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EBOLA VIRUSBY- SHREYA AHUJAROLL NO. 11

HOW DID EBOLA COME INTO BEING?The virus family Filoviridae includes 3 genera: Cuevavirus, Marburgvirus, and Ebolavirus.

Three species of Ebolavirus have been found so far, Bundibugyo ebolavirus, Zaire ebolavirus, and Sudan ebolavirus. The virus causing the 2014 west African outbreak belongs to the Zaire species

Ebola virus disease (EVD) first appeared in 1976 in 2 simultaneous outbreaks, one in Sudan, and the other in Zaire. The latter occurred in a village near the Ebola River, from which the disease takes its name.

In 1989, Veterinary staff in Virginia had first noticed numerous deaths in monkeys in one animal room and suspected it to be Simian Hemorrhagic Fever (SHF). Tested samples were shown to contain a filovirus in high concentrations along with SHF virus. This was later identified as a strain of the Ebola virus and was clearly responsible for much of the morbidity.

A recent study suggests that the virus may have passed into its first human victim, a child, from a small insect-eating bat, an animal so diminutive that it is hunted by children but not by adults.

THE STRUCTURE OF EBOLA VIRUSEbola virions are pleomorphic, appearing as either long filamentous forms or in shorter U-shaped or circular configurations.

Virions have a uniform diameter of 80nm and vary greatly in length (up to 14,000nm)

They are composed of a helical nucleocapsid, a membrane envelope derived from host cell plasma membrane, and a surface projection layer composed of 10nm long peplomers.

Ebola virus infectivity is stable at room temperature and is destroyed in 30mins at 60C. Infectivity can also be destroyed by ultraviolet and gamma radiation, lipid solvents, and phenolic disinfectants.

GlycoproteinMatrixProteinNucleoproteinNucleocapsidCapsomeres

MOLECULAR STRUCTURE AND ORGANISATION OF GENOME

The nucleocapsid in the center of the virion is made of a RNA wound helically with the proteins NP, VP35, VP30, and last but not least L. The viral proteins VP40 and VP24 are found in the viral tegument or the area between the envelope and nucleocapsid.

Each virion of Ebola virus has a single stranded, negative sense RNA linear genome. There are seven sequentially arranged genes contained in a single molecule approximately 19kb in length.

The virus codes for seven structural proteins and one non-structural protein.

Having a negative sense polarity, the virus RNA structure begins in the 3 position and ends in the 5 position. Following the 3 position is a non-transcribed region known as the leader.

The transcriptional signals of Filoviruses contain a common sequence, 3UAAUU at the 5 end of start sites and 3end of stop sites, a property that is so far unique to these viruses.

The 5end of the genome has a transcriptional stop (Polyadenylation) site. Termination of transcription occurs at a series of five or six Us where repeated copying by the polymerase results in addition of long poly (A) tails to the transcripts.

UAAUUCUUUUU

Processing of GP protein

Glycoprotein: It makes up the virion surface spikes that mediate virus entry into susceptible host cells through receptor binding.

VP40: This protein is believed to have a matrix protein function and maintains viral structure integrity. It may facilitate the budding of the virion at the plasma membrane by facilitating interaction between nucleocapsid proteins (NP and VP30) and envelope proteins (GP and VP24)

VP24: Membrane associated protein which may function in the un-coating of the virion during infection.

Nucleoprotein: It is the primary structural protein associated with Filovirus nucleocapsids. Interacts with matrix protein during virus packaging and assembly.

VP30 (Also known as minor nucleoprotein): It is tightly bound in the nucleocapsid and it can bind to the RNA non-specifically. It is involved in transcription activation and re-initiation.

Polymerase (L protein): It is the RNA Dependent RNA Polymerase required for transcription and translation. Caps and poly-adenylates mRNAs. Uses +ssRNA as templates for synthesizing copies of ssRNA.

VP35: It acts as a cofactor for L polymerase. It is responsible for binding a host double stranded RNA and inhibiting the host cells alpha/beta interferonVIRAL PROTEINS AND THEIR ROLES

Ebola virus multiplication in host cell

SO WHAT MAKES EBOLA SO DEADLY??

The virus isn't what ends up killing you. It's your own immune system

Ebola virus symptoms and its effects on the human body

Fever, headache and chills are caused by an overt cytokine response to virusBleeding from the eyes, ears and mouth is caused by loss of vascular integrity due to endothelial cell death and dysregulation of clotting factors because of hepatocellular necrosisImpaired kidney and liver function due to death (apoptosis) of renal and hepatic cellsVirus evades the immune response by causing rapid destruction of lymphoid tissue, including B and T cells, and disrupting the interferon responseWatery diarrhea, nausea, vomiting and abdominal pain is due to necrotic infection of the gastrointestinal tract - loss of the ciliated cells required for absorption of nutrients - gastrointestinal bleedingMuscle pain and weakness is likely due to the cytokine response to the virusHaemorrhagic rashes, bruising, and oozing caused by loss of vascular integrity due to endothelial cell death and hepatocellular necrosis

EBOLA VIRUS DIAGNOSISThe methods used are:

ELISAEbola Virus lysates used Four dilutions 1/4, 1/16, 1/64, 1/256

RT-PCRPrimer used: 5-GAGACAACGGAAGCTAATGC-330 min at 50C to allow RT followed by 2 min at 94C to allow enzyme inactivation and denaturation. This is then followed by 38 cycles of denaturing at 94C for 30 s, annealing at 50C for 30 s, and elongation at 68C for 1 min.

PLAQUE ASSAYDilutions of virus are adsorbed to a monolayer of cells and incubated. The monolayers are overlaid with 1% agarose and the virus plaques were counted after incubation

NUCLEOTIDE SEQUENCINGDNA products purified after PCR are sequenced and analyzed

REDS Rapid Ebola Detection StripsNanoparticles linked to antibodies which recognize Ebola antigen

Recent Advances in Treatment of EBOLASUB-UNIT VACCINESEBOV genes inserted into a DNA plasmid can be injected directly into a patients muscle, where expression of the antigen can elicit an immune response to the corresponding virus particle.GP, NP, VP40, VP35 used Low survival rates

VECTOR BASED VACCINESViruses can be used as vaccine vectors when genes encoding antigens of EBOV are inserted and expressed from the viral carrier.Vaccinia Virus, Adenovirus, Vesiculovirus, VEE virus have been used Adenoviruses have shown success in non-human primates and are undergoing phase 1 trial

HIV DRUG used out of sheer desperationLamivudineDr. Gobee Logan, treated 15 Ebola patients with this drug and 13 survivedLamivudine is a Cytidine analog reverse-transcriptase inhibitorThe HIV-1 RT enzyme recognizes and incorporates Lamivudine Triphosphate in the place of Cytidine Triphosphate.Cytidine (C) is present in both DNA chains (base pairs GCAT) and RNA chains (GCAU)Lamivudine competes with Cytidine as a nucleic acid base for Ebola RNA Replicase in human macrophages

There is no proven cure for Ebola at this time

Recent Advances in Treatment of EBOLA..contd.FAVIPIRAVIR6-fluoro-3-hydroxy-2-pyrazinecarboxamide is an antiviral drug that selectively inhibits the RNA-dependent RNA polymeraseThe drug was found effective in patients with low to moderate Ebola infection, however it had no effect in people who were heavily infected

TKM-EBOLAThis drug is based on RNAi therapyTargets viral proteins L, VP35 and VP24Deliver the siRNA through stable nucleic acid lipid particles (SNALPs)It has been granted emergency use approval by the US Food and Drug Administration

HEMOPURIFIERIt filters the blood to remove virus particles when connected to a dialysis machine.The United States Food and Drug Administration (FDA) granted approval in January 2015 for a clinical trial of a device

TETRANDRINEModifies pore channels in the host cell membrane needed for Ebola infectionMost potent compound tested with low cytotoxicity

CONCLUSIONWhat makes Ebola so different than other viruses is its ability to attack and overcome its host so efficiently. It quickly migrates throughout the lymph nodes and the blood stream to infect the parenchyma of most organs. The only cells it will not infect are skeletal/cardiac muscle and bone. Although this is a truly morbid subject, Ebola serves as one of the most efficient viruses in nature. So efficient and so deadly, that frequently contamination wipes out the host population before the virus can spread to a wider demographic.

One of natures most deadly killing machines!!

http://www.who.int/mediacentre/factsheets/fs103/en/

http://news.nationalgeographic.com/news/2014/12/141230-ebola-virus-origin-insect-bats-meliandou-reservoir-host/

http://www.operonlabs.com/?q=node/7

http://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-ebola-virus-disease

https://www.internationalsos.com/ebola/index.cfm?content_id=442&language_id=ENG

http://www.npr.org/blogs/goatsandsoda/2014/08/26/342451672/how-ebola-kills-you-its-not-the-virus

Jonathan S. Towner et al (2004) Rapid Diagnosis of Ebola Hemorrhagic Fever by Reverse Transcription-PCR in an Outbreak Setting and Assessment of Patient Viral Load as a Predictor of Outcome. J. of Virology. 78:8,4330-4341

Jason S. Richardson (2010) Recent advances in Ebolavirus vaccine development. Human Vaccines 6:6, 439-449

Fields Viology

References

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