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Award Title: Biology BSc (Hons)

Student Registration Number: Date of Birth: 09/05/95

Module Code & Title: HUMAN BIOCHEMISTRY AND PHYSIOLOGY : BIOL50435

Assignment Description: Disease State Report

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Viral Haemorrhagic Fever.

An insight into the biochemical and physiological dysfunctions which are commonly associated.

Viral Haemorrhagic fever (VHFs) is a name given referring to a group of illnesses that are caused by several families of viruses, this term is used to describe a severe multisystem syndrome in which many organs within the body are effected. There are four main taxonomic families of viruses which may cause a viral haemorrhagic fever, these are represented in figure 1.

Virus Family; (Viruses within Family) Fatality rate Filoviridae; (Ebola and Marburg viruses)

Ebola: 50% to 90%, Marburg: 23% to 70%

Arenaviridae; (New World haemorrhagic fever and Lassa fever)

New world haemorrhagic fever: 15% to 30%,Lassa fever: >1%

Bunyaviridae; (Rift Valley fever, Crimean-Congo fever)

Rift Valley fever: 14% to 17%Crimean-Congo fever: 10% to 40%

Flaviviridae; (yellow fever, Omsk haemorrhagic fever, Kyasanur Forest disease)

Yellow fever: >50%Omsk haemorrhagic fever: 0.5% to 3%Kyasanur Forest disease: 3% to 10%

As you can see, depending on the responsible Virus the severity, with regards to fatality rate, varies from up to 90% to lower than 1%. The reason for this requires an understanding of the different challenges that each virus introduces to the human body once an infection has occurred. Depending on the causative virus, the symptoms and clinical features that occur within the body vary. The severity of the altercations to the body’s normal biochemistry and physiology generally correlates to the virus’s fatality rate. Due to the high fatality rate I will focus on the Ebola virus; the causative virus of Ebola Haemorrhagic Fever (EHF), and the challenges the human body is presented with upon and during an infection.

General Clinical features associated with (EHF).Clinical Features Laboratory findings due to EHF

vomiting, headache, prostration, fatigue (< 1 week)

Chest pain, Diarrhoea, Acute onset of fever, cough, pharyngitis

Maculopapar rash Haemorrhagic manifestation Pancreatitis, hepatitis, conjunctival

inflammation CNS dysfunction Shock with DIC and organ failure ( < 7

days after symptoms begin) Sequelae: ocular disease, hearing loss,

pericarditis, arthralgia

Leukopenia Leukocytosis Thrombocytopenia Elevated liver enzymes Elevated amylase Lab features of DIC

It is necessary to note that there are 5 species of Ebola virus; Zaire, Bundibugyo, Sudan, Reston and Taï Forest. Depending on the species the symptoms may vary, so to avoid confusion this report will concern the general biochemical and physiological dysfunctions that may occur. EHF may last for up to 21 days, after the 4-10 day incubation period, a multitude of biochemical and physiological altercations can occur during this time period (see figure 2), however detrimental effects post-infection have been reported by Formenty (1999) to last up to 3 months.

FIGURE 1. Represents viruses responsible for causing viral haemorrhagic fever with corresponding fatality rate. (S.F. Dept Public health, 2008).

According to Lee & Sapphire (2009), Symptoms of EHF generally begin to occur within a 4-10 day incubation period, however in fatal cases, death can occur as early as 6-9 days after onset as a result of uncontrolled viral replication and multiple organ failure. To understand how some of the symptoms within table 2 become apparent within the human body, it is necessary to understand the initial stages of EHF. Many studies have been conducted to determine the initial target cell of the Ebola virus (EBOV), and according to Ströher et al (2001) morphological studies on post-mortem material suggests that the primary target for viral replication are; dendritic cells and macrophages via CD4 and CD8 receptors. These cells are then thought to circulate via the lymphatic system to regional lymph nodes where viral replication can continue, and the target cell-associated virions are able to spread. However EBOV is able to effect other cells indirectly, the virus does this by using infected cells almost like a biochemical weapon, to cause dysfunction to cells which don’t appear to be adequate hosts.

Lymphocytes are not directly infected by EBOV however, studies indicate that lymphocyte apoptosis is a prominent feature of infection (Bradfute et al, 2010). It is therefore hypothesised that factors secreted or expressed by target cells are responsible for triggering the cell death within lymphocytes. Evidence supporting this hypothesis can be derived from a study conducted by Geisbert et al (2003), data obtained from this study suggest that dendritic cells and macrophages increase the expression of tumour necrosis factor-α (TNFα) (see figure 3), as well as apoptosis-inducing ligand and Fas transcript during an EHF infection. This data is supported by several other reports stating similar results (Formenty et al, 1999), and may help to explain why Leukopenia typically occurs during the early stages of EHF. As a result of this the innate immune response may become dysfunctional, leaving the host with little time to adaptively respond to the infection. Although damage focuses within the lymphatic system initially, it is also stated by Geisbert el al (2003a) that when EBOV enters the blood system, the virus exhibits tissue tropism for the; liver, adrenal gland and spleen.

The Liver appears to be an important target of EBOV, data obtained by Geisbert et al (2003) suggests that Kupffer cells are an adequate host cell thus; viral replication continues within the Liver. Initial infection of the liver occurs within the early stages of the infection causing damage directly to hepatocytes and other cells found within the liver. Reports have shown that enzymes; Aspartate aminotransferase, and Alanine aminotransferase are both elevated (see figure 4). This supports that the virus causes damage to liver tissue either directly via replication or as a result of a biochemical imbalance due to factors released upon cell lysis.

As a result of the damage it may be difficult for the liver to continue its function both biochemically and physiologically, the liver plays vital roles within a wide range of systems within the body, therefore progressive liver dysfunction can be catastrophic to the body’s normal homeostatic procedures (see figure 5).

FIGURE 3. Displays the increase of Tumour Necrosis Factor during an EHF in Cynomolgus macaques, data obtained by Geisbert et al, (2003a).

POSSIBLE BIOCHEMICAL DYSFUNCTION ASSOCIATED WITH LIVER DAMAGE; WITH RELEVANCE TO EHF.

EFFECTS ON BODY DUE TO DYSFUNCTION.

- INADEQUATE SECRETION OF PRECURSOR MOLECULE ANGIOTENSINOGEN.

- As the precursor to hormone Angiotensin, inadequate amounts of angiotensinogen may inhibit the vasoconstriction process thus preventing vasoconstriction; a process crucial for raising blood pressure and slowing down haemorrhage/acute blood loss. This may play a role in the amplification of the haemorrhagic feature associated with this infection.

- Inadequate secretion, via the liver, of glycoprotein hormone; thrombopoietin.

- Responsible for stimulating Megakaryocytopoiesis, an inadequate amount of thrombopoietin may lead to a reduced platelet production thus directly effecting thrombus formation; causing thrombocytopenia and massive altercations to the clotting cascade.

- Inability to secrete/synthesize sufficient quantities of glycogen

- Insufficient quantities of Glycogen may cause the body to enter a hypoglycaemic state. This dysfunction would render the liver useless an unresponsive within the feedback systems implemented for blood glucose homeostasis.

FIGURE 4. Biochemical values during EHF; note elevated AST and ALT. ALT-Alanine aminotransferase, AST-Aspartate aminotransferase in comparison to reference standard. Data obtained by Formenty et al, (1999)

- DISRUPTION TO THE LIVER NORMAL METABOLIC PATHWAYS

- Inability for the liver to carry out its normal metabolic functions is massively detrimental to the body, as a result of such dysfunctions many systems within the body would be effected. In particular the Central Nervous System would not receive sufficient nutrients to support function thus causing CNS dysfunction.

In relation to the liver, it has been reported that numerous organs are manifested with EBOV during EHF, for example Geisbert et al (2003) constructed a graph, shown in figure 6, which represents such activity.

In relation to the liver, the adrenal gland appears to be directly affected by EHF, data obtained by Geisbert et al, (2003a) indicates that on day 4 of an infection EBOV was detected in adrenal cortical cells within the zona fasciculate and zona reticularis. EBOV was also detected within stroma cells in the adrenal cortex and within fibrocytes in the adrenal capsule, this then further developed into

FIGURE 5. Shows examples of biochemical dysfunctions that may be caused by liver damage; an organ that is largely damaged during EHF.

FIGURE 6. Shows mean infectivity of 6 Cynomolgus macaques; note the multi-organ involvement. LN-Lymph Node. (Geisbert et al, 2003a)

multifocal congestion of the adrenal cortex and multifocal degradation of the adrenal cortical cells (Geisbert et al, 2003a). It is know that the adrenal cortex plays an important role within the renin-angiotensin-aldosterone system which regulates blood pressure, figure 7 represents this feedback system.

Adrenocortical necrosis, as a result of EHF, has been reported in humans by Formenty et al, (1999), and in non-human primates by Geisbert et al, (2003a), thus rendering the organ unresponsive within the negative feedback system represented in figure 7a. As a result of such dysfunction the adrenal gland is be unable to synthesize sufficient aldosterone, this prevents the kidney from regulating Na+

and K+ and, as a result of this, ultimately chronic blood volume; and thus blood pressure (Booth et al, 2002). It is reported by Feldman (2011) that impaired secretion of aldosterone leads to hypotension and Na+ loss with hypovolaemia, therefore impairment of adrenocortical function could have an important role in the shock that typically occurs in later stages of EHF. There are many other dysfunctions that occur within the body, the coagulation cascade also seems to be largely effected during EHF.

A prominent manifestation of EHF is disseminated intravascular coagulation (DIC), this is the formation of microthrombi via activation of the clotting cascade. These microthrombi may restrict blood supply therefore may be a contributing factor to the multiple organ dysfunction characteristic of EHF. In fact pathological data obtained by Gando (1999) suggests that fibrin deposition is a factor that contributes to multiple organ failure. However before acute DIC can become apparent there must be sufficient stimulus to overwhelm the natural anticoagulation processes (Feldmann & Geisbert, 2011). Anticoagulation factors which are mainly synthesized primarily within the liver Parenchymal cells, which have most likely already been damaged at this point due to viral assault, act to inhibit the formation of microthrombi, however if the rate at which these factors are consumed exceeds rate of synthesis; levels of inhibitors within plasma will decline (Feldmann & Geisbert, 2011). For example Geisbert et al (2003a) reported that activated Protein C, an important substance which prevents thrombus formation, concentration drops significantly during EHF (see figure 8); this goes to show that the virus is able to overcome the body’s natural anticoagulation systems.

During DIC, the extrinsic coagulation pathway, which is Tissue Factor (TF) dependent, is the dominant route for thrombin production and thus fibrin deposition (Geisbert et al, 2003b). The factor that induces DIC within EHF is most likely an increase of TF, the major initiator of the coagulopathy witnessed within EHF, however in order to explain how this occurs we need to begin with the initial target of EBOV; monocytes/macrophages. Data obtained by Geisbert et al, (2003b) suggests that the expression of TF was only observed in monocytes/macrophages that show

FIGURE 8. Shows concentrations of Protein C during EHF, note the concentration only returns to 60% when compared to pre-infection concentration. Data obtained by Geisbert et al, (2003b).

FIGURE 9. Represents the increase of TF during EHF. Indirect fluorescent antibody analysis was performed for TF from peripheral blood mononuclear cells of an EBOV-infected rhesus macaque at day 2 (B) and at day 5 (C) (Geisbert et al, 2003b).

evidence of EBOV replication, thus suggesting that expression of TF may be directly increased by EBOV infection (Geisbert et al, 2003b).

As you can see in figure 9, the concentration of TF within the blood increases during the progression of an infection, this is hypothesized by Geisbert et al (2003b) to be an important factor in the diagnosis of DIC within EHF. DIC results in widespread activation of the clotting cascade and thus the formation of multiple fibrin thrombi within small vessels throughout the body.

As a result of this, blood flow is restricted throughout the body and organs receive inadequate blood supply, this then leads to and/or hemorrhagic shock, which appears to be a lot like septic shock, multiple organ failure and finally death of the host (Geisbert et al, 2003a); all features which are reported by S.F. Dept Public health (2008) in figure 2.

Around day 4-5, after incubation, of EHF it may become clear whether the host is more likely to survive the infection, production of antibodies, or whether the infection will be fatal, progression to hypercytokinemia, referred to as the “cytokine storm”; possibly the final event of EHF. It is necessary to note that this may not occur in all cases and dysfunctions which precede this stage may be a significant factor in the death of the host. Like many of the symptoms associated with Ebola, they mostly all originate from dysfunctions caused to the primary target cells. According to Pérez et al (2014), macrophage cells are mainly responsible for the increased secretion of anti and pro- inflammatory cytokines during EHF, this report also indicates that EBOV glycoprotein (GP) are the likely route used for activation of the cytokine cascade via macrophages, this is shown in figure 10.

As you can see, endothelial cells appear to be affected due to the release of cytokines which are activated and secreted due to EBOV GP. It has been reported by Wahl-Jensen et al (2005) that EBOV

FIGURE 10; Role of shed GP during EHF. It is reported further by Pérez et al (2014) that EBOV GP are secreted from infected cells, as well as replicated virions, these GP then bind to further cells using TLR-4, where they then activate the cells ability to secrete various cytokines. Image from Pérez et al (2014).

causes a deregulated inflammatory cytokine response, which is responsible in the decreased endothelial cell barrier function associated with this infection.

In fact studies have shed further light on this area, Geisbert et al (2003c) emphasized the involvement of Tumor Necrosis Factor-α (TNFα) within the loss of vascular integrity. TNFα is now considered to be the major cytokine responsible for increased permeability of vascular tissue, and data acquired by Geisbert et al (2003c) supports this. In relation to this EBOV GP is able to infect endothelial cells directly, this produces cytopathic effects and direct damage to the cells barrier, via viral replication. This accompanied by the cytokine effects on endothelial cells leads to the loss of vascular integrity, and as a result large quantities of small breaks occur throughout the circulatory system amplifying; and further reducing blood pressure. In contrast to this pressures, due to fluid leakage, within organ cavities may increase, causing excessive stress to organs which, at this stage, may already be dysfunctional, causing further damage and amplifying physiological damage. In addition to this it has been reported by Geisbert et al (2003c) that within endothelial cells, small molecular weight protein Albumin shows a significant decrease in value when the cell become infected (see figure 11), causing a vast net decrease of Albumin throughout the body. Since it is know that this protein has a major function in the control of colloid osmotic pressure, an excessive reduced amount would reduce plasma osmotic pressure, this dysfunction could then result in edema. Further swelling to the body could induce further CNS dysfunction and possibly lead to permanent physiological dysfunction of organs such as the brain (Geisbert et al, 2003c).

It is also thought that during the late stages of EHF, Nitric oxide (NO) levels begin to elevate (see figure 12), possibly as a response to the heavy virus load or may result from cytokine-induced overexpression of inducible NO synthase in phagocytic cells throughout the body (Sanchez et al. (2004). An increase of NO within the blood can cause some serious physiological dysfunctions, for example Sanchez et al (2004) reported that fatal cases, which exhibit high NO concentrations, are associated with cardiac stress and heart failure. It also suggested that elevated NO may contribute to the hypotension characteristic of EHF, as well as the possibility of this factor forming toxic molecule which may further stress the host.

To summarize EHF is capable of causing a wide range of biochemical dysfunctions to a host, which results in a wide range of physiological dysfunctions within the human body; and non-

Figure 12; Shows a comparison of fatal and non-fatal NO levels in peripheral blood of Ebola-S virus-infectedPatients. (Sanchez et al, 2004)

Figure 11; Shows total serum protein and albumin values post-infection in Cynomolgus monkeys. Note the decrease of protein Albumin. Data obtained from Geisbert et al, (2003c).

human primates (Geisbert et al, 2003a). Examples within this report are accompanied with many other dysfunctions that occur during EHF.

The initial attack appears to be on monocytes within the immune system, the virus is able to transcribe and synthesize various factors and proteins which allow it to recruit further cells for viral replication. In this process biochemical imbalances are created, causing other cells, such as lymphocytes, to become effected. Transportation, via the lymphatic system, to the spleen is the likely route used to enter the vascular system, where the virus is then able to affect organs such as the adrenal gland and the liver where necrosis occurs causing large amounts of physiological/biochemical dysfunction (Feldman et al, 2011). As a result of this further organs such as the kidneys may become effected indirectly by even further biochemical imbalances due to necrosis within other organs. For example the kidneys inability to regulate K+ and Na+ due insufficient Aldosterone synthesis within the zona glomerulosa of the adrenal gland, subsequently causing hypotension Na+ loss. In fact Geisbert et al, (2003a) found that almost all organs are effected either directly or in-directly, this highlights the level of dysfunction, both physiological and biochemical, which occurs during EHF. DIC also occurs which results in inadequate blood supplies to most organs throughout the body, resulting in multiple organ failure and eventually death. With little treatment available, therapies only appear to rehydrate patients in a hope that their immune system will produce antibodies.

If the host survives during the multiple organ failure associated with EHF then the infection may progression into its preliminary phase, which presents a massive cytopathic attack on numerous cells throughout the body. This is combined with a body wide activation of mass cytokine secretion, causing considerate fluid loss and uncontrollable bleeding; death most likely follows this phase. Although activation of the cytokine cascade is an immune response which usually occurs during an infection caused by a new or particularly harmful pathogen; it also inflicts a large amount of damage to the host. In the unlikely situation that the host survives then subsequent conditions such as deafness and hair loss have been reported by Formenty et al, (1999).

So to truly conclude; it’s not the virus that kills the host, it’s the hosts own biochemical functions and immune responses which are hijacked during EHF that are the most likely cause of death.

References:

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