA

activation of plasminogen. uPA binds to urokinase plasminogen activator receptor on cells to activate

plasminogen. Upon activation, plasmin cleaves cross-linked fibrin to generate fibrin degradation products

(FDPs), such as D-dimer. (55) tPA and uPA are inhibited by plasminogen activator inhibitor (PAI)-1 and PAI-

2. PAI-1 has a higher affinity for tPA, while PAI-2 has a higher affinity for uPA. Plasmin is inhibited by α2-

antiplasmin. Thrombin also activates thrombin activatable fibrinolysis inhibitor, which cleaves plasminogen

binding sites in fibrin. (55)

Section 3: Mechanisms of hemostatic derangement in viral hemorrhagic fever

In this section, possible mechanisms leading to bleeding during VHF are discussed. Many different biomarkers

have been measured in both humans and non-human primates (NHPs) during VHF. Unfortunately, most human

data can be thought of as a single snapshot that may or may not accurately represent the total infection course.

Additionally, most time points from humans are based on symptom onset. Because many of the initial

symptoms of VHF can be mild and non-specific, pinpointing the exact timepoint during infection when a

sample was acquired from an infected individual can be difficult and are estimates at best. Thus, data from NHP

models have been key to exploring and elucidating the mechanisms underlying bleeding during VHF. Tables 3

and 4 summarize various hemostatic-related parameters and biomarkers in both humans and NHPs infected

with the different HFVs, respectively.

Bleeding can arise from either consumption of coagulation factors and platelets and/or inadequate

synthesis of coagulation factors and platelet production. Coagulation activation is part of the host response to

viral infection and is thought to limit pathogen dissemination and prevent plasma leakage resulting from

damage due to infection. (129) However, viral infection can lead to DIC that results in the consumption of
coagulation factors (consumptive coagulopathy) and platelets (thrombocytopenia), leading to bleeding. The

International Society on Thrombosis and Hemostasis recently updated the scoring criteria for the clinical

diagnosis of DIC (Table 5). (130) An increase in the prothrombin time and a decrease in fibrinogen indicate the

consumption of coagulation factors. A low platelet count indicates thrombocytopenia, whereas D-dimer is a

marker of both coagulation activation and fibrinolysis. Severe coagulopathies, including DIC, are life

threatening. (130)

The coagulation system is activated during viral hemorrhagic fever

Coagulation activation is a feature common to nearly all HFV infections. The TF/FVIIa complex is the primary

activator of coagulation in vivo, and TF expression is increased in many diseases. (48) TF is a transmembrane

protein that localizes FVII/FVIIa to the surface of cells and extracellular vesicles (EVs). Measuring TF in

plasma is difficult due to its low abundance. (131) Importantly, activity-based assays are more sensitive than

antigen-based assays. (132)

A landmark study demonstrated that EBOV increases TF expression in peripheral blood mononuclear

cells (PBMCs, notably the monocyte) in vitro and in vivo using a NHP model. (98) Whether other cell types

also upregulate TF following EBOV infection is unknown. Additionally, a recent study showed that there is

increased EV-TF activity in plasma from EBOV-infected NHPs. (128) Further, TF expression is central to

EBOV pathogenesis, as administration of the TF/FVIIa inhibitor NapC2 reduced disease (e.g., delayed

appearance of characteristic rash and lower D-dimer) and prolonged and increased survival in EBOV-infected

NHPs. (119)

Infections with MARV in a NHP model also led to increased TF expression in PBMCs compared to

controls. (121) However, the administration of NapC2 was not protective in a MARV NHP model. (121) The

authors noted that the virus isolate used was more virulent than other strains previously used in the same NHP

model. (121) While inhibition of the TF/FVIIa complex was not effective in preventing severe disease in this

MARV model, it does not preclude the possibility that TF/FVIIa inhibition may be effective for less virulent

MARV strains.
 Several studies have measured plasma levels of TF antigen in DENV-infected individuals. It is difficult

to compare these studies because they use different assays to measure TF and samples from patients with

different disease severities. One study found increased plasma TF in severe DF compared to DF. (101)

Interestingly, the healthy control group and the DF group did not differ significantly. This could be due to the

timing of sample collection, as samples were collected 1-15 days post symptom onset, which on the extreme

end may be past the window of induction. (101) A study examining TF expression in the plasma of children

with severe DF at different times during disease observed children with severe DF had high levels of plasma TF

at hospital admission that decreased over time. (99) Additionally, PBMCs from individuals with severe DF had

increased TF protein expression compared to those with DF or healthy controls. (100) A fourth study found a

trend towards increased EV-TF activity in individuals with DF compared to healthy controls. (133) However,

the point during disease that the samples were collected is unclear. (133)

Individuals with LASV had higher levels of plasma TF protein compared to non-LASV febrile controls.

(80) In fatal cases of RVFV, plasma TF antigen is also increased. (88) An increase in plasma EV-TF activity

was also observed in individuals infected with SNV. (81) However, the cellular source of TF during LASV,

RVFV, and SNV infections remains to be explored. An increase in EV-TF activity in the plasma of individuals

infected with Puumala virus, a close relative of HTNV, has also been noted and associated with intravascular

coagulation. (134)

TF levels during other HFV infections have not been measured. Additionally, because all the HFVs

examined in this review are enveloped, the new virions derived from TF-positive host cells may incorporate TF

into the viral envelope as has been shown for several herpesviruses. (135) However, this remains to be

established for HFVs. In summary, increased TF on PBMCs (likely monocytes), other cell types, and EVs is

likely the primary activator of the coagulation cascade observed in VHF ( Figure 3). Understanding the triggers

that activate coagulation in VHF may identify new therapeutic targets to prevent coagulation activation during

VHF.

Viral hemorrhagic fever is associated with liver damage

VHF is associated with liver damage in humans and NHP models, as evidenced by increased aspartate

aminotransferase and alanine aminotransferase and liver pathology. (58, 61-63, 67-70, 74, 77, 78, 80, 88, 91-95,

97, 115, 136-144) The liver is the primary site for synthesis of most coagulation factors, including prothrombin

and fibrinogen. Hepatocytes are also the primary source of the anti-coagulant protein C. Indeed, the level of

protein C decreased in humans and NHPs infected with HFVs (EBOV, MARV, LASV, YFV, and DENV). (2,

70, 73, 98, 109, 111, 119, 121, 122) Several of the HFVs (EBOV, MARV, LASV, RVFV, CCHFV, and YFV)

are hepatotropic, meaning they directly infect hepatocytes (Table 1). Some of these viruses are also cytopathic

in cultured hepatocytes (EBOV, LASV, CCHFV, YFV) while others are not (MARV). (145-149) From

examination of pathological specimens, hepatocytes stain positive for viral antigen and show signs of apoptosis

and necrosis. However, whether this is due to viral replication or immune-mediated mechanisms cannot be

determined. In addition to directly killing hepatocytes, these viruses may also lead to generalized hepatocyte

dysfunction upon infection, which is further increased by severe inflammation. For the non-hepatotropic viruses

(HTNV, SNV, and DENV), general liver dysfunction or pathology likely arises through immune-mediated

mechanisms. Ultimately, both processes lead to a reduction in coagulation factor and protein C synthesis

(Figure 3).

Thrombocytopenia and platelet dysfunction during viral hemorrhagic fever

Thrombocytopenia can be due to decreased platelet production, increased platelet activation and consumption,

or a combination thereof. The role of platelets during HFV infection is best characterized for DENV. (150)

Both decreased platelet production and increased activation and consumption contribute to thrombocytopenia

during DENV infection. Transient bone marrow suppression and infection of megakaryocytes may contribute to

decreased platelet production early during infection. (151-153)

During the toxic phase of DENV infection in which thrombocytopenia is a key feature, the bone marrow

is normal, suggesting increased platelet clearance and/or consumption is underlying thrombocytopenia. Platelets

isolated from individuals infected with DENV are activated, exhibiting increased surface expression of active

αIIbβ3, P-selectin, and phosphatidylserine (PS). (154-157) Platelets from individuals with DENV are also less
responsive to in vitro agonist stimulation compared to those from convalescent individuals or healthy controls.

This suggests that platelets from infected individuals may be exhausted and have reduced function, and as such

they cannot adequately respond to stimuli. (156) Additionally, some platelets isolated from individuals with

DENV are apoptotic. (154, 155)

Platelet activation likely occurs through several mechanisms during DENV infection. Both viral RNA

and antigen have been detected in isolated human platelets. (157, 158) This observation in combination with

human platelets supporting DENV replication in vitro suggests that DENV may replicate to some extent in

platelets in vivo. (157-159) However, whether platelets produce new virions in vitro or in vivo is hotly debated

and platelets likely do not contribute to viremia. (160) The viral protein NS1, which is released into the

circulation during infection, can also activate platelets directly. (161, 162) The levels of NS1 are positively

correlated to disease severity and the development of thrombocytopenia. (162) Indeed, in vitro NS1-stimulated

platelets have increased levels of PS and P-selectin surface expression, as well as pro-longed aggregation

following in vitro agonist stimulation. (163) In a DENV mouse model that develops thrombocytopenia, genetic

deletion of NS1 prevented thrombocytopenia. (163). Activated platelets may also bind to activated ECs. (163)

EC-bound platelets are removed from the circulation and cannot be measured during routine blood counts,

ultimately contributing to the observed thrombocytopenia.

Activated platelets express various receptors that mediate their clearance. (164) Both increased PS

exposure and the coating of platelets with antibodies or complement enhance engulfment by phagocytes. Both

anti-platelet antibodies and antibody- and complement-coated platelets have been isolated from individuals with

DENV. (157, 165) Moreover, increased platelet-phagocyte aggregates have been isolated from individuals with

DENV; additionally, phagocytosis of platelets isolated from individuals with DENV compared to healthy

controls was increased in vitro and this correlated with disease severity. (154) Exposure of platelets to NS1 also

led to increased phagocytosis in vitro, suggesting a mechanism by which phagocytosis may be stimulated in

vivo. (163) Platelets may also be removed by phagocytes through opsonization or lysed via complement

activation and subsequent formation of the membrane attack complex. (165, 166)
 Platelets can also contribute to inflammation during DENV infection via the secretion of various

cytokines, which can activate other cell types and increase vascular permeability. (150) DENV-infected

platelets may also be an important source of secreted NS1, which directly contributes to increased vascular

permeability in addition to its role in platelet activation. (167)

Thrombocytopenia occurs mainly during severe LASV infection and rarely in non-severe infection. In

NHPs, platelet survival time is normal. However, platelets from both humans and NHPs with severe LASV

infection exhibit aggregation defects in response to in vitro agonist stimulation. (80, 110, 168) In a follow up

study, human plasma from severe LASV cases inhibited aggregation of platelets collected from healthy donors.

Platelet aggregation activity was restored following washing of the platelets, suggesting the presence of a

platelet inhibitor in the plasma of individuals with severe LASV. (169) Aggregation curves also show that

platelets from individuals with LASV disaggregate, which may be suggestive of defects in granule release,

which is known to sustain platelet aggregation over time. (80, 168) Improved aggregation responses also

coincided with clinical improvement, suggesting that platelet inhibition contributes to the pathology of LASV.

(168)

Pathogenic Orthohantaviruses (e.g., HTNV and SNV) bind the inactive form of β3 integrins. (170)

This may lead to defective platelet aggregation and adhesion responses. Indeed, regardless of illness severity,

platelets from individuals with HFRS exhibit aggregation and granule release defects in response to in vitro

agonist stimulation that improve as illness resolves. (171) However, the defects in severely ill individuals were

worse compared to moderately and mildly ill individuals. These properties appear to be intrinsic to the platelets

themselves, rather than due to their environment (e.g., plasma), as mixing normal platelets with HFRS plasma

led to normal aggregation. Additionally, quiescent platelets bind infected ECs via cell-surface displayed viral

glycoproteins in vitro, which may lead to reduced circulating platelet numbers. (170)

Individuals with CCHFV have an increased immature platelet fraction and mean platelet volume

compared to healthy individuals. (172) This suggests that the bone marrow is likely not suppressed and that

thrombocytopenia in CCHFV is due to increased activation and consumption rather than decreased production.
However, studies on bone marrow specimens show conflicting results, with some showing a normal marrow

with respect to megakaryocyte numbers and platelet production and others showing decreased megakaryocyte

numbers. (66, 173, 174) It remains unclear if platelets are activated or if they exhibit defective aggregation

responses during CCHFV infection.

The state of platelets during EBOV and MARV infections is unclear. Excessive coagulation activation

was thought to be the primary driver of thrombocytopenia during EVD and MVD for many years. This was

because very little data exists on platelet function during EVD and MVD in humans or NHPs. One study using

an NHP model of EBOV infection showed platelet aggregation defects in response to in vitro agonist

stimulation that coincided with increased levels of plasma platelet factor-4 in vivo. (117) These results suggest

increased platelet activation and degranulation during infection. Effects of EBOV on the bone marrow are

conflicting, with a singular report from an unspecified number of patients suggesting normal bone marrow

cellularity, including megakaryocyte number; in contrast, a more recent and thorough investigation on the bone

marrow from NHPs infected with EBOV suggests EBOV can infect megakaryocytes and induce bone marrow

suppression. (136, 175)

Mechanisms underlying thrombocytopenia in YFV and RVFV infections remain to be elucidated.

Infection with the hemorrhagic fever viruses induces endothelial cell activation and

increased vascular permeability

HFV infection leads to EC activation, which is broadly characterized by a loss in anti-thrombotic properties,

expression of pro-coagulant, pro-adhesive, and pro-inflammatory molecules, and increased vascular

permeability. This can occur directly through viral infection, by the action of specific viral proteins, or through

inflammatory mediators released in response to viral infection. These mechanisms likely co-occur for many of

the HFVs and can lead to disrupted barrier function (i.e., the loosening of junctions between cells), increased

vascular permeability (the loss of EC barrier function in vivo), and increased vascular leak (the leakage of cells

and proteins from the blood into the tissue).

 Regardless of whether ECs are direct targets or sustain any outward damage, infection with HFVs leads

to EC activation and disruption, marked by increases in the circulation of 1 or more of the following proteins:

vWF, soluble (s)-ICAM-1, s-VCAM-1, and s-P-selectin. Viral antigens from most of the HFVs have also been

found in ECs from different tissues, and all of them can replicate in cultured ECs. However, only some of the

viruses have evidence of replication in ECs in vivo (e.g., EBOV, MARV, CCHFV, HTNV, SNV, YFV, DENV)

and even fewer (e.g., MARV, RVFV, and DENV) are directly cytopathic in ECs in vitro. (8, 176-179) This

suggests there are alternative mechanisms leading to the observed increases in vascular permeability during

VHF.

How each virus alters ECs ultimately shapes the observed pathology. For example, severe HTNV

infection manifests as HFRS, and concordantly the strongest viral antigen staining is observed in the kidney,

particularly renal capillary ECs. (180) In contrast, SNV infection presents as HCPS, and the ECs lining the lung

capillaries and other small vessels stain the strongest for viral antigen. (8) Indeed, pathogenic

Orthohantaviruses utilize β3 integrins in the inactive conformation as entry receptors, including αIIbβ3 and αVβ3,

expressed by endothelial cells and platelets. (181) Importantly, neither HTNV nor SNV kill ECs directly but

drastically increase vascular permeability. (8, 180) Markers of EC activation (s-E-selectin, s-ICAM-1, s-

VCAM-1, and VEGF) and a marker of degradation of the EC glycocalyx (syndecan-1) are elevated in the

circulation in individuals with HFRS or HCPS (Table 3). (106, 182) In vitro work suggested several possible

mechanisms by which HTNV and SNV may increase vascular permeability. The first mechanism suggested that

virus binds to the surface of the endothelium and inhibits binding of host proteins to β3 integrins. β3 integrin

inhibition then leads to increased VEGF-induced vascular permeability in ECs. (183, 184) The second

mechanism builds on of the first and posits that increased VEGF combined with hantaviral inhibition of αVβ3

integrin, leads to internalization of VE-cadherin and a redistribution of the junctional protein ZO-1, resulting in

decreased barrier function. (15, 184-189) However, a later study using a more sophisticated in vitro capillary

system did not observe changes in VE-cadherin or VEGF levels. (190) Rather, in the third proposed

mechanism, increased permeability was the result of increased FXII binding and autoactivation and increased
kallikrein, which together led to increased bradykinin and increased vascular permeability. (190) Whether 1 or

any of these mechanisms occur in vivo remains unclear. However, all ultimately would lead to loosened EC

junctions, resulting in decreased barrier function in vitro and increased vascular permeability in vivo without

cell death.

For other viruses, such as EBOV, and likely MARV, ECs are targets late in infection but appear to have

minimal damage in vivo. (136, 191) The viral glycoprotein GP is known to induce EC activation and changes in

EC barrier function. (192-195) These changes are further enhanced by the presence of TNFα, which is present

in the blood at high levels during EBOV and MARV infections. (2, 196, 197) There are increased levels of

various markers of EC activation in the circulation, including s-thrombomodulin, s-P-selectin, s-PECAM-1, s-

ICAM-1, with some being associated with severe disease (s-thrombomodulin, s-P-selectin, s-PECAM-1, s-

ICAM-1), hemorrhage (s-ICAM-1, s-thrombomodulin) or death (s-thrombomodulin) (Table 3). (84, 85)

There is evidence for multiple mechanisms of decreased EC barrier function and increased vascular

permeability and leak in DENV and YFV infections. DENV NS1, a viral protein, induces decreased EC barrier

function directly. (198) While DENV NS1 reduced the barrier function of ECs of various origins in vitro, YFV

NS1 decreased the barrier function of only liver ECs. (199) Data suggests that both DENV and YFV NS1 also

induce glycocalyx disruption in vitro. (198, 199) Indeed, sydecan-1 is elevated in individuals with severe

DENV and YFV infection (Table 3). (107, 108) There is also evidence that DENV may replicate in ECs.

However, similar evidence for YFV is lacking. (176) Flavivirus NS2 and NS4 have also been suggested to have

viroporin-like activities, which may contribute further to increased permeability, but this remains to be

established. (200) Both DENV and YFV infections are associated with increased levels of various cytokines,

which can further enhance the vascular permeability induced by viral proteins and viral replication. (201, 202)

For CCHFV and LASV, there is histopathological evidence of outward EC damage, but it remains

unclear if this is virus- or immune-mediated. (13, 203) CCHFV activates ECs via infection in vitro in a dose-

dependent manner, and the viral protein GP38 decreased EC barrier function in vitro by disrupting the EC

glycocalyx. (204, 205) GP38 also increased vascular permeability and leak in vivo in a CCHFV mouse model.
(205) During CCHFV infection plasma levels of vWF and s-ICAM-1 are significantly elevated compared to

healthy controls (Table 3). (104) In a separate cohort, s-ICAM-1, s-VCAM-1, and VEGF were higher in

individuals that died from CCHFV infection compared to those who survived ( Table 3). (104, 105) LASV

infection is also associated with increases in plasma EC activation markers (e.g., vWF, s-ICAM-1, and s-

VCAM-1) in the plasma compared to non-LASV febrile controls or healthy controls ( Table 3). (80)

Additionally, in vitro data suggests LASV is not cytopathic to ECs. (80, 206)

Markers of EC activation (VEGF, s-PECAM-1, s-E-selectin) are moderately elevated in the circulation

of individuals infected with RVFV compared to healthy individuals (Table 3). (87) In RVFV infection, no

staining for viral antigen is observed in ECs from infected domesticated ruminant or mouse tissues, which

suggests an immune-mediated mechanism of EC activation. (207, 208)

While increased vascular permeability and leak contribute to the shock and multi-organ failure observed

in end-stage VHF, increased vascular permeability also causes cells underlying the vascular endothelium to

become exposed to the circulation. Importantly, some of these cells are a source of high levels of TF. When

exposed to the circulation at the scale occurring during VHF, this likely contributes to coagulation activation. In

addition, activation of ECs leads to down regulation of anti-coagulant proteins, exacerbating the

hypercoagulable state.

Phosphatidyl serine exposure and viral mimicry

PS is normally located on the inner leaflet of the cell membrane. During damage or cellular death, PS is flipped

to the outer membrane. Normally, this serves as an “eat me” signal to phagocytes and results in clearance of the

damaged cells. (209) In addition to dying cells, activated platelets also have increased PS exposure. (209) PS

also contributes to coagulation as PS binding to TF leads to a conformational change in TF that enhances the

pro-coagulant activity of the TF/FVIIa complex. (210) Additionally, PS provides a surface for the assembly and

function of coagulation proteases/cofactor complexes. (48)

During viral infection, both dying cells and activated platelets are important sources for PS.

Additionally, PS may be incorporated into both the viral envelope of new virions and into EVs released from
PS-positive cells. Indeed, EBOV, MARV, LASV, YFV, and DENV have all been shown to incorporate PS into

their viral envelopes, facilitating viral entry into host cells using receptors that bind PS (e.g., T cell

immunoglobulin and mucin receptor family protein and TYRO3–AXL–MER (TAM) family proteins). (211)

This mechanism of entry not only increases the ability of the virus to enter phagocytes, a preferred target of

infection for many HFVs, but may also promote coagulation. However, coagulation activation directly by viral

particles remains to be established for HFVs as has been done for other viruses (Cytomegalovirus and Herpes

Simplex Virus-1 and -2). (135, 212).

Fibrinolysis during viral hemorrhagic fever

Dysregulation of the fibrinolytic system may contribute to bleeding in VHF. Indeed, D-dimer is elevated to

some extent in all VHFs considered herein. Additionally, tPA is elevated in humans or NHPs infected with

EBOV, LASV, RVFV, SNV, and DENV, and levels of plasmin anti-plasmin complexes are elevated in

individuals with severe DF (Tables 3-4)(2, 15, 62, 63, 66, 67, 70-74, 79, 81-91, 98, 102, 110-112, 117, 119-

122, 73) However, PAI-1 is also increased in humans with the same viruses, which may limit the activation of

the fibrinolytic system by tPA (Table 3). (72, 73, 80, 84, 86, 87, 102, 103)

To better understand the kinetics of when these elevations occur, animal models are essential. In a NHP

model of EBOV infection, D-dimer begins to rise at 1-day post-infection and continues to increase over the

course of infection. Both tPA and uPA increased by day 4 post-infection. (98) Unfortunately, PAI-1 could not

be measured in this model because the available assays were incompatible with NHP samples. (98) In 2 other

studies using transcriptomics and a multiplex bead-based assay on whole blood from NHPs infected with a

different EBOV strain, PAI-1 and tPA both increased. (123, 213)

Very few studies have examined biomarkers of fibrinolysis other than D-dimer or total PAI-1. While

these biomarkers can suggest increased fibrinolysis, other biomarkers such as plasmin anti-plasmin complexes

or tPA/PAI-1 complexes, may be better metrics of fibrinolytic activity. As such, it is presently unknown

whether fibrinolytic dysregulation contributes directly to bleeding in VHF. Thus, more studies on the

fibrinolytic system in both humans and NHPs are needed.

Summary and perspectives

VHF is a catch-all term, encompassing several different diseases caused by viruses from distinct families.

Bleeding is the symptom for which the term VHF was coined, but bleeding is not necessarily a feature in all

cases. Regardless, bleeding is a severe manifestation of the spectrum of diseases caused by HFVs. Herein, we

propose some potential common mechanisms underlying bleeding in VHF (Figure 3). Viral infection activates

coagulation via inducing increased TF expression on infected cells. The activity of the TF/FVIIa complex is

further enhanced by increased PS exposure, resulting in even more coagulation activation. It is possible that the

intrinsic pathway also contributes to the activation of coagulation in VHF. In parallel, there is decreased clotting

factor synthesis by the liver due to damage during infection. Activation of platelets and decreased platelet

production lead to thrombocytopenia. In addition, platelet dysfunction occurs in VHF. Viral infection also

activates ECs, causing decreased activity of the activated protein C anti-coagulant pathway, conversion to a

prothrombotic surface, and increased vascular permeability. EC activation also leads to increased tPA release,

which ultimately results in increased plasmin activity and enhanced fibrinolysis. We propose that a combination

of these pathways leads to bleeding during VHF.

The pathways highlighted above are based on data primarily from individual viruses, as a complete

picture for any singular virus is missing. Thus, it still remains unclear if the mechanisms discussed herein

extend to all the HFVs. Some pathways, such as fibrinolysis, also remain largely unexplored. More studies are

needed to better understand the complete picture of the hemostatic dysregulation during VHF caused by

different viruses. While human biomarker data has been helpful in opening avenues of investigation, human

data alone at a singular time point after an undefined amount of time after infection cannot provide the full

picture. In vitro data also cannot capture the complex interplay of the coagulation and immune systems. Thus,

animal models have been and will continue to be essential in advancing our understanding of mechanisms

underlying bleeding in VHF. A better understanding may open new therapeutic avenues to treat and prevent

bleeding during VHF.

ARTICLE INFORMATION
Affiliations

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill (M.V.P.). UNC

Blood Research Center, Department of Medicine, University of North Carolina at Chapel Hill (M.V.P., N.M.).

Division of Hematology, Department of Medicine, University of North Carolina at Chapel Hill (N.M.).

Acknowledgements

None.

Sources of funding

This work was supported by funding from the National Institutes of Health National Heart, Lung, and

Blood Institute R35HL155657 (N.M) and the John C. Parker professorship (N.M), and the American Heart

Association 25PRE1375748 (M.V.P.).

Disclosures

None.

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Highlights

• Viral hemorrhagic fever is a term collective referring to several different diseases caused by

various viruses.

• Both direct effects from infection and the host immune response contribute to hemostatic

dysregulation.

• We discuss herein the potential mechanisms underlying bleeding in viral hemorrhagic fever,

including increased tissue factor expression, thrombocytopenia, platelet dysfunction, decreased

coagulation factor synthesis, and endothelial cell activation and damage.

Table 1. Information on the viruses that cause viral hemorrhagic fever.

EBOV MARV LASV RVFV CCHFV HTNV SNV

Arenaviridae, Nairoviridae, Hantaviridae, Hantaviridae,
Virus family, Filoviridae, Filoviridae, Phenuiviridae,
Mammarenavi Orthonairovir Orthohantavir Orthohantavir
genus Ebolavirus Marburgvirus Phlebovirus
rus us us us
Hepatocytes
Primary Hepatocytes Endothelial Endothelial
Neurons
cellular targets Myeloid (1) Myeloid (2, 3) Myeloid (4) Endothelial Myeloid (7) Myeloid
Myeloid
of the virus (6) (7, 8)
(5)
Liver, spleen, Liver, spleen,
secondary secondary
Organ(s)
lymphoid lymphoid Liver, spleen
primarily Liver (14) Liver (6) Kidney (7) Lung (15)
organs, others organs, others (12, 13)
affected
later in later in
infection (1) infection (3)
Geographic Sub-Saharan Sub-Saharan Western Africa, Saudi Africa, Middle Asia (7, 20) North America
distribution Africa (1) Africa (3) Africa (17) Arabia, Yemen East, Southeast (7, 20)
(18, 19) Asia,
Southern/Easte
r Europe (6)
 Contact with
infected Contact with
Contact with
rodent/rodent infected
infected
secretions Mosquito bite, Tick bite rodent/rodent
Contact with Contact with rodent/rodent
excretions contact with secretions
Mechanism of infected bodily infected bodily secretions
(e.g., urine, infected bodily Contact with excretions
transmission fluids/tissues fluids/tissues excretions
feces) fluids/tissues infected bodily (e.g., urine,
(1) (3) (e.g., urine,
(19, 21) fluids (6) feces) (7, 20)
feces) (7, 20)
Contact with
infected bodily
fluids (17)

Culex and Apodemus Peromyscus

Hyalomma
Vector N/A N/A N/A Aedes spp. agrarius (7, maniculatus
spp. (6)
(19, 21) 20) (7, 20)

Aedes spp.,
rodents, wild
Unknown – Rousettus Apodemus Peromyscus
Multimammat ruminants, bats Hyalomma
Reservoir host bats suspected aegyptiacus agrarius (7, maniculatus
e rat (17) (19) tick (6)
(1) (23) 20) (7, 20)

EBOV, Ebola virusCCHFV, Crimean Congo hemorrhagic fever virus; MARV, Marburg virusDENV, dengue virus; LASV, Lassa

fever virusEBOV, Ebola virus; RVFV, Rift Valley fever virusHTNV, Hantaan virus; CCHFV, Crimean Congo hemorrhagic fever

virusLASV, Lassa fever virus; HTNV, Hantaan virusMARV, Marburg virus; SNV, sin nombre virusRVFV, Rift Valley fever virus;

YFV, yellow fever virusSNV, sin nombre virus; DENV, dengue virusYFV, yellow fever virus

Table 2. Case presentation of viral hemorrhagic fever caused by different viruses.

EBOV MARV LASV RVFV CCHFV HTNV SNV YFV DENV

Variable Variable Variable
Global Estimated Estimated
– usually – usually and Estimated >100,000,
incidence 100,000- 10,000- 200,000
small small outbreak >10,000 30 (20) 000**
(cases/year 300,000 15,000** (10)
<300* <300 (3, dependent (20, 27) (11)
) (17) (26)
(3, 24) 25) (18)
Case
fatality 60 (24) 50 (28) 1 (29) <1 (30) 5-40 (31) ~1 (7, 20) 35 (20) >20 (10) <1 (11)
rate (%)
Cases 60-85 - 20 (17, <5 (30) 12 (33) ~20 (34) >50 (35) 12 (36) 0.5-5 (11)
having
severe
(32) 29)
manifestati
ons (%)
Cases
having
<50 34-83 <30
hemorrhag 40 (17,
(variable (variable) <1 (30) 3.6-6 (33) (variable) N/A 12 (36) 0.5-5 (11)
ic 29)
) (37) (38) (34, 39)
manifestati
ons (%)
Case
fatality
rate for
30-60
cases with 15-70
- - <50 (30) - 5-15 (7) >50 (35) (36, 40, Variable†
hemorrhag (17, 29)
10)
ic/severe
manifestati
ons (%)
Outward Maculop See Bleeding Macular Petechiae Petechiae No Jaundice Petechiae
signs of apular EBOV. from the rash outward
hemorrhag rash mouth, signs of Liver and Bruising
ic disease nose, bleeding renal
rectum, Bruising Bruising Bruising dysfuncti Bloody
and (7, 20) on vomit/sto
vagina ol
Petechiae
Bloody
conjuncti (16)
Petechia Blood Noseblee va Bruising
e gums, d
(17) stools, or Bloody
Bleeding vomit Coughing gums or
/bruising up blood Bloody vomit
stools,
vomit, or (9, 10)
(21) urine

Bloody (7, 20)

Bloody stools,
vomit/sto vomit, or
ol urine
 (33)
Bloody/
red
conjuncti
va

(3, 37)

Approved Only for

Yes (36,
vaccine Yes (41) No (25) No (42) livestock No (6) No (20) No (20) No‡
9, 10)
available (14, 19)

* The 2013-2016 outbreak was the largest on record (>28,000 cases).

** Many cases are asymptomatic, making true incidence difficult to estimate.

†Mortality for severe dengue can be up to 20 percent, but with proper supportive care, the mortality can be as low ~1 percent (11).

‡ There are 2 approved vaccines and 1 vaccine in phase 3 trials – however, the safety and efficacy of such vaccines is hotly debated
(16, 43).

EBOV, Ebola virusCCHFV, Crimean Congo hemorrhagic fever virus; MARV, Marburg virusDENV, dengue virus; LASV, Lassa
fever virusEBOV, Ebola virus; RVFV, Rift Valley fever virusHTNV, Hantaan virus; CCHFV, Crimean Congo hemorrhagic fever
virusLASV, Lassa fever virus; HTNV, Hantaan virusMARV, Marburg virus; SNV, sin nombre virusRVFV, Rift Valley fever virus;
YFV, yellow fever virusSNV, sin nombre virus; DENV, dengue virusYFV, yellow fever virus
Table 3. Hemostatic-related parameters and biomarkers in humans infected with different viruses causing viral

hemorrhagic fever.

Parameter EBOV MARV LASV RVFV CCHFV HTNV SNV YFV DENV
Increase
Increase Increase Increased Normal** Increase Increased Normal
PT - d *†
d (56) d (61) * (62-66) (67, 68) d (8, 15) (69, 70) (71-73)
(57-60)
Increase Increased
Increase Increase Increase Increased* Increase Increased Increased
aPTT d (74- * (62-66,
d (56) d † (60) d (61) * (67, 79) d (8, 15) (70) (71-73)
76) 77, 78)
Normal Increase Increased
TAT - - - - - -
(80) d (81)** (73, 82)
D-dimer/FDP Increase Increase Increase Increase Increased Increased* Increase Increased Increased
 * (62, 63,
d (83- d (87, d (15, (71, 73,
d (74) d (86) 66, 89, * (67, 79) (70, 91)
85)** 88) 81) 82)
90)
Yes (62,
No* (57, Yes(61,
Thrombocytopeni Yes(56, Yes(74- 65, 66, Yes** (67, Yes (15, Yes (69, Yes (72,
59, 60, 88, 94,
a 92) 76) 77, 78, 68, 79) 96)** 70) 73, 97)
93) 95)
89, 90)
Increase Increase Increase Increased
TF - - - - -
d (98)** d (80) d (81) (99-101)
Increase Increase Increase Increase Increased
tPA - - - -
d (84) d (86) d † (87) d (102) (72, 73)
Increase Increase
Normal Increased
PAI-1 - - d (80, - - d (102, -
† (87) (72, 73)
86) 103)
s-TM
s-TM s-ICAM-
Elevated EC s-ICAM-1
s-ICAM- 1
activation or EC s-vWF (104,
1 s- s-VEGF Syndecan Syndecan
glycocalyx - s-VEGF 105) -
s-P- VCAM- (106) -1 (107) -1 (108)
disruption (87) s-vWF
selectin 1
markers (104)
(85) s-vWF
(80)

* Minority of studies show normal values or opposite result

**Not all separated by viral strain/serotype

† Limited patient number

‡ Modest changes

PT, prothrombin timeaPTT, activated partial thromboplastin time; aPTT, activated partial thromboplastin timeEC, endothelial cell;
TAT, thrombin anti-thrombinFDP, fibrin degradation product; FDP, fibrin degradation productICAM-1, intercellular adhesion
molecule-1; TF, tissue factorPAI-1, plasminogen activator inhibitor-1; tPA, tissue plasminogen activatorPT, prothrombin time; PAI-1,
plasminogen activator inhibitor-1s, soluble; EC, endothelial cellTAT, thrombin anti-thrombin; s, solubleTF, tissue factor; TM,
thrombomodulin; vWF, von Willebrand factortPA, tissue plasminogen activator; ICAM-1, intercellular adhesion molecule-1VEGF,
vascular endothelial growth factor; VEGF, vascular endothelial growth factorvWF, von Willebrand factor
Table 4. Hemostatic-related parameters and biomarkers in non-human primates infected with different viruses

causing viral hemorrhagic fever.

Parameter EBOV MARV LASV RVFV CCHFV HTNV SNV YFV DENV**
Increase
Increased Increased Increased Increased* Increased
PT - - d (91, -
(109) (2) (110, 111) (112-114) (115)
116)
Increased
Increased Increased Increased Increased Increased Increase
aPTT (109, - -
(2) (110, 111) (112-114) (118) (115) d (116)
117)
TAT - - - - - - - - -
Increased
Increased Increase
(98, 117, Increased Increased Normal
D-dimer/FDP (110, 111, - - d (91, -
119, (2, 121) (112) (115)
122) 116)
120)
Yes (98,
109, No* (2, No* (110,
Thrombocytopeni Yes (112, Yes (118, Yes
117, 121, 111, 122, - No (115) -
a 114) 127) (116)
120, 124) 125, 126)
123)
Increased
Increased
TF (98, 119, - - - - - - -
(121)
128)
Increased
tPA - - - - - - - -
(98, 123)
Increased
PAI-1 - - - - - - - -
(123)

*Represents consensus of the majority of studies

** Does not cause overt clinical manifestations in non-human primates

PT, prothrombin timeaPTT, activated partial thromboplastin time; aPTT, activated partial thromboplastin timeFDP, fibrin degradation

product; TAT, thrombin anti-thrombinPAI-1, plasminogen activator inhibitor-1; FDP, fibrin degradation productPT, prothrombin

time; TF, tissue factorTAT, thrombin anti-thrombin; tPA, tissue plasminogen activatorTF, tissue factor; PAI-1, plasminogen activator

inhibitor-1tPA, tissue plasminogen activator;

Table 5. Disseminated intravascular coagulation scoring criteria according to the International Society on

Thrombosis and Hemostasis.

 Parameter Values Score

<50 2
Platelet count (x 109/L)
50-100 1

Increased ≥6 2
Prothrombin time (seconds)
Increased 3-6 1

Fibrinogen (mg/dL) <100 1

Strong increase 3
D-dimer
Moderate increase 2

Figure 1. Overview of the phylogenetic relationship between viruses causing viral hemorrhagic fever. An

abbreviated phylogenetic relationship of the different viruses causing viral hemorrhagic fever is shown by virus

family with each individual species shown below. Figure made with [Link].

Figure 2. Geographic distribution of the different hemorrhagic fever viruses. Inverted triangles colored

coded to represent each individual virus are shown on the map where the virus is known to circulate: Ebola

virus (EBOV, pink), Marburg virus (MARV, red), Crimean Congo hemorrhagic fever virus (CCHFV, purple),

Rift Valley fever virus (RVFV, orange), yellow fever virus (YFV, dark blue), dengue virus (DENV, light blue),

Hantaan virus (HTNV, dark green), sin nombre virus (SNV, light green), and (LASV, yellow green). The

vectors and/or reservoirs for each virus are also shown, as the distribution of the viruses follows that of their

vector and/or reservoir.

Figure 3. Potential mechanisms underlying bleeding during viral hemorrhagic fever. Viral infection

induces tissue factor (TF) expression in host cells, resulting in coagulation activation and consumption of

clotting factors. Liver infection leads to decreased production of clotting factors. Coagulation activation and

viral infection lead to platelet activation and clearance, resulting in thrombocytopenia. Viral infection also

activates endothelial cells (ECs), leading to increased vascular permeability and decreased protein C anti-

coagulant activity. Activated ECs also release tissue plasminogen activator (tPA), resulting in increased plasmin

and enhanced fibrinolysis. Cumulatively, these mechanisms result in bleeding during viral hemorrhagic fever.

For each potential mechanism, a colored dot corresponding to each virus that has evidence supportive of that
mechanism is shown: Ebola virus (EBOV, pink), Marburg virus (MARV, red), Crimean Congo hemorrhagic

fever virus (CCHFV, purple), Rift Valley fever virus (RVFV, orange), yellow fever virus (YFV, dark blue),

dengue virus (DENV, light blue), Hantaan virus (HTNV, dark green), sin nombre virus (SNV, light green), and

(LASV, yellow green). APC, activated protein C; TM, thrombomodulin; EPCR, endothelial protein C receptor;

PT, prothrombin time; aPTT, activated partial thromboplastin time. Figure made with [Link].