1. Introduction

Rift Valley fever phlebovirus (RVFV) is a significant veterinary and public health threat that has caused widespread outbreaks of disease in livestock and humans in most countries in Africa and since 2000 in the Arabian Peninsula, specifically Yemen and Saudi Arabia [1]. It is caused by a mosquito-borne RNA virus of the order Bunyavirales, family Phenuiviridae,. genus Phlebovirus [2]. During years of abnormally high rainfall, vast swarms of mosquitoes, mainly of the genera Aedes and Culex, emerge from standing water, and with sufficient numbers of susceptible unvaccinated livestock in the same area as RVFV-infected mosquitoes, epidemics commence [3]. Besides transmission by mosquito bite, exposure to blood and tissues of infected animals can transmit RVFV to humans ^[4][5][6][4–6].

Rift Valley fever (RVF) mainly affects sheep but cattle, goats, camelids, and other wild ruminants;, particularly African buffaloes, are also particularly susceptible ^[7][28]. The onset of disease in cattle and sheep is marked by the onset of pyrexia, and may be accompanied by anorexia, weakness, listlessness, a nasal discharge, diarrhoea and occasionally haematochezia and haematuria ^[8][29]. Peracute disease occurs in lambs and calves less than two-weeks-old, with estimated mortality as high as 90% to 100% in lambs and 10% to 70% in calves ^[9][30]. Mature livestock are significantly less susceptible to fatal disease with mortalities of approximately 10% to 30% in sheep and 5% to 10% in cattle ^[9][30]. In camels, RVF can cause abortions and neonatal deaths, whereas infected wild ruminants are usually clinically asymptomatic ^[9][10][30,31].

Epidemics are also characterised by near-simultaneous abortions of pregnant domestic ruminants, African buffaloes, and camels ^[8][11][12][29,32,33]. Whereas reports regarding abortions in cattle, camels, and African buffaloes infected with RVFV are scant, multiple studies have demonstrated a wide variety of outcomes for pregnant ewes and their foetuses ^{[12][13][14][15][16]}[32,34–37]. Large numbers of pregnant ewes abort (90%–100%) ^[9][30]. Ewes in the later stages of pregnancy may be more susceptible to lethal disease and die before aborting, whereas ewes in the earlier stages of pregnancy may abort or resorb the foetus and survive the infection ^{[12][13][17][18]}[32,34,38,39]. Occasionally, RVFV infected ewes show no clinical signs, do not seroconvert, do not have a detectable viraemia and lack lesions typical of RVF ^[13][34]. Similarly, in approximately 20% of ovine foetuses, the rapid progression of placental necrosis causes foetal mortality before foetal organs become infected ^[16][19][37,40]. Therefore, it may not be possible to rule out RVF in individual cases even if sampling is adequate.

Most human infections with RVFV present as an uncomplicated acute febrile illness. However, in a minority of patients, severe hepatic disease with haemorrhagic manifestations, renal impairment, encephalitis, and ocular lesions can complicate illness ^[20][41]. Additionally, RVFV can replicate in the syncytiotrophoblast layer of the human placenta and its vertical transmission correlates with an increased risk of miscarriage in humans ^{[20][21][22][23]}[41–44].

2. Aetiological Agent

Rift Valley fever virus is an enveloped, single-stranded RNA virus with only one serotype and multiple lineages ^[24][25][26][48–50]. The viral genome consists of three segments, large, medium, and small (L, M, and S) ^[27][51]. Similar to other arboviruses such as bluetongue and epizootic haemorrhagic disease virus, RVFV’s genome has low substitution rates, with genomic diversity largely driven by reassortment ^[25][49]. The L RNA segment codes for the viral RNA-dependent RNA polymerase while the M segment codes for the two envelope glycoproteins, carboxy-terminus glycoprotein (GC) and amino terminus glycoprotein (GN), as well as two non-structural proteins, NSm1 and NSm2 ^[28][52]. The S segment codes for the viral nucleocapsid protein (N) and the nucleus-associated non-structural (NSs) protein ^[27][29][51,53]. The virus’ NSs protein has many biological functions in infected cells, which include modulating the interferon response, facilitating efficient viral translation, and acting as a general inhibitor of transcription ^[30][72]. The NSm protein of RVFV suppresses apoptosis in target cells ^[31][80].

Dendritic cCell-sSpecific iIntercellular adhesion molecule-3-gGrabbing nNon-integrin (DC-SIGN) expressed on the surface of dermal dendritic cells possibly plays a critical role in the initial transmission of RVFV following an insect bite or mucosa exposure ^[32][61]. Heparan sulfate (HS) has also been shown to facilitate RVFV entry into cells ^[33][63] and liver/lLymph-sSpecific iIntercellular adhesion molecule-3-gGrabbing nNon-integrin (L-SIGN) might play a role in the liver tropism of RVFV ^[34][35][36][64–66]. However, L-SIGN is expressed on liver sinusoidal endothelial cells, not hepatocytes, and acts as an attachment/capture receptor rather than an endocytic receptor. Therefore, the details of RVFV entry into hepatocytes remain to be clarified.

Recent studies demonstrated that the cellular tropism of RVFV coincides with the distribution of class AI scavenger receptors (SCARA1) in tissues ^[37][38]. Following the initial transmission of RVFV by dendritic cells in the skin, SCARA1 might play a secondary role in further rounds of RVFV infection in other cells and tissues.

Recent studies demonstrated that the cellular tropism of RVFV coincides with the distribution of class AI scavenger receptors (SCARA1) in tissues [45,67]. Following the initial transmission of RVFV by dendritic cells in the skin, SCARA1 might play a secondary role in further rounds of RVFV infection in other cells and tissues.

3. Macro- and Microscopic Lesions and Virus Tropism in Natural Infections

3.1. Overview

Descriptions of the lesions in natural infections of RVF are similar in humans and ruminants except for encephalitis and retinitis that occurs in humans but has not been described in ruminants ^{[39][40][41][42][43][44][45]}[32,35,37,45–47,82–88]. While lesions can be seen throughout the body, multifocal, randomly distributed hepatic necrosis is the most commonly observed lesion in both ruminants and humans. Furthermore, detailed studies of statistically relevant numbers of sheep and observations in other ruminants reveal that lesions in neonatal ruminants (<1-month-old) and foetuses differ from those observed in adult ruminants, with the most prominent differences noted in the liver, kidneys, and lymphoid tissues ^[37][46][47][1,37,45,46,89]. While there is no clear explanation for these differences, they may be due to age-related susceptibility of the primary target cells of RVFV ^[47][1,37,45,46,89].

Immunohistochemistry based examination of the tissue and cellular tropism of RVFV in naturally infected sheep reveals viral antigen-positive hepatocytes, renal tubular epithelial cells, renal juxtaglomerular and extraglomerular mesangial cells, adrenocortical epithelial cells, cardiomyocytes, Purkinje fibres, skeletal muscle cells, epidermal keratinocytes, endothelial cells, vascular smooth muscle cells, tissue macrophages, and neutrophils ^[37][46][37,45,46].

In adult ruminants and neonates, lesions suggestive of vascular endothelial injury include mild hydropericardium, hydrothorax and ascites, marked pulmonary congestion and oedema, congestion and oedema of lymph nodes, and haemorrhages in many tissues ^[37][46][45,46]. Conversely, in foetuses, haemorrhages are minimal but plasma leakage with effusions in body cavities, accompanied by severe brain and lung oedema, is common ^[16][37]. Occasionally, the rapid progression of placental necrosis causes foetal mortality before other foetal tissues can be infected ^[19][40]. Notably, foetal malformations have not been reported in natural cases of RVF or in experimental cases using wild-type virus ^{[13][16][17][19]}[32,34,37,38,40]. Instead, ill-advised use of live attenuated vaccines in ruminants in the first or second trimester of pregnancy should be investigated if foetal malformations are reported ^{[48][49][50][51]}[90–93].

3.2. Liver

In adult cattle and sheep, innumerable petechia may be present on the parietal and cut surfaces of the liver and occasionally, there are mural haemorrhages and intraluminal blood in the gall bladder ^[37][43][32,35,45,86]. Histologically, foci of necrosis are situated irregularly throughout the lobule in adult ruminants and might involve up to two-thirds or more of the lobule.

In calves and lambs less than a month-old, pale pinpoint subcapsular petechiae and foci of necrosis may be present in the liver and virtually all hepatocytes undergo necrosis ^[46][32,35,46]. In foetuses, the liver usually does not have any discernible macroscopic lesions ^[16][37]. However, microscopically there is random dropout of hepatocytes from the reticulin framework which ranges from minimal to nearly diffuse hepatocyte necrosis.

In all age groups, injured hepatocytes oin all age groups often have features of apoptosis ^[46][39][32,35,37,45,46,82]. Features of early apoptotic cells (also referred to as Councilman bodies or acidophilic bodies) include dissociation of cells, cellular shrinkage and rounding, hypereosinophilic cytoplasm, pyknosis (nuclear chromatin condensation) and karyorrhexis (nuclear fragmentation) ^[37][45]. Early apoptotic bodies fragment into multiple smaller (late) apoptotic bodies that are eosinophilic fragments of cytoplasm of varying sizes, which may or may not contain nuclear fragments ^[37][45].

Amongst the Phenuiviridae, RVFV is unique in that the NSs protein forms ribbon-like filaments in the nucleus even though the virus replicates in the cytoplasm of host cells ^[29][53]. In haematoxylin-and-eosin-stained tissue sections these rod-shaped and eosinophilic intranuclear inclusions are a significant diagnosis-specific indicator ^[37][46][35,37,45,46].

A very distinctive feature in ruminant neonates and foetuses is discrete randomly distributed foci of liquefactive hepatic necrosis (also referred to as primary foci) against a background of diffuse hepatocellular death ^[39][40][35,37,46,82,83]. Primary foci vary in size and number and consist of lysed hepatocytes and infiltrating neutrophils and macrophages that undergo degenerative changes and likewise disintegrate. This results in myriads of small nuclear and cytoplasmic remnants within a collapsed reticulum framework.

The hepatic lesions in humans are similar to those in ruminants. In humans, foci of hepatic necrosis are associated with haemorrhage and involve the mid to central zones of the hepatic lobule, and often extend peripherally to the portal tracts ^[45][88]. Hepatic necrosis may also be diffuse ^[52][94]. Councilman bodies (early apoptotic bodies) and a mild inflammatory infiltrate of predominantly lymphocytes and macrophages with a few neutrophils, and a lack of bile stasis are also described in RVF hepatic pathology in humans ^[45][52][88,94].

In sheep, immunohistochemistry with an anti-RVFV nucleoprotein antibody reveals viral antigen in the cytoplasm of injured hepatocytes, and in cytoplasmic fragments within the sinusoids and central veins ^[37][46][37,45,46].

3.3. Kidney

Macroscopically, in the kidneys a few small cortical haemorrhages may be present [32,45,82]. Microscopically, adult sheep, cattle, and calves frequently have a severe nephrosis ^[53][54][32,35,45,95,96]. Conversely, in young lambs (< 1-month-old) and foetuses, the kidney lesion rarely progresses beyond tubular epithelial cell degeneration ^[46][39][32,37,46,82].

Similarly, in human patients that died from RVF, subcapsular renal haemorrhages, degeneration of proximal tubular epithelial cells, and a slight infiltrate of cells in the glomeruli are described ^[45][88]. Moreover, focal renal tubular epithelial necrosis with interstitial inflammation and intratubular casts were reported for a single human kidney sample ^[44][87].

In adult sheep, immunolabeling for RVF viral antigen is most often present in the renal cortex within glomerular and interstitial capillaries, tubular epithelial cells, and vascular smooth muscle cells ^[37][45]. In ovine neonates and foetuses, immunolabelling within blood vessels and capillaries is more extensive, with viral antigen also often present in the medullary interstitial capillaries ^[16][46][37,46]. Immunolabelling is also present at the vascular pole of the glomerulus opposite the macula densa, within a small group of cells that are likely juxtaglomerular and extraglomerular mesangial cells, also referred to as Lacis cells.

3.4. Adrenal Gland

In the adrenal glands, necrosis varies from individual cells to aggregates and is found predominantly in the zona fasciculata, although occasionally cells in the zonae glomerulosa and reticularis are also involved ^[37][46][39][35,37,45,46,82]. In sheep, irrespective of age, immunolabelling may be present in areas of necrosis or in single cells or small groups of apparently viable cells ^[37][45]. In ovine neonates and foetuses, viral antigen may also be present in capillaries in the periadrenal adipose tissues or in blood vessels in the capsule ^[16][46][37,46]. Lesions and viral antigen are absent from the adrenal medulla in all age categories in sheep.

3.5. Lymphatic Organs

Typically, RVFV-infected spleens are not enlarged and occasionally they have sub-capsular petechiae ^[12][37][42][32,45,85]. Occasionally, the spleen is also congested ^[39][40][82,83]. Microscopically, there are varying degrees of lymphocytolysis in the white and red pulp, generally giving specimens a paucicellular appearance ^[53][54][35,37,45,46,82,95,96]. Similar lesions have been reported in humans fatally infected with RVFV, including karyorrhexis and karyolysis of lymphocyte nuclei in the spleen and lymph nodes as well as atrophy of the white pulp ^[41][45][84,88].

Macroscopically, lymph nodes are often enlarged and oedematous with scattered haemorrhages in the cortex and medulla ^[39][42][32,35,82,85]. In the intestinal tract, lymphoid depletion in the lamina propria is most severe in the distal jejunum and ileum and includes moderate oedema, pyknosis, and karyorrhexis of mononuclear cells in the submucosa, and a mild neutrophilic infiltrate ^[37][39][45,82].

In the spleen and lymph nodes, immunolabelling for RVFV is mainly in cellular debris or macrophages, including tingible body macrophages ^[37][45]. In adult sheep, positive labelling for viral antigen is observed more readily in the white pulp, and particularly in the marginal zone of the spleen or the sinusoids of the lymph nodes ^[37][45]. In young lambs and foetuses, labelling is also prominent in the subcapsular red pulp and smooth muscle cells within the capsule of the spleen as well as small blood vessels [46].

3.6. Lung

In ruminants, marked lung oedema and congestion is a consistent macroscopic finding irrespective of age ^[39][35,37,45,46,82]. Multifocal haemorrhages, or blood in the trachea and bronchi, may also be present in sheep and cattle ^[39][43][45,82,86]. Microscopically, intra-alveolar and interstitial oedema with atelectasis, emphysema, and occasional haemorrhages are present in adult ruminants and neonates. In foetuses, oedema expands the connective tissue surrounding blood vessels, bronchi, or bronchioles and is also present in the pulmonary septa ^[16][37]. In fatal human RVF cases, there is frank haemorrhage in the lungs, with microscopic alveolar oedema and haemorrhage ^[45][88].

In the lungs of sheep, viral antigen is present in pulmonary intravascular macrophages or in the capillaries associated with cellular debris ^[37][46][37,45,46]. Immunolabelling is also present in endothelial cells and vascular smooth muscle cells, but this is more prominent in young lambs and sheep foetuses ^[16][46][37,46].

3.7. Heart

Epi- and endo-cardial haemorrhages are present in most cases in ruminants and humans ^{[37][46][39][45]}[35,45,46,82,88]. However, histomorphological lesions definitively attributable to RVFV infection are not present in the cardiac parenchyma ^[37][46][45][35,37,45,46,88].

Immunolabelling for RVFV is rare in adult sheep and typically associated with endothelial cells or vascular smooth muscle cells ^[37][45]. In neonates—and especially foetuses—labelling in the heart is widespread and involves cardiomyocytes, Purkinje fibres, endothelial cells or cellular debris in small blood vessels and capillaries, as well as in vascular smooth muscle cells of small blood vessels ^[16][46][37,46]. In contrast, dDiffuse labelling of the myocardium and intense subepi- and endocardial labelling is occasionally observed in foetuses ^[16][37].

3.8. Gastrointestinal Tract

Occasionally in cattle and sheep, marked congestion of the mesenteric and omental vessels is present accompanied by petechiae and ecchymoses in the serosa along the entire course of the gastrointestinal tract ^[39][43][35,45,82,86]. Haemorrhages may also be present in the mucosa and submucosa of the abomasum or the intestines ^[37][39][35,45,82]. Fresh or partially digested blood is also frequently present in the lumen of the abomasum or intestines ofin ruminants ^[14][39][35,82]. Histologically, small necrotic foci are occasionally present in the lamina propria of the small intestine ^[37][39][45,82]. In humans, intestinal haemorrhage, foci of necrosis in the mucosa and microscopic haemorrhages in the muscularis and subserosa, hasve been reported ^[45][88].

In adult sheep, immunolabelling for RVFV is most often present in necrotic foci in the small intestine, either associated with cellular debris or on rare occasions in the cytoplasm of macrophages ^[37][45].

3.9. Subcutis and Skin

Subcutaneous haemorrhages are especially prominent on the abdomen, in the axillary region, the medial aspect of the hind limbs and the lower portions of the extremities ^[39][40][43]. Immunolabelling for RVFV in sheep is often present in keratinocytes or in superficial dermis in association with cellular debris or vascular endothelial cells ^[16][37][46].

Subcutaneous haemorrhages are especially prominent on the abdomen, in the axillary region, the medial aspect of the hind limbs and the lower portions of the extremities [32,35,45,46,82,83,86]. Immunolabelling for RVFV in sheep is often present in keratinocytes or in superficial dermis in association with cellular debris or vascular endothelial cells [37,45,46].

3.10. Nervous System

Other than oedema, no lesions have been reported in any tissues from the central nervous system of natural cases in cattle or sheep ^{[37][46][39][40][43]}[32,35,45,46,82,83,86]. Histologically oedema is especially prominent in foetuses ^[16][37]. In humans, focal areas of necrosis with an infiltrate of lymphocytes and macrophages may be present in the brain ^[45][88]. Perivascular cuffing indicative of encephalitis is also present in humans ^[45][88].

In sheep, viral antigen is mainly present in vascular endothelial cells or cellular debris in capillaries and small blood vessels in the meninges. Occasionally, viral antigen is present in capillaries in the white or grey matter but not within the brain parenchymal cells.

3.11. Reproductive Organs

Haemorrhages may be present in the testis of sheep ^[37][45]. While immunolabelling for RVFV is present multifocally within the connective tissue surrounding the seminiferous tubules, efferent ductules, and duct of the epididymis, and also in vascular smooth muscle, endothelial cells, macrophages, and fibroblasts, it is absent from the reproductive parenchyma ^[37][45]. Similarly, in the uterus of sheep haemorrhages are occasionally present in the perimetrium and myometrium yet viral antigen is confined to the blood vessels, and is always absent from the endometrium ^[37][45].

3.12. Placenta

Placental lesions attributable to natural RVFV infection have only been described in sheep ^[16][37]. Macroscopic lesions include intercotyledonary oedema with congestion and necrosis of the cotyledons. The most significant histological lesion is necrosis of trophoblasts and endothelial cells in the chorioallantois. In the cotyledonary villi, necrosis of trophoblasts is generally diffuse with multifocal cellular debris between the villi ^[16][37].

Immunolabelling for viral antigen is predominantly in trophoblasts and cellular debris in the cotyledonary chorioallantois ^[16][37]. Occasionally binucleate and multinucleated maternal syncytial cells are also viral antigen positive. Viral antigen is also present in vascular endothelial cells, intravascular cellular debris, or non-cell associated ^[16][37].

4. Comparison of Contemporary Knowledge of RVF Disease with the Pathogenesis of Other Viral Haemorrhagic Fever Viruses

4.1. Model of the Pathogenic Mechanism

All VHFs are characterized by a broad spectrum of clinical manifestations ranging from asymptomatic cases to mild and severe symptomatic cases with malaise, fever, vascular permeability, decreased plasma volume, coagulation abnormalities and varying degrees of haemorrhage ^[55][101]. Common amongst the VHFs are liver damage, lymphocyte depletion, and abundant pro-inflammatory cytokine and chemokine production leading to systemic inflammatory response syndrome ^{[56][55][57][58]}[100,101,103,104]. This may result in increased endothelial cell permeability with oedema, impairment of the coagulation system (as evidenced by thrombocytopenia, consumption of clotting factors, increased levels of fibrin degradation and bleeding), hypotension and multiorgan failure, culminating in circulatory shock in the terminal stages of disease ^[55](Figure 1) [101].

Detailed models representing the current understanding of VHF have been proposed ^[59][56][55]prior [99–101]. Studies in nonhuman primates and rodents experimentally infected with EBOV, CCHFV, and DHFV, suggest that some of the antigen-presenting cells are early targets of these viruses ^[60][61][105,106]. Antigen-presenting cells are specialists at capturing microbial antigens, breaking them into small peptides, and displaying these to the appropriate T lymphocytes thereby inciting the adaptive immune response ^[62][107]. Antigen-presenting cells include dendritic cells of the dermis, spleen, and lymph nodes, Langerhans’ cells in the epidermis, macrophages, B lymphocytes, and type II and type III epithelioreticular cells of the thymus ^[62][107]. It is thought that early in the infection the virus is endocytosed by antigen-presenting cells in the mucosa or the skin ^[55][57][101,103]. The vVirus gains entry via the bite of an infected insect (e.g., RVFV, CCHFV, or DHFV), breaks in the skin (e.g., EBOV), or through exposure to excreta of infected rodents (e.g., Lassa fever virus) ^[1][55][60][1,101,105]. Virus replicates in the cytoplasm of macrophages or dendritic cells at the site of viral entry and is then conveyed to the lymph nodes and parenchymal cells in the liver, kidney, adrenal cortex, and other organs. Fragmentation of many cells via necrosis or apoptosis, with phagocytosis of cell remnants, promotes further systemic dissemination of virus ^[55][57][101,103]. Presumably, tissue macrophages and vascular endothelial cells become secondarily infected and systemic inflammatory response syndrome, accompanied by microcirculatory dysfunction and shock follows ^[57][103].

Figure 1. Model of the pathogenic mechanism underlying RVFV infection. Following a mosquito bite, the virus is endocytosed by antigen-presenting cells. Suppression of type I IFN production and necrosis of infected macrophages and dendritic cells cause wide dissemination of viruses. This systemic spread leads to necrosis in a variety of tissues and cells together with suppression of both the innate and adaptive immune responses. Apoptosis of lymphocytes might occur through mediator effects and loss of dendritic cell support, exacerbating the failure of the immune response. An excessive pro-inflammatory cytokine and chemokine response follow, resulting in increased microcirculatory dysfunction through the action of inflammatory mediators. Impairment of the coagulation system results in widespread haemorrhages. Fatal outcomes result from multiorgan failure, oedema in many organs (including the lungs and brain), hypotension and circulatory shock. DC, dendritic cell. NO, nitric oxide. (Illustration adapted from Bray M, 2005).

4.2. The Role of the Liver and Kidney

Hepatocellular death is common in all VHFs ^[57][103]. In some cases of VHF, fulminant hepatic failure follows, however, hepatocellular lesions are often not significant enough to cause death ^[57][103]. Acute renal dysfunction may also play a role in the pathogenesis of VHFs with a fatal outcome. In the recent EBOV outbreak in Sierra Leone, patients had evidence of both hepatocellular damage and impaired kidney function and these were characterized by increased levels of liver enzymes, creatinine, blood urea nitrogen, and other markers ^[63][112]. Increased deviation from normal values for blood urea nitrogen (BUN), aspartate aminotransferase, and creatinine predicted a fatal outcome. In Marburg haemorrhagic fever, renal dysfunction presenting as proteinuria, with pale swollen kidneys, is frequently observed in fatal cases ^[64][113].

Similar findings are reported in RVF for both humans and ruminants. In a clinical study of severe illness due to RVFV infection in 165 human patients in Saudi Arabia, 69 were diagnosed with hepatic failure alone, 55 with both hepatic and renal failure, and 13 with renal failure alone, of which respectively 12, 39, and 3 patients died ^[20][41]. Acute renal failure associated with RVF was also described in hospitalized human patients in Sudan where 85 of 194 patients had signs and symptoms of renal failure without hepatic involvement ^[65][114]. Similarly, research comparing the susceptibility of three breeds of Nigerian sheep to experimental RVFV infection revealed increased BUN values in study participants starting on day three post-infection and continuing until the animals died ^[66][115]. Another experimental study also reported increased BUN values and histopathological changes indicative of renal injury in several sheep and cattle ^[53][67][95,98].

Renal dysfunction is also occasionally a serious complication of fulminant liver failure secondary to the development of portal hypertension, which then leads to splanchnic and systemic vasodilatation ^[68][116]. Vasodilatation, mediated by nitric oxide and other vasodilators, causes relative hypovolaemia and reduced effective central blood volume ^[68][116]. However vascular damage with plasma leakage causing non-dependant oedema and fluid sequestration in the body cavities may also exacerbate hypovolaemia and contribute to the development of renal failure.

Hypovolaemia may also be further exacerbated by reduced synthesis of albumin by the liver and impaired secretion of steroid synthesizing enzymes by virus-infected adrenal cortical cells ^[57][103]. Adrenal cortical cells are permissive to infection by many VHF viruses including RVFV ^[53][54][37,45,46,95,96]. Reduced albumin leads to reduced plasma oncotic pressure and contributes to plasma leakage, whereas reduced levels of steroid synthesizing enzymes from the adrenal glands may contribute to hypotension and sodium loss ^[57][103]. Finally, renal impairment may also occur because of direct virus-related injury tof kidneys, which has been demonstrated in both ovine and human RVFV infections ^[53][54][69][32,41,82,87,95,96,119].

4.3. Coagulation Disorders

VHFs also cause a diversity of coagulation disorders that present either as widespread bleeding or as thrombosis ^[56][100]. Humans and animals with severe illness due to RVFV may have bleeding from the gums, haematemesis, haemoptysis, epistaxis, melaena, haematuria, vaginal bleeding, petechial rashes and ecchymoses of the skin, or bleeding from venipuncture sites ^[70][71][29,41,45,86,120,121]. Conversely, thrombosis is described in earlier studies in ruminants, wherein mention is made of thrombosis of the central veins of the liver in 3 of 34 lambs, and fibrin thrombi in 6 of 30 adult cattle and calves ^[14][39][35,82].

The mechanisms of widespread bleeding in VHFs have often been attributed to direct viral infection or damage of vascular endothelial cells and thrombocytopenia ^[56][100]. Thrombosis has been ascribed to the release of pro-inflammatory cytokines and chemokines from virus-infected endothelial cells and monocytes/macrophages ^[56][100]. Disseminated intravascular coagulation (DIC) possibly follows, because of the activation of the coagulation cascade and a reduction in the production of coagulation factors due to severe hepatic necrosis. However, DIC can be classified as either enhanced-fibrinolytic-type DIC (fibrinolytic DIC) or suppressed-fibrinolytic-type DIC (thrombotic DIC) ^[72][123]. In fibrinolytic DIC, microthrombi are histologically difficult to demonstrate due to enhanced fibrinolytic activation ^[72][123]. Laboratory findings include a steep increase in PIC, but only a mild increase in the activity of plasminogen activator inhibitor, the fibrinolytic inhibitory factor [123]. Additionally, levels of TAT, D-dimers and FDPs, which reflect the dissolution of microthrombi, are also elevated. Bleeding symptoms in enhanced fibrinolytic DIC are severe, and life-threatening bleeding may occur ^[72][123].

The occurrence of DIC in human RVF cases in Saudi Arabia has been reported but was inadequately characterized to enable classification as either fibrinolytic or thrombotic ^[5][20][5,41]. More recently though, a study of 3 human RVF cases in Uganda demonstrated elevated D-dimer and tissue plasminogen activator levels, which is consistent with increased fibrinolysis ^[73][124]. In DHF, it was demonstrated that levels of FDPs are not elevated to a degree consistent with thrombotic DIC ^[74][117]. Instead, fibrinolytic DIC occurs in DHF wherein degradation of fibrinogen prompts secondary activation of procoagulant homeostatic mechanisms ^[75][125]. In sheep, experimentally infected with RVFV, thrombocytopenia and prolonged prothrombin and clotting times are present and plasma fibrinogen levels fluctuate during the period of RVFV infection [115]. In a wild-type RVFV rhesus macaque infection study, 3 of 15 animals had haemorrhagic disease accompanied by thrombocytopenia, prolonged prothrombin and clotting times and significant decreases in FDPs and fibrinogen levels, again consistent with fibrinolytic DIC ^[76][126].

4.4. Suppression of the Immune System

Dysregulation of the inflammatory response also contributes to a fatal outcome in VHF. Wild-type RVFV infection of human monocyte-derived macrophages can lead to a productive infection and inhibition of the innate immune response via decreased expression of IFN-α2, IFN-β, and TNF-α ^[77][127]. In human patients infected with RVFV, interleukin-8 (IL-8) and interleukin-6 (IL-6) levels were increased and were at similar levels in fatal cases and survivors ^[73][78][124,128]. Interleukin-8 is a pro-inflammatory chemokine produced by macrophages in response to infection, while IL-6 is an important mediator of fever ^[78][128]. Serum levels of monokine-induced-by-gamma-interferon (MIG), interferon-gamma-induced-protein-10 (IP-10), and interleukin-10 (IL-10) were significantly increased while the chemokine RANTES (regulated-upon-activation-normal-T-cell-express-sequence) was significantly decreased in fatal human RVFV cases relative to survivors and controls ^[78][128]. This suggests an imbalance in the immune response in fatal RVFV cases, because MIG, IP-10, and RANTES are pro-inflammatory chemokines whereas IL-10 is immunosuppressive. This imbalance would contribute to the failure of adaptive immune responses to clear the infection in fatal cases and explain why RVFV replicates to significantly higher levels in patients with fatal outcomes, but the details of tsuchis pathological mechanism a model remain to be determined ^[78][79][128,129].

4.5. Lymphocytes

Interestingly none of the VHF viruses infects lymphocytes. However, their rapid loss by apoptosis is a prominent feature of these diseases and lymphocytolysis is often noted ^[55][58][80][101,104,130]. In Ebola subtype Zaire, MARV and RVFV infections, lymphoid depletion affects the centres of B-cell follicles in lymph nodes ^[37][80][45,130]. The cause of lymphocytolysis in VHF’s is unknown and further study of the pro-inflammatory cytokine milieu and its potential relationship to the observed lymphocytolysis caused by natural RVFV infections is warranted, because laboratory animal models such as the mouse have produced contrary findings ^[78][128].

4.6. The Central Nervous System

Encephalitis, a complication of RVF in humans, has been experimentally reproduced in mice and rats, but has not been described in sheep or any other ruminants ^[81][82][41,131,132]. Neurologic disease has also been described in other VHFs such as Lassa fever, DHF, and Marburg haemorrhagic fever [113,133,134]. Meningoencephalitis associated with RVFV infection was described in 7 of 165 patients in Saudi Arabia and accompanied by either retinitis (n = 5), hepatitis (n = 3), or kidney failure (n = 1) ^[20][41]. In experimental infection studies in marmosets and African green monkeys, RVFV also causes encephalitis [135]. Previously, in fatal human RVF cases, focal necrosis with an infiltrate of mononuclear cells and perivascular cuffing in the central nervous system (CNS) has been described ^[45][88]. This finding together with the delayed-onset encephalitis and/or retinitis in humans suggests an inadequate adaptive immune response ^[20][41,136]. Additionally, an increased mortality rate is seen in human patients infected with both RVFV and human immunodeficiency virus (HIV), and all these patients present with CNS symptoms [137].

Notably, it has not been established exactly how RVFV crosses the blood-brain barrier. Aerosol or intranasal exposure might be necessary since encephalitis occurs in laboratory animals exposed to RVFV via this route rather than via subcutaneous injection ^[83][108,139,140]. Furthermore, although RVFV is mosquito-transmitted, consuming or handling products from sick animals, including slaughtering sick livestock, and handling dead foetuses is associated with severe disease or death ^[4][5][6][4–6]. Therefore, RVFV might infect peripheral sensory and motor neurons in the eyes or oronasal mucosa directly, causing encephalitis and retinitis via these routes ^[84][141].