Filoviruses(causative agents of Ebola hemorrhagic fever): An overview

▶The History of Ebolavirus Discovery and Current Epidemiological Situation

Severe hemorrhagic fevers caused by filoviruses (Lat. filum – thread) are the endemic diseases of West and Equatorial Africa, where they were met long ago.

The first filovirus was isolated in 1967 in Marburg (Germany) by R. Siegert and coworkers after the laboratory outbreak of hemorrhagic fever in Yugoslavia and Germany. Primarily infected laboratory personnel operated with monkey cell cultures, delivered from Uganda (Africa). From 31 affected people 7 died.

The isolated agent was designated as Marburg virus and the disease was referred to as “Marburg hemorrhagic fever”.

Subsequently the limited outbreaks of the disease were repeatedly registered in Africa, the last occured in 2014.

In 1976 a great outbreak of a new hemorrhagic fever emerged in Sudan and Zaire that involved more than 600 people with 430 fatality cases. Outbreak onset was located in Yambuku village near the Ebola River.

Marburg virus, initially presumed as the causitive agent of epidemic, was further rejected after the thorough study of a novel isolated pathogen termed as ebolavirus (S. Pattyn and coworkers, Belgium, 1976; K. Johnson and coworkers, USA, 1976).

In 1982 both Marburg and Ebola agents were placed into newly formed viral family Filoviridae. Later it has become known that Ebola disease is caused by closely related several species of ebolaviruses.

Since 1975 the outbreaks of Ebola hemorrhagic fever were regularly registered in Central and West Africa.

In December 2013 a new epidemic of Ebola virus disease emerged in Guinea; the infection rapidly spread to the neighbouring countries (Liberia, Sierra Leone and others) and moved outside the initial region. The single cases of infection were diagnosed in United States, European countries and in a number of African states).

Due to its serious community health threat in 2014 WHO constituted Ebola disease outbreak as Public Health Emergency of International Concern (PHEIC).

By April 2016, more than 28,800 cases of Ebola virus disease were indicated (above 15,000 of them were laboratory confirmed) that resulted in 11,325 death cases. Therefore, outbreak lethality reached almost 40%.

Only after intensive united efforts of international organizations and national state authorities the Ebola epidemic was terminated. Last cases of the disease were officially registered in March, 2016.

On March 29, 2016, WHO ended the state of the Public Health Emergency of International Concern for the Ebola outbreak in West Africa.

▶Classification of Filoviruses

The family Filoviridae pertain to the order Mononegavirales.

The causative agents of Ebola virus disease (EVD) and Marburg virus disease (hemorrhagic fevers) pertain to genera Ebolavirus and Marburgvirus, respectively. Genetic similarity between two genera is moderate – less than 50%.

Marburgvirus genus has a single viral species Marburg virus.

Genus Ebolavirus harbors 5 closely related viral species Zaire ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, Taп Forest ebolavirus, and Reston ebolavirus, where first four species cause hemorrhagic fevers in humans. The most severe disease is related with Zaire ebolavirus.

▶Structure of Ebolaviruses

All ebolaviruses contain linear single-stranded negative-sense non-segmented RNA. The viruses look like long cylindrical thread-like structures about 80 nm in breadth and 600-1000 nm in length.

These viruses are covered with the external lipid envelope. Viral nucleocapsid displays the helical symmetry.

Ebolaviruses contain nucleocapsid proteins bound to viral RNA. Internal protein L develops RNA polymerase activity.

GP glycoproteins are the outer structural components of the viral envelope. During reproduction, GP proteins are largely produced in soluble form (sGP).

The matrix viral proteins (VP) support envelope structure

▶Virion Resistance

The viability of ebolaviruses is moderate. Within aerosol droplets they retain infectivity at least for 1-2 hours, when dried – for several days. Sunlight and UV radiation readily inactivate them.

These viruses can withstand low-temperature exposure. Dried blood spots with ebolaviruses remain viable at 4oC for 3-4 weeks.

Heating at 60oC for 30-60 min and boiling for 5 min irreversibly inactivates virus.

Viruses are sensitive to most of conventional disinfectants (halides, aldehydes, phenol, detergents, ethanol, etc.) For ebolaviruses WHO recommends surface disinfection with household bleach (sodium hypochlorite).

▶Viral Replication Cycle

The target cells for ebolavirus replication are all the cells of monocyte/macrophage lines, dendritic cells, endotheliocytes, adrenal cells, hepatocytes.

Viral attachment to the host cells is mediated via GP proteins binding to numerous membrane receptors (lectins and many others)

Specific binding induces viral penetration by endocytosis. Acidification of endosome facilitates viral uncoating and RNA release.

The replication of ebolaviruses occurs in the cytoplasm of infected cells.

At first viral mRNAs are transcribed on genomic (–) RNA matrix. mRNAs are next translated on cellular ribosomes resulting in viral proteins.

New genomic (–) RNAs are reproduced by viral RNA polymerase via the step of (+) RNA intermediate that serves as the template for progeny genome synthesis.

Nucleocapsid assembly is performed at the inner part of cytoplasmic membrane. Maturating virions migrate across the cell membrane, where they are covered with lipid envelope. Finally ebolaviruses are released from the infected cells by budding.

Filoviruses are characterized with high reproduction rate. Massive egress of nascent virions results in destruction of infected cells.

Culture of ebolaviruses is performed only in specially organized and highly equipped national centers (laboratories) that maintain biocontainment precautions at biosafety level 4 (BSL-4) as the highest level of biosafety precautions.

BSL-4 is created for rapidly transmitted microbial pathogens, which cause diseases with highest fatality rate (like ebolaviruses).

In BSL-4 conditions ebolaviruses can be readily cultured within continuous cell lines (Vero cells or others), or by inoculation in laboratory animals (guinea pigs, hamsters, mice, or primates).

▶Pathogenesis, Clinical Findings and Immunity in Ebola Virus Disease

Hemorrhagic Marburg and Ebola fevers are extremely dangerous, highly contagious and fatal zoonotic viral infections.

Both agents stay in WHO Risk Group 4 pathogens and US list of “Biological Select Agents or Toxins” being present in Tier 1 of this list (the highest rank of public threat).

The sources of infection in cases of Ebola disease outbreaks are sick animals (primates, swine, or certain species of antelopes) or sick humans, primarily taking care on patients with Ebola.

Fruit bats and less likely rodents seem to be the animal reservoirs that harbor ebolaviruses in natural conditions.

The infection is transmitted predominantly via direct or indirect contact of human susceptible host with infected excretions of sick animals or humans.

Also the disease is contracted by alimentary route after the ingestion of infected meat.

High concentration of viruses is observed in patient’s blood, and in sufficient amounts in feces, vomits, breast milk, or sperm that is enough for transmission. In this vein Ebola infection can be transmitted by sexual intercourse still over 3 months after clinical recovery.

Aerosol spread of infection is only possible in cases of great concentration of virus in droplets. Direct air droplet human-to-human transmission of Ebola disease is not registered.

The virus is not transmitted by arthropod vectors.

Medical workers treating patients with Ebola, as well as other caregiving persons are at the highest risk of infection. In Africa the infection spread is maintained by traditional burial rituals with unprotected contacts with the deceased.

Incubation period varies from 2 to 21 days (an average of 6-10 days).

The causative agent usually enters the body through skin lesions or cuts. Also it can penetrate conjunctiva. The virus propagates in regional lymph nodes. Hematogenous and lymphogenous dissemination spreads ebolaviruses throughout the body; they appear in all inner organs and tissues.

The main targets for ebolaviruses are the cells of innate immunity (monocytes, macrophages, dendritic cells, neutrophils), endothelial cells, and the cells of parenchymatous organs.

Extensive viral reproduction results in massive cell death, activates systemic inflammation with parallel deep suppression of antiviral immune responses.

The viruses activate apoptosis of lymphocytes, monocytes and macrophages.

Soluble sGP protein arrests neutrophil activation. Structural VP proteins inhibit the secretion of both types of interferons.

In 2-3 days the viruses affect vascular endothelium all over the body’s tissues resulting in generalized vasculitis.

Hard damage of hepatocytes and endothelial cells leads to disseminated intravascular coagulation (DIC) that entails hypotension and collapse. As the result of systemic infection, hemorrhagic and necrotic lesions emerge in all organs and tissues. This is followed by massive internal bleedings and tissue edema resulting in hypovolemic shock with possible fatal outcome.

The infection has acute onset with fever above 38oC and extensive pain syndrome (headaches, abdominal and chest pain, muscular and joint pain). At 5-7 days of the disease about 50% of patients exhibit skin rash, followed by internal bleedings and mucosal hemorrhages.

Hematemesis, hemoptisis, and post-injection bleedings can be observed.

The development of hemorrhages and hypovolemic shock seriously worsenes the prognosis of Ebola virus disease.

The lethality of Ebola outbreaks is very high – it varies from 20 to 90% (average at 50%).

The recovery is slow, the convalescents produce long time shedding of virus. Hearing and vision disorders are common.

Post-infectious humoral immunity renders the high levels of specific antiviral antibodies but their role in protection against reinfections remains unclear.

Due to their prominent virulence, rapid and severe disease course, and high fatality of infection, ebolaviruses are generally ascertained as the potential agents of bioterrorism and biological warfare.

▶Laboratory Diagnosis of Ebola Virus Disease

Taking into account the extreme danger of Ebola infection all the manipulations with ebolavirus agents should be performed in laboratories maintaining biosafety level 4 (BSL-4) as the highest grade of biocontainment precautions.

The specimens are taken from patient’s blood and autopsy materials

Laboratory diagnosis of infection is verified by RT-PCR and other molecular genetic tests detecting viral nucleic acids.

Viral antigen is determined by immunochromatography.

Isolation of ebolaviruses is not routinely used because of high demands to biosafety. The virus is cultured in various cell lines with its further identification by PCR.

For serological diagnosis ELISA test is elaborated detecting antiviral IgM and IgG antibodies.

▶Principles of Prophylaxis and Treatment of Ebola Virus Disease

A substantial threat of Ebola virus disease requires strict measures in order to prevent the emergence and spread of the infection. It is compulsory to keep professional and public awareness, heightened infection surveillance with rapid case validations, patient isolation and management. Additional measures should be directed to control travellers visiting the areas of disease outbreaks.

Vaccines for specific prophylaxis of Ebola infection will be soon introduced into clinical practice as several genetically engineered vaccines from Russia, USA, and Great Britain manufacturers are starting clinical trials now.

There is still no specific antiviral treatment for Ebola disease. Positive result is achived by administration of the sera of convalescent patients with high titers of specific antiviral antibodies.

Extensive supportive treatment greatly amends the disease prognosis. The prevention of hypovolemic shock and DIC development, the maintenance of vital body functions including active fluid resuscitation with correction of electrolyte disbalance and coagulation disorders favor the outcome of Ebola infection.