Ultrastructural pathology of human liver in Rift Valley fever

Abstract

Rift Valley fever (RVF) is a zoonotic disease that primarily affects ruminant animals and can also cause fatal disease in humans. In the current report, we present the ultrastructural changes in the liver of a man aged 60 years who died from RVF in the Aseer Central Hospital, Abha, Saudi Arabia. The main hepatic changes by transmission electron microscopy included the presence of 95–115 nm electron-dense particles consistent with RVF virions, nuclear condensation, vacuolar degeneration, lipid droplet accumulation and mitochondrial damage and dilation. There were also viral inclusion bodies with electron-dense aggregates, dilation of intercellular spaces, damage of sinusoidal microvilli with widening of space of Disse, dilation of bile canaliculi and increasing number of phagolysosomes.

Background

Rift Valley fever (RVF) is a rare disease caused by a virus, mainly affecting ruminating animals but can cause fatal disease in humans. In humans, it involves multiple organs with liver being the primary site of viral replication. In the hepatocytes, many electron microscopic changes are seen that are important in early identification of disease, in limiting the spread and initiating appropriate treatment of RVF-afflicted patients.

Case presentation

A man aged 60 years presented to the Aseer Central Hospital, Abha, Saudi Arabia, with RVF. The disease was confirmed by the presence of viral antigen and anti-RVF IgM as measured by quantitative real-time PCR and ELISA. The patient died after 6 days from admission. Liver specimens were obtained in 2.5% glutaraldehyde and sent to the Department of Pathology, College of Medicine, King Khalid University, Saudi Arabia, for transmission electron microscopic (TEM) examination. All procedures performed in the current study were in accordance with the ethical standards of King Khalid University Committee, Saudi Arabia, and have been performed in accordance with ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments, or comparable ethical standards.

The specimens were trimmed, fixed in glutaraldehyde solution in 0.1 M sodium cacodylate buffer, pH 7.2, and placed in a thermal box cooled to 4°C for 2 hours. They were postfixed in 1% osmium tetroxide in a sodium cacodylate buffer and then dehydrated in an ascending series of ethyl alcohol and embedded in Spurr's resin. Ultrathin sections stained with uranyl acetate and lead citrate were examined by TEM (Jeol 100 CXII; Japan) operated at 80 kV.

TEM examination revealed the presence of 95–115 nm electron-dense particles consistent with RVF virions (figure 1A) and inclusion bodies with electron-dense aggregates in the cytoplasm of hepatocytes (figure 1B). Most hepatocytes showed apoptotic changes, most notably, cell shrinkage and chromatin condensation (figure 1C). There were also vacuolar degeneration, lipid droplet accumulation and increasing number of phagolysosomes (figure 1D). Mitochondria in the majority of hepatocytes appeared damaged (figure 2A) and had cisternal segmentation and vesiculation (figure 2B). Moreover, examined specimens showed dilation of intercellular spaces (figure 1D), damage of sinusoidal microvilli, dilation of space of Disse with the presence of deposited collagen (figure 2C) and dilation of bile canaliculi (figure 2D).

Figure 1.

Representative micrograph showing ultrastructural changes in hepatocytes from a RVF-infected patient. (A) Electron-dense, 95–115 nm particles consistent with RVF virions (arrow). (B) Inclusion bodies (arrow) with electron-dense aggregates (white asterisks). (C) Nuclear shrinkage and chromatin condensation (white asterisk). (D) Vacuolar degeneration (black asterisks), lipid droplet accumulation (white asterisk), numerous phagolysosomes (white arrows) and dilation of intercellular space (arrow heads).

Figure 2.

Representative micrograph showing ultrastructural changes in hepatocytes from a RVF-infected patient. (A) Mitochondrial damage (arrows). (B) Cisternal segmentation and vesiculation (arrows). (C) Damage of sinusoidal microvilli (arrow heads), red blood cells (white asterisks) and an eosinophil (arrow). (D) Dilation of bile canaliculi (asterisks) and vacuolar degeneration (arrows).

Discussion

RVF is a viral zoonosis affecting humans and a wide range of ruminant animals, mostly, sheep, goat and cattle.¹ The disease was first described in 1930 along the shores of Lake Naivasha in the greater Rift Valley of Kenya.² Then, afterwards, the disease became endemic in Africa probably due to climatic changes such as episodes of heavy rainfall in eastern and southern regions of Africa.³

RVF is caused by a virus belonging to Bunyaviridae family. The virus is primarily transmitted by mosquitoes to and among animals. The virus is transmitted transovarially in mosquitoes. There are more than 30 mosquito species that are capable of transmitting the disease. They belong to seven genera, of which, Aedes and Culex are considered the important vectors.³ In addition to infection by mosquito bites, ruminating animals are the main infectious route for humans through contact with body fluids such as blood during slaughtering and butchering, and foetal membranes and amniotic fluid of viraemic animals.⁴ Direct human-to-human transmission has not been reported. Transplacental transmission of RVF virus (RVFV) may occur in vertebrates including humans.⁵

Since its identification in 1931, major epidemics have occurred in African countries most notably South Africa in 1951,⁶ Egypt in 1977 and 2003,⁷ Kenya in 1997, 1998 and 2006–2007,⁸ Tanzania in 2007,⁹ Sudan in 2007,¹⁰ Mayotte in 2008¹¹ and Mauritania in 2010–2012^{12 13} In 2000, RVF cases were confirmed in Saudi Arabia and Yemen, making the first report of the disease outside Africa and raising concerns of possible extension to other parts in Asia and Europe.¹⁴

RVF epidemics usually lead to severe economic losses and hundreds of human deaths.^{10 15} For instance, South Africa epizootic in 1951 caused losses over 100 000 sheep and half million livestock abortions.⁶ In Egypt, epizootic in 1977, there were a notable human epidemic causing about 600 deaths and significant livestock abortion and mortalities.¹⁶ The first RVF outbreak in Saudi Arabia in 2000 was reported to affect 800 individuals with a case death rate of about 25%.¹⁷

In humans, the majority of infections with RVFV are subclinical or associated with moderate to severe, non-fatal, febrile illness that is followed by full recovery after 2 weeks.¹⁸ However, some cases develop ocular disease, encephalitis or haemorrhagic fever. Ocular disease occurs 1–3 weeks after the onset of initial symptoms. It can affect one or both eyes leading to macular oedema with/without retinal haemorrhage, vasculitis, infarction or vitreous haze. Some patients also show retinal detachment, uveitis or arterial occlusion. Complete recovery of vision does not occur, however, some individual show partial improvement after several months of infection.¹⁹ Encephalitis occurs 1–4 weeks after initial symptoms in <1% of patients. In which, patients show a variable number of neurological manifestations , including convulsions, ataxic gait, persistent hemiparesis, meningeal irritation, confusion, visual hallucinations, hypersalivation and coma.²⁰ Haemorrhagic fever is fatal and occurs suddenly in about <1% of overall RVF patients. It is characterised by symptoms of haemorrhagic diathesis with hepatitis, and death occurs in 3–6 days after the onset of symptoms. Postmortem examination shows diffuse necrosis of hepatocytes in the centrilobular areas.²¹ Moreover, some patients die from renal failure or disseminated intravascular coagulation (DIC).^{22 23}

Liver constitutes the primary site of RVFV replication in human patients.²⁴ Al-Hazmi et al¹⁴ reported that the hepatic form of RVF was the predominant organ manifestation and occurred in 88% of patients in the Saudi outbreak during September–November in 2000. Unlike histopathological picture that prominently consists of extensive hepatocellular necrosis, reports in ultrastructural changes of hepatocytes in RVF-infected patients are so limited in the literature. In our current report, electron-dense particles consistent with RVF virions were seen in the cytoplasm of degenerated hepatocytes in the RVF-infected patient. RVF viral particles were reported to be rarely seen in hepatic tissues. This may be due to that hepatocytes undergo abortive infection or apoptosis before release of mature RVF virions.²⁵ Additions to the presence of viral particles, large inclusion bodies consisting of electron-lucent large bodies with electron-dense aggregates in intact hepatocytes were seen. The presence of these inclusions in intact hepatocytes indicates an early infection of these cells.²⁵ Ellis et al²⁶ reported that RVF's inclusions formed of either fine or coarse materials.

Similar to our findings that include cellular shrinkage and nuclear condensation, Narayanan et al²⁷ reported widespread apoptosis in RVFV-infected hepatocytes. The authors attributed these apoptotic changes to increase reactive oxygen species (ROS) followed by proapoptotic genes. Vacuolar degeneration, lipid droplet accumulation and numerous phagolysosomes were apparent in the examined specimens. In consistent, Reed et al²⁵ found that RVFV-infected mice maintained many fat vacuoles, pale cytoplasm and numerous phagolysosomes. Examination of mitochondria revealed mitochondrial damage, mostly, in the form of cisternal segmentation and vesiculation.²⁵ In this context, Narayanan et al²⁷ reported that the RVF viral protein accumulated in the mitochondria of RVFV-infected hepatocytes and contributed to mitochondrial damage and production of ROS. On the other hand, Reed et al²⁵ did not report any mitochondrial damage in RVFV-infected mice. Examined specimens showed also sinusoidal damage with dilation of space of Disse and dilation of bile canaliculi.²⁵ Likewise, Reed et al²⁵ found that space of Disse was dilated with vacuoles, fibrin and collagen in RVFV-infected mice. The authors reported intact bile canaliculi.²⁵

In conclusions, the most prominent pathological findings in our current report in the RVFV-infected patient were the presence of viral particles in degenerated hepatocytes, large inclusion bodies in intact hepatocytes, apoptotic changes, mitochondrial damage and blood sinusoidal alterations.

Learning points.

• Rift Valley fever (RVF) is a zoonotic disease mainly affecting ruminant animals and may lead to fatal illness in humans.

• In humans, multisystemic involvement is seen with the major replication site of virus being the hepatocytes.

• The main hepatic changes seen in TEM are the presence of 95–115 nm electron-dense particles consistent with RVF virions, nuclear condensation, vacuolar degeneration, lipid droplet accumulation, and mitochondrial damage and dilation.