Virology tidbits

Monday, 24 March 2014

Molecular aspects of Ebola and other Filoviruses

In the last few days news organizations reported an outbreak of Ebola in West Africa, leading (as of March 24th) to a death of 59 out of 80 infected people.

The Ebola virus and its variant Marburg virus are known to be one the most lethal viruses infecting humans. Once infected patients die of hemorrhagic fever, a painful and agonizing death characterized by fever, excessive blood loss and diarrhea.

There is no effective therapy, except replacing fluids, blood, coagualnts as well as relatively generic measures such as the administration of Immunoglobulin or antiviral pharmaceuticals such as Ribavirin or S--adenosylhomocysteine (SAH) hydrolase inhibitors. Antibiotics are also given to prevent secondary infections should the patient survive. Novel treatments include the use of antisense RNA, which has been shown to treat infections in non-human primates under experimental conditions. The application of a recombinant vesicular stomatitis virus expressing the Glycoprotein from Ebola has been developed as well and might prevent death of the patient.

In order to prevent the infection from spreading into the community, patients have to be quarantined and caregivers have to wear protective clothing. The mortality is high, varying from 90% in the case of Ebola Zaire to 34% to Ebola Bundibugyo; a special case might be Ebola Reston, which did not cause any fatality among humans –although it is not clear if humans can get infected in the first place.

The following strains of Ebola virus have been identified during past epidemics:

Ebola Zaire (1976)
Ebola Sudan (1976)
Ebola Côte d'Ivoire (1994; also known as Tai Forest Virus and may only be a close relative to Ebola)
Ebola Reston (1994; causing simian hemorrhagic fever, not infectious for humans)
Ebola Bundibugyo (2007)

A relative of Ebola, Marburg Virus (closely related to Ebola but distinct from) was identified in the 1960s to be the causative agent of a small epidemic of hemorrhagic fever among animal care workers in Marburg/Germany and Yugoslavia with a fatality rate of 23-90%.

In general the first symptoms of disease include a general malaise with Influenza-like symptoms, including fever/chills, chest pain, and phryngitis. If the central nervous is affected symptoms include severe headache, depression, confusion, fatigue, and coma. The most visible symptoms include hemorrhagic symptoms - such as bleeding at injection sites, and hematomas. Death generally occurs because of low blood pressure, tissue necrosis and multiple organ dysfunction.

Ebola and Marburg virus’ belong to the order of the Mononegavirales -an order which includes other viruses of interest, Hendra and Nipah Virus, both which cause serious diseases in their own right- and the family of Filoviridiae.

Organisation of the Filovirus genome

As such, the viruses have a negative strand RNA genome with a length of approximately 19kb, encoding for seven genes (NP, VP35,VP40,(s)GP, VP30, VP24 and L) each gene is flanked by a Non Translated (NTS) 3’ leader sequence and a 5’ trailer sequence (see figure), a feature shared with Nipah and Hendra viruses. Furthermore a non-coding region of varying length separates most genes. Five of the proteins encoded are shared with other negative ssRNA viruses (including the RNA dependent RNA Polymerase or L-protein and the Glycoprotein (GP)), whilst the Viral Proteins (VP) 35 and 40 are unique to Filoviruses.

Ebola viruses, similar to other Filoviridiae, infect not only human and non-human primates but other animals such as pigs and some species of fruit bats as well, in addition to a wide variety of cell lines. Cell types susceptible to Filovirus infection include but not limited to adrenal cortical cell, hepatocytes, endothelial cells, fibroblasts, dendritic cells, monocytes, and macrophages, thus explaining the wide range of symptoms in infected individuals.

Following exposure to both Ebola and Marburg virus, early targets of the virus include cells constituting the immune system -macrophages, dendritic cells and monocytes. This allows the virus to be spread to other parts of the body via the lymphatic and blood system, reaching the liver and the intestine. In addition, non-infected lymphocytes are depleted by probably via the induction of bystander apoptosis rather than infection with nascent virus.

Functions of the proteins

As mentioned above the L gene encodes the RNA dependent RNA Polymerase that is required for the conversion of the negative ssRNA into the positive strand RNA which serves as a template for negative ssRNA (to be incorporated into the genome of newly synthesized viral particles) as well as the synthesis of seven monocistronic mRNA species.

Simplified illustration of the Filovirus replication cycle

The glycoprotein is incorporated into the membrane of viral particles and –upon infection- binds to the receptor of the host cell, whilst the function of the soluble Glycoprotein (which is secreted and not incorpor-ated into the nucleocapsid and unique to Ebola virus) is unknown.

The Nucleocapsid (NP) and VP 35/24 proteins are required for the assembly of the viral particle whereas VP 30 is required for viral budding – the release of the virus particle from the host cell. In addition of its function in the assembly of the viral particle the Nucleocapsid protein might also be involved in the transport of the pre-assembled particle to the surface of the infected cell along the microtubuli. Prior to its incorporation in the virus particle the Glycoprotein is modified within the Golgi.

Because of the mortality of Ebola and Ebola related viruses, outbreaks are self limiting and relatively small in terms of numbers of people infected. The virus is also mostly transmitted by close contact with infected patients in addition to poor sanitary conditions and contaminated water and food. The virus is believed to have originally been limited to infect primates and fruit bats – although the natural host has not been identified with absolute certainty. It crossed the species barrier only when humans started to explore those areas. Ebola and Ebola-related diseases are considered to be zoonotic –of animal origin. In the case of Ebola, larger epidemics have been avoided so far, probably thanks to the high mortality this virus exhibits.

Interactions between viral proteins and the host cell

In recent years detailed analysis has revealed a number of cellular proteins which are interacting with different viral proteins during the infectious cycle of Ebola virus.

Most notably this work has identified not only the cellular receptors required for viral entry but also proteins required for the release of the viral genome into the cytoplasm of the host cell. As it turns out both the fusion of Filoviruses and the release of the genome into the cytoplasm closely resemble the mechanism found in Influenza virus and other pathogens. It should be noted that there is no bona fide receptor for Ebola or Marburg virus, but it seems that some might function as co-receptor or that the virus can bind different receptors. Viral entry itself is mediated by lipid raft dependent mechanisms and macropinocytes, a specialized subtype of endocytosis. Following entry into the cytoplasm, the viral genome is released in a Cathepsin B and L dependent process similar to the release of the Influenza virus genome, although this varies between cell lines (in Vero cells, Cathepsin L is required but is dispensable in human dendritic cells).

Studies using different cell lines confirmed the presence of a clear cytopathic effect (CPE) , although with a different severity which depends not only on the virus but also on the cell line used. Interestingly primary human cells -macrovascular or microvascular endothelial cells as well as macrophages and monocytes derived from normal peripheral blood -infected with Ebola do not exhibit any CPE. Cell death of other cell types infected -such as hepatocytes- seems to be non-apototic although Ebola virus does not antagonize apoptotic signaling pathways.

More recent results enabled to visualize the formation of nascent viral particles in distinct inclusion bodies by live cell microscopy. In theory it might be possible now to almost observe viral infection "live as it happens", thus leading to new insights into the biology and pathogenesis of diseases caused by Filoviruses.

Friday, 21 March 2014

Influenza and the Great War: a contribution to the centenary of the Great War

This year marks the centenary of the beginning of the Great War -or outside of Great Britain more commonly known as World War 1 - so I think it is time to look at a chapter of the war often neglected. Instead of focusing on the beginning of the war, I would like to draw attention to the end of the war in 1918 – in particular on Germany whose defeat in 1918 coincided with the Influenza epidemic.

The influenza epidemic of 1918/1919 became widely known as the “Spanish Influenza”, a title misleading since the first cases of the disease were not reported in Spain but in the USA; Spain however was a neutral country and press reports were uncensored so cases were reported to the public in contrast to those parties involved in the war. The disease (known as “la grippe”) crossed the Atlantic with the soldiers bound for the battlefields of France and Belgium and would soon lead to a worldwide pandemic - the “single worst demographic disaster of the twentieth century” as one author in 2003 would label it. In contrast to this standard view, there is some

evidence that the 1918 virus (or its precursor) occurred first in western Europe – in army camps located at Aldershot (UK) and Étaples (France), causing local outbreaks between 1915 and 1917. It is there that pigs, geese, horses and duck as well as vulnerable soldiers in large numbers were mixing; a contributing factor might have been the presence of poison gas victims among the soldiers as well as the wet weather. Indeed the army camp at Étaples was suffering from a high number of Influenza cases resembling those in the second wave of the latter epidemic were reported between December 1916 and March 1917, preceding cases in the US by one year as were areas in England. Those cases however were not diagnosed as Influenza but as pneumonia, severe bronchiolitis or severe respiratory disease. Additionally the affected soldiers were predominately within 25 and 35 years of age - again an age not associated with severe cases of during "common" Influenza epidemics.

It is commonly accepted that the 1918/1919 epidemic occurred in three to four waves, with 22 to 50 million people dead, more than both World Wars combined. The first wave of the wave swept the US in the early months of 1918 almost unnoticed, indistinguishable from the annual wave. The second wave however started in mid-late August simultaneously in West Africa, France and on the east coast of the US, climaxing in October/November 1918 with a higher mortality than the previous wave.

This wave was followed by a third (less lethal) wave early 1919 and maybe by a fourth wave at the beginning of 1920. Contemporaries in the 1920s claimed that Encephalitis lethargica, a disease claiming hundreds of thousands of victims between 1920 and 1925, was linked to the Influenza epidemic in 1918/1919, thus representing a fifth wave (there is no proof for this claim).

Influenza hit the western front in the last phase of the Great War and is considered to have been transmitted to the German Army by French prisoners of war, starting at the end of May and reaching its zenith in June/July 1918. It was due to this disease that the Oberste Heeresleitung (OHL/German High Command) decided to postpone of would be the last German offensive of the war. Similar measures were taken by the French army - the virus did not distinguish between the invaders and the defenders.
Despite the low mortality of the first wave, soldiers were left weakened and ordered to reconvalescence in field hospitals behind the front and at home. It was because of those transports -soldiers on leave and prisoners of war transported to camps into the Reich- that the disease would travel from the west to the east, from the western front into Germany. The second wave hit German soldiers in late August 1918, and would soon reach Germany with a mortality rate similar to those observed in other countries. Unlike the first wave however, it seemed that the second wave appeared simultaneously throughout Germany, first affecting urbane centers in the west followed by rural areas and -to a lesser extent- cities in the east. At the same time the US army ceased to recruit soldiers due to the spread of the disease among soldiers, so there is some speculation that Germany might have been able to prolong the war in the west beyond November 1918. Soldiers however fared better than civilians since they received better medical care. Inside the Reich, it was left to the municipal authorities to combat the disease. In order to prevent a nationwide panic however measures to close schools, restaurants, and theaters or to prohibit other public gatherings were not introduced. In some places, even the care for Influenza patients had to be organized by the Protestant and Catholic Church. The press in France, Britain and Germany would blame enemy spies for introducing the disease. Once the epidemic was over, the German public would forget about it altogether, replaced by fears of a Bolshevik revolution and the defeat in the west.

Given that Germany suffered from the Flu epidemic the same way as France or Great Britain did, the virulence cannot be attributed to the special circumstances of Germany. Nonetheless, at the time the public linked the Influenza pandemic in 1918 to the starvation induced by the naval blockade imposed by the British naval forces. The Influenza epidemic did however increase the willingness to end the war - among soldiers and civilians alike.