Ebola (continued)

Ebola Virus----CDC microbiologist Cynthia Goldsmith----Electron Micrograph

Ebola Virus—-CDC microbiologist Cynthia Goldsmith—-Electron Micrograph

Ebola Virus Size

Glance at a standard metric ruler marked in centimeters and millimeters; those are 100 and 1000 parts of a meter, respectively. One inch has 25.4 millimeters. Now focus your mind on one millimeter— it is tiny and you need to stare quite intensely to see and comprehend it. For comparison, the unaided human eye resolution ability for distinguishing between two tiny objects is roughly 0.1 mm. That’s one tenth of a millimeter and standardized at normal reading distance of 16 inches, bright light, etc. An average  human hair is about that same size — one tenth of a millimeter. This means that if we take the smallest dot the human eye can see and place two of them any closer than 0.1mm, you will see them as one dot. 

Now the jump into imagination land.

One millimeter is broken into 1000 parts called micrometers— millionths of a meter, the symbol for that unit being μm. Do vague thoughts come into your brain from the many times you learned or tried to learn the Metric System? Putting the μm into perspective, remember a human hair is on average 100 μm in diameter (one tenth of a millimeter). Bacteria are between 0.5 and 5 μm in length and require a microscope to see (a few species are up to 0.5 mm and visible to the human eye). Stop and re-read this paragraph if you need to!

Finally we arrive at the virus world by dividing a micrometer into 1000 parts called nanometers (n) each a billionth of a meter. Viruses are usually between 20 and 300 nanometers in size, but in the Ebola family Filoviridae  they can have very long filaments. This statement is from the Centers for Disease Control web site:

Structurally, filovirus virions (complete viral particles) may appear in several shapes, a biological features called pleomorphism. These shapes include long, sometimes branched filaments, as well as shorter filaments shaped like a “6”, a “U”, or a circle. Viral filaments may measure up to 14,000 nanometers in length, have a uniform diameter of 80 nanometers, and are enveloped in a lipid (fatty) membrane. 

Another reference states the Ebola virus is typically 800 nm in length.

Structures in the nanometer range are so small that light waves used in the normal light microscope for visualization (visible light having wave lengths between 400-800 nm) cannot be used for imaging viruses. An electron beam must  be bounced off or shot through the object with magnetic fields doing the  “focusing” of the electron microscope. The colored diagram at the top of this post on this page is an electron micrograph of the Ebola virus. 

Ebola Genetics and Classification

The diagram below is a quick reminder or lesson in basic molecular genetics as it occurs in an animal or plant cell; it is verbally summarized below. This is useful for understanding viral genetics.

DNA transcription and translation [Image by Atheists, Agnostics and Freethinkers of Waterloo, Canada]

Diagram 1: FROM GENETIC MATERIAL TO PROTEIN– A Generalized Diagram of DNA Transcription and Translation in an Animal or Plant Cell
[Image by Atheists, Agnostics and Freethinkers of Waterloo, Canada]

  •  DNA, deoxyribonucleic acid, or RNA, ribonucleic acid, function as the chemicals carrying genetic information in organisms and viruses; those molecules carry a code for the manufacture of proteins, the final product of a gene. The actual code is the sequence of four nucleotides which are the building blocks in the single strand of DNA or RNA, with each 3 sequential nucleotides specifying one amino acid out of the 23 which are “protein-building.” Diagram 2 below shows the basic structure of nucleotides and how they are attached to each other.
  • Most species have DNA as the genetic material.  Viruses have either DNA or RNA ; most
    RNA General Structure: Shown are two nucleotide building blocks of RNA. Each consists of a base, a ribose sugar and a phosphate group. The lower nucleotide shows the structure of one of the four bases in RNA which are adenine, guanine, uracil and cytosine

    Diagram 2: RNA Building Blocks: Shown are two nucleotide building blocks of RNA. Each consists of a base, a ribose sugar and a phosphate group. The lower nucleotide shows the structure of one of the four bases in RNA which are adenine, guanine, uracil and cytosine. (credit: : Narayanese at the English language Wikipedia)

    viruses have RNA as does Ebola.

  • In animals and plants the code in DNA first must be transcribed into another molecule called messenger RNA, using DNA as a template. This mRNA is then translated into protein with the assistance of yet another type of RNA called transfer RNA The latter molecules transfer amino acids to the assembly area in the cytoplasm of the cell. (see diagram 1 above). 
  • The Ebola virus particle enters an animal cell with only seven genes  The virus lacks an energy system and many other physical components for transcription and translation of its genetic material. It carries several essential proteins and its RNA Genome described below for starting the process of replicating itself but must “take over” much of the energy and protein production system of its host in order to complete it. It must also “deceive” the host cells defensive systems in order to enter the cell and carry out its own replication. 

 For better visualization of an RNA genome, below is a more detailed diagram of an RNA molecule showing four nucleotides. The actual size of the Ebola genome is about 19,000 nucleotides in a sequence coding for its seven proteins and start/stop “instructions.” 

RNA-structure-diagram-wcpd

Diagram 3: RNA molecule showing four nucleotides. The phosphate groups and the sugar ribose molecules can be thought of as the “backbone” of the RNA. The sequence of bases cytosine, guanine, adenine and uracil carry the triplet genetic code. The Ebola virus genome is a strand of about 19,000 nucleotides.(Credit:Public Domain diagram))

 

Should you want more detail and/or diagrams of these genetic processes,  look through this panoply of diagrams on the topic.

There are two main classification systems for viruses and they are usually used in conjunction with each other. One system, the Baltimore System, classifies them by (1) the type of genetic material (DNA or RNA); (2) whether that chemical is single stranded or double stranded; and (3) in the case of single stranded RNA viruses, whether the RNA structure is negative sense or positive sense.  Positive-sense viral RNA is similar to mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus first must be converted (transcribed) to positive-sense RNA by the virus RNA polymerase enzyme before translation. (the word “transcribed” is often used only for DNA to RNA transcription and “copied” used for negative to positive RNA but “copied” implies making an exact duplicate which of course is not the case–so we will use “transcribe” meaning using one stand as a template and making a “complementary” strand)

Based on the above brief description, viruses are placed into seven groups. Ebola is in Group Five which have single stranded and negative sense RNA meaning the RNA can’t be directly translated into protein product. Group Five viruses have an RNA polymerase enzyme which uses the negative sense genome RNA as a template to produce positive sense RNA, which is then used for translation of the code into proteins. Ebola is placed into the Family Filoviridae which includes 3 genera: Cuevavirus, Marburgvirus, and Ebolavirus.

Here is detail in terms of classification and related data from Wikipedia:

The family and CDC Filoviridae is the taxonomic home of several related viruses that form filamentous infectious viral particles (virions), and encode their genome in the form of single-stranded negative-sense RNA. Both viruses, and some of their lesser known relatives, cause severe disease in humans and nonhuman primates in the form of viral hemorrhagic fevers. All ebola viruses and marburg viruses are Select Agents, World Health Organization Risk Group 4 Pathogens (requiring Biosafety Level 4-equivalent containment), National Institutes of Health/National Institute of Allergy and Infectious Diseases Category A Priority Pathogens, Centers for Disease Control and Prevention Category A Bioterrorism Agents and listed as Biological Agents for Export Control by the Australia Group.

According to the World Health Organization, five species of Ebolavirus have been identified: (1) Taï Forest (formerly Ivory Coast), (2) Sudan, (3) Zaire, (4) Reston and (5) Bundibugyo. Ebola-Reston is the only known Filovirus that does not cause severe disease in humans; however, it can still be fatal in monkeys and it has been recently recovered from infected swine in South-east Asia.  Ebola-Zaire is the most dangerous of the known EVD-causing viruses, and is responsible for the largest number of outbreaks.

The 2014-2015 outbreak in west Africa, (first cases notified in March 2014), is the largest and most complex Ebola outbreak since the Ebola virus was first discovered in 1976. There have been more cases and deaths in this outbreak than all others combined. See World Health Organization for more detail. 

 Detail of Ebola Structure

 

Ebola Virus diagram indicating general structure and showing location of the seven gene products

Ebola Virus diagram indicating general structure and showing location of the seven gene products. The blue outer portion represents the outer “viral envelope” or lipid bilayer

Ebola has seven genes which produce seven structural proteins. The generalized diagram above indicates the location of those proteins relative to the structure of the virus.The proteins and their functions in brief are:

  • Nucleoprotein (NP)—Part of a spiral structure which includes the RNA, in the center of the virion.
  • Virion Protein 35 (VP35) — A minor protein which acts against interferon, the natural protein in animal cells which normally functions to destroy viruses.
  • Transcription activator (VP30) —Triggers the process of Transcription.
  • Glycoprotein (GP) — Forms spikes on the outer surface of the virion; it mediates attachment and entry into the host cell. The gene for this protein is now being used in Ebola vaccine development and, as of December 2014, is in phase 1 clinical trials. 
  • Virion Protein 24 (VP24) — Like VP 40, VP 24 is a matrix protein crucial for virus budding out of the host cell. Both VP 40 and 24 also maintain the shape of the virion.
  • Virion Protein 40 (VP40) — Like VP 24, is a matrix protein. A matrix is simply an inner portion of a cell or virion. Also a very appropriate name here because “matrix” is derived from  the Latin word for womb (in turn from mater or mother); both proteins are Involved in virus “reproduction.”
  • RNA Polymerase (L)—A large virus protein enzyme which, in the host cell, assembles copies of the virion RNA genetic material from positive copies of that material and transcribes it into messenger RNA in preparation for translation. This enzyme is sometimes called RNA Replicase for reasons just stated; It is technically called RNA Dependent-RNA Polymerase.

Below is another diagram of Ebola with layer structure emphasized:

Diagram of Ebola showing layers. The outermost "Viral Envelope" is a lipid bilayer which the virus particle "picks up" from the animal cell as it buds off from that cell. [credit: http://www.torontosun.com/2014/07/30/what-you-need-to-know-about-ebola]

Diagram of Ebola showing layers. The outermost “Viral Envelope” is a lipid bilayer which the virus particle “picks up” from the animal cell as it buds off from that cell. [credit: http://www.torontosun.com/2014/07/30/what-you-need-to-know-about-ebola]

Ebola Life Cycle

The diagram below is a brief pictorial summary of the Ebola life cycle:

Credit: http://www.bbc.com/news/world-africa-26835233

Credit: http://www.bbc.com/news/world-africa-26835233

 As noted earlier, the Ebola virus has a genome of only seven RNA genes and this RNA is of the negative type which must  be transcribed to the complementary positive strand which is usually called messenger RNA ( mRNA). The transcript mRNA will then be used to translate its “message” into proteins for insertion into new virions. The virus brings its own RNA Polymerase enzyme with it into the host cell to carry out the transcription. The polymerase enzyme creates the mRNA transcript by the standard complimentary base pairing construction process, moving along the negative genome strand, “reading it” and creating the new mRNA strands by taking new nucleotides from the host cell and joining them. Later, for placement into new virion units, the polymerase enzyme will use positive strands of RNA it has created from the genome negative strands to make negative genome strands also for placement into new virions.

To help visualize this see a detailed diagram below of these life cycle processes in a host cell.

Ebola life cycle-simple1

Detail of the Ebola invasion of a human host cell. See detailed explanation below (credit: https://web.stanford.edu/group/virus/filo/class.html/ Stanford University/Tara’s Ebola Site/Honors thesis)

Key to above diagram:

  1. Virion attaches to cell membrane (not shown) and once inside releases RNA negative genome (blue).
  2. The  negative genome is transcribed into positive mRNA by RNA Polymerase and then:;
  3. Translated into 7 proteins;
  4. The negative viral genome RNA is used as a template by RNA Polymerase to create positive RNA strands which are then used by the Polymerase to:
  5. Create negative viral genome RNA strands which are:
  6. Placed into new virion units “under construction” with proteins translated earlier.
  7. The new units bud out of the cell host membrane.
  8. And as they bud out they “steal” or “wrap themselves” in the cell membrane which becomes their protective viral envelope (black). Grey glycoprotein “spikes” are shown protruding from the virus surface; they were one of the proteins produced earlier in the cycle and are critical for chemical reactions which allow the virion entrance into a host cell (see below.

 A Final Ebola Life Cycle Diagram with More Detail

Key to diagram

1.  Ebola virus (EBOV) binds to attachment factors and receptors on the cell surface through

Ebola Life Cycle from White and Shornberg 2012 http://www.nature.com/nrmicro/journal/v10/n5/full/nrmicro2764.html

Ebola Life Cycle: from White and Shornberg 2012.  http://www.nature.com/nrmicro/journal/v10/n5/full/nrmicro2764.html

its spike glycoprotein (GP) and causes an infolding (pinocytosis) of the host membrane resulting in the virus being moved inside the cell wrapped in a piece of the cell membrane. Essentially the virus is “tricking” the cell to incorporate it. Normally the cell uses this process to take in needed nutrients or substances .

2-3-4 The virus is then placed into and transferred to various compartments in the cell called macropinosomes, endosomes and lysosomes in which it is processed in an attempt by the cell to keep the virus in a “trash can” and digest it. But the virus “tricks” its way out of the late endosome. Endosome enzymes called CatB and CatL “digest” the virus spikes of GP into several pieces. This triggers a series of events, some unknown, which allows a fusion of the virus membrane with the endosomal membrane and release of the virus into the cell.

5The viral nucleocapsid (four proteins—NP, VP35, VP30 and L (RNA Polymerase) and the viral genome) is released into the cytoplasm, where the genome is replicated by RNA Polymerase.

6.  The viral genome is transcribed into mRNA with the aid of the viral proteins VP35, VP30 and L (RNA Polymerase).

7.  Viral mRNAs are then translated into their respective proteins using the cell energy and “machinery.”

8.  mRNAs encoding GP are brought to the endoplasmic reticulum (ER), where GP is synthesized and modified.

9.  GP is further modified in the Golgi and delivered to the plasma membrane in secretory vesicles. If you need a review or lesson on these cell structures go here.

10.   At the plasma membrane the ribonucleoprotein complex (RNA plus nucleoprotein (NP)) and associated viral proteins assemble with the membrane-associated proteins (matrix proteins VP24 and VP40, and GP), and the resultant virions bud from the cell surface.

11.  Other forms of GP, including soluble GP (sGP), are also secreted.

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Ebola Adaptation

We will discuss Ebola mutation below but It is worth noting the incredible adaptations which the virus has evolved to overcome the normal human cellular defenses. (That is not a very scientific statement; it is teleological and sounds like the virus knows what it is doing. That is Walt Disney like and good science does not speak that way. The point of course is that evolution is a powerful process and a blind one at that; mutations arise which will be selected if they allow one organism or genetic unit to better survive, replicate and pass on their genome.)

 Some adaptations were stressed above, the little “tricks” (speaking anthropomorphically again) the virus uses to get into the cell and then out of the enclosure. Here are several other evolved “tricks” discussed on the excellent web site on Ebola by Operon Labs: 

A notable feature of Ebola is that underneath the GP spikes, there is a lipid-bilayer, making Ebola an enveloped virus. The lipid bilayer is derived from infected cell membranes as the virus buds from the cell.  Thus, Ebola membranes contain not only ‘normal’ lipid bilayer, they also contain a fair amount of other proteins and lipid rafts which were derived from the virus’s previous host cell.  A viral envelope (as seen in Ebola) can help many viruses evade the immune system, because most of the virus’s antigenic components besides GP are not exposed to scrutiny  (they are ‘cloaked’ by a real human-cell derived lipid bilayer).   

And another statement from Operon dealing with interferon proteins — which are produced by animal cells when invaded by microorganism — to repel the “invaders.”  

Interferon [in human cells] is categorically one of the most important ‘master’ antiviral genes that exists.  Interferon is the ‘switch’ for a whole constellation of gene products (called Interferon Stimulated Genes), most all of which cause the cell to ‘double check’ and ‘inspect’ everything, making it nearly impossible for a virus to properly replicate.   The power of Interferon is why Ebola (and other dangerous viruses) target it so ruthlessly.  Ebola must absolutely destroy and disable the host Interferon response in order to be able to succeed in it’s prime directive — replicate at all costs. Ebola’s VP35 gene prevents this [interferon protection] process from happening.

Final Questions to be Reviewed Here

  1.  Does the Ebola virus mutate?
  2. How is Ebola involved with bats and monkeys?
  3. Is there a vaccine against Ebola?
  4. How to they damage the body?
  5. Has the virus  been on Earth for 35 million years?
  6. Why do modern times seem to have accelerated the emergence/reemergence and spread of viruses such as AIDS, Ebola, chikungunya fever, avian flu, dengue fever, norovirus gastroenteritis, hepatitis E, etc. And why are most of these viral diseases caused by RNA genome viruses?
  7. What is the etymology of the name Ebola?
  8. What are hypotheses for the origin of the first viruses? What did they evolve from?

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