Virology tidbits

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.
ResearchBlogging.org




Further reading:

Mühlberger E (2007). Filovirus replication and transcription. Future virology, 2 (2), 205-215 PMID: 24093048 

Huggins, J., Zhang, Z., & Bray, M. (1999). Antiviral Drug Therapy of Filovirus Infections: S‐Adenosylhomocysteine Hydrolase Inhibitors Inhibit Ebola Virus In Vitro and in a Lethal Mouse Model The Journal of Infectious Diseases, 179 (s1) DOI: 10.1086/514316 

Takada A (2012). Filovirus tropism: cellular molecules for viral entry. Frontiers in microbiology, 3 PMID: 22363323 

Kiley MP, Bowen ET, Eddy GA, Isaäcson M, Johnson KM, McCormick JB, Murphy FA, Pattyn SR, Peters D, Prozesky OW, Regnery RL, Simpson DI, Slenczka W, Sureau P, van der Groen G, Webb PA, & Wulff H (1982). Filoviridae: a taxonomic home for Marburg and Ebola viruses? Intervirology, 18 (1-2), 24-32 PMID: 7118520

Nanbo A, Watanabe S, Halfmann P, & Kawaoka Y (2013). The spatio-temporal distribution dynamics of Ebola virus proteins and RNA in infected cells. Scientific reports, 3 PMID: 23383374 

Hoenen T, Shabman RS, Groseth A, Herwig A, Weber M, Schudt G, Dolnik O, Basler CF, Becker S, & Feldmann H (2012). Inclusion bodies are a site of ebolavirus replication. Journal of virology, 86 (21), 11779-88 PMID: 22915810

Iwasa A, Halfmann P, Noda T, Oyama M, Kozuka-Hata H, Watanabe S, Shimojima M, Watanabe T, & Kawaoka Y (2011). Contribution of Sec61α to the life cycle of Ebola virus. The Journal of infectious diseases, 204 Suppl 3 PMID: 21987770


Hofmann-Winkler H, Kaup F, & Pöhlmann S (2012). Host cell factors in filovirus entry: novel players, new insights. Viruses, 4 (12), 3336-62 PMID: 23342362

Sayama Y, Demetria C, Saito M, Azul RR, Taniguchi S, Fukushi S, Yoshikawa T, Iizuka I, Mizutani T, Kurane I, Malbas FF Jr, Lupisan S, Catbagan DP, Animas SB, Morales RG, Lopez EL, Dazo KR, Cruz MS, Olveda R, Saijo M, Oshitani H, & Morikawa S (2012). A seroepidemiologic study of Reston ebolavirus in swine in the Philippines. BMC veterinary research, 8 PMID: 22709971

3 comments:

  1. Greetings, I linked your blog to twitter and one of my followers @stgoldst (PhD student of virology) mentioned some clarifications. He mentioned that Marburg and Ebola aren't 'related' nor is Marburg a prototype of Ebola. Per: "The prototype of Ebola, Marburg Virus (closely related to Ebola but distinct from)..." Though they are in the same family of viruses they are quite different species on a similar level as SARS and MERS was his comparison; so I guess it just depends on what you define as closely related. Perhaps it's just more accurate to say they are in the same viral family and cause similar symptoms perhaps? Relatedness might have suggested genetics and they are actually quite different genetically. And you mentioned: "Ebola Reston (1994; Simian Hemorrhagic Fever Virus, not infectious for humans)", he said simian hemorrhagic fever virus is in a different viral family from Ebola Reston...Arteriviridae. Did you mean that Ebola was also discovered in 1994 during an SHFV outbreak? Just thought I'd toss in some thoughts, I work on Flavis...but I've only started working the infectious disease field since 2010, prior to that I was microbial/viral ecology (environmental, not health related). Cheers. Feel free to chat up @stgoldst on twitter if you want :)

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    Replies
    1. Dear Mel,
      Thanks for your comment.
      You are correct in your statement that Simian hemorrhagic fever virus is a positive strand RNA virus belonging to the Arteriviridae within the order of the Nidovirales – but Ebola Reston is (in contrast to the other Ebola viruses) the causative agent of simian hemorrhagic fever. It is a case where a different virus causes a similar disease (similar in the symptoms). According to Dalgard et al. (1992) and Fischer- Hoch et al. (1992) both viruses (Ebola Reston and SHFV) were isolated during the outbreak at the animal facility in Reston and it was only in subsequent laboratory studies that Ebola Reston was shown to be highly pathogenic for monkeys. Ebola Reston was also postulated to have infected swine in the Philippines in 2008, so it seems that (similar to other Ebola viruses) not only infects monkeys but swine as well (Sayama et al., 2013).
      Regarding the closeness of both Marburg and Ebola, so it seems that it partly depends on the sequence of individual genes on the definition of closeness. From an antigenic point of view both are certainly cousins, and hence are classified in different genera. This is different from MERS-CoV and SARS-CoV, where both are of the same genus (Betacoronavirus) but of different lineages (de Groot et al., 2013)
      You might find that I decided to clarify these points in my post. I hope this anwers your questions.

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  2. I am a novice and do not work in the medical field but wonder if you can tell me if hydrogen bonding appears in the molecular structure and if so would you describe its function?

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