Molecular

Molecular Characteristics

Family: Flaviviridae
Genus: Flavivirus

• Spherical viruses that range from 40-60 nm long
• Single stranded positive-sense RNA strand
• The genome is surrounded by a nucleocapsid in the lipid bilayer of the host cell
• The envelope is derived from the host cell and is composed of 2 viral proteins. The viral proteins are called protein E and protein M. Protein E is a type of hemagglutinin and is the primary target of host's immune response. Protein E is also known as the antigen of the virus. Protein M is a stabilizing factor that protects protein E during viral assembly. It also protects protein E during transmission of the virus from cell to cell. Protein M starts as protein prM, which is the inactivated state before viral replication.
• Protein C
• This protein is known as the Core protein of JEV (Japanese Encephalitis Virus). This protein interacts with the host cell membranes during the assembly of the virus.
• The genome is 11kb long
• It contains a 5' cap, but 3' poly(A)-tail
• Having no poly(A)-tail will cause the mRNA to be less stable and degrade faster. This could mean that this virus could have problems going from mRNA to amino acid codons during translation.

Figure 1: The Japanese Encephalitis virus's protein configuration and genome.


Infection

Clathrin-mediated Endocytosis
(AKA: Receptor-mediated Endocytosis)

• Most common method of entry for biological molecules and in some instances viruses
• Process:
• The virus binds to a specific receptor on the plasmid membrane, which then signals clathrin molecules to accumulate on the inside of the plasma membrane. Adaptor proteins bind to clathrin and these proteins direct the assembly of the clathrin complex and invagination process. Adaptor proteins have three binding sites: one for clathrin, one for the plasma membrane and one for the receptor on the plasma membrane. During inward budding of the virus, the clathrin and adaptor protein complex surrounds the newly formed vesicle and pinches it off into the cell. There is a link to the video on how the process works using a visual animation.
• https://www.youtube.com/watch?v=-ZFnO5RY1cU

Figure 2: Clathrin-mediated endocytosis animation.

• Other pathways into the cell include: cholesterol-dependent, caveolae-mediated and phagocytosis.

Immune Response

• Viral replication occurs in the cytoplasm in the skin's dendritic cells
• Virus travels through the lymphatic system and into the brain
• Humoral immunity through antibodies IgM and IgG that protect the CNS. IgM is known as the first responder of antibodies and one of the ones that is most plentiful, along with IgG. IgG is the only antibody that we have that can cross the placenta.
• Innate immune response through cellular secretion of interferons. Interferons give a boost to the immune system and increase inflammation. Interferons themselves do not kill individual cells or viruses.
• The JAK-STAT pathway (Janus Kinas Signal Transducer and Activator of Transcription) is the human body's way of fighting off viruses. Interferon induces a phosphorylation process that in tern induces transcription of a particular set of genes. These genes produce proteins that have antiviral, anti-tumor and immunomodulatory effects. Unfortunately, JEV has a mechanism that inhibits the JAK-STAT pathway. Researchers do not know the exact mechanism on how the virus inhibits this pathway; if they did there would be better treatments out there for JEV.

Potential Treatments

Methyl-Beta-Cyclodextrin

• Another popular way that viruses enter the cell is through lipid rafts. Lipid rafts are parts of the plasma membrane with high concentrations of cholesterol and sphingomyelin. The virus attaches to these lipid rafts to gain entry through the porous membrane, caused by the high concentration of cholesterol. Lipid rafts function as the center of signaling molecule assembly.
• One of the best pharmacological agents out there can disrupt lipid raft composition
•  MBCD (methyl-beta-cyclodextrin) selectively extracts membrane cholesterol, therefore stopping the passage way for the virus to enter. In an experiment that tested MBCD's effect on JEV was conducted and measure through immunofluorescence staining. Cells were treated with MBCD before infection. Looking at Figure 3(A), comparing the control to the 10mM MBCD, the amount of cells infected with JEV decreased significantly. The blue cells in these pictures represent cells without JEV and the green cells are the ones infected with JEV. Part D of Figure 3 shows the amount of cell viability comparing the treatment and control groups. This tested to see if MBCD had an effect on healthy, normal cells in the infected individual. The bar graph shows that there was no significant difference between the control and treatment group. This means that MBCD does not harm or affect healthy cells.

Figure 3: Immunofluorescent diagrams in a MBCD study.

Chlorpromazine

• Is another drug that is being experimented on to potential treat JEV. Chlorpromazine inhibits clathrin-mediated endocytosis. Chlorpromazine is commonly used as an antipsychotic drug and to treat schizophrenia. But when treating JEV, chlorpromazine effects the distribution of clathrin and prevents them from assembling with adaptor protein molecules to prevent the coat-assembly. If clathrin and the adaptor protein can't bind to each other, the virus will no longer be able to infect the cell. 
• In a study using Vero cells, chlorpromazine was used and it decreased the chance of JEV infection by 80%. This study was only done as a pretreatment (vaccine-like) and the effect of it this drug working after JEV infection is still unknown.

Figure 4: Chemical structure of Chlorpromazine.

Eric's Biological Integration Statement

           Japanese Encephalitis (JE) is an extremely deadly viral disease. 25% of people that become infected with it succumb to the disease and 50% of those that survive have permanent brain damage. JE is an arbovirus that is transmitted through mosquitoes causing symptoms in humans like severe headaches, high fevers, comas, tremors, convulsions and encephalitis. The worst case scenario that could happen is that ISIS causes a biological war against the United States, using JE infected mosquitoes to infect the population. Since the majority of the U.S. doesn't vaccinate against the disease, this could potentially wipe out a fourth of the country's population. Our goal for this project is to increase awareness about JE and how described how it works to the general public. XXXXX
In the molecular category of our Blogger I went over the classification of JE virus and it's genome. It is part of the family Flaviviridae and the genus Flavivirus. It has a single stranded positive-sense RNA virus that is 11kb long. The envelope surrounding the viral genome is made up of protein E (a hemagglutinin) and protein M (protects protein E during transport and replication). JE virus enters the cell through clathrin-mediated endocytosis. The virus binds to a specific receptor, clathrin molecules aggregate with adaptor protein molecules and inward budding occurs. The human's immune response to viruses is to activate the JAK-STAT pathway through interferon release, but JE has a mechanism that stops this. One of the reasons why there isn't a successful treatment yet. Potential treatments are methyl-beta-cyclodextrin which degrades membrane cholesterol inhibiting lipid raft entry and chlorpromazine which is a drug that inhibits clathrin-mediated endocytosis.
For the organismal category written by Brandon he went over how the virus is transmitted between hosts. JE virus is a vector-borne illness, that vector being mainly mosquitos of the Culex species. Reservoirs and amplifying species were pigs, warding birds and some bats. JE is zoonotic diseases that passes between animals and humans. Humans, along with cattle, sheep and chickens, are called dead-end hosts. Dead-end hosts mean that they can not transmit the disease, the infection stops at them. The virus can be spread by mosquitos and coming into contact with blood, saliva, urine or fecal matter from an infected animal. A risk factor that increases the risk of getting infected is the type of season we are in.
For the ecological category written by Greg he went over where the virus is spread. Mature pigs are the main reservoir hosts and the next common are birds. The pig will become infected, allowing the virus to multiply and become stronger. Then a vector will take the blood from the pig and transmitted the virus to another species. The JE virus is commonly found in agricultural areas, specifically near rice farms and standing water. Pigs are the most commonly infected and they experience abortions, convulsions and still births. Horses when infected do not show side effects most of the time, but when they do they get swelling in their brain and the inability to walk. Dog, cats and birds can become infected, but won't show any symptoms at all.
For the public health category written by Ryan he went over the types of vaccinations and the U.S.'s game plan in case of a JE virus outbreak. In Asia there are 68,000 new cases of JE a year and with the population of Asia that puts 3 billion people at risk. There are four main types of vaccinations: inactivated mouse brain-based vaccines, inactivated cell-based vaccines, live attenuated vaccines and live chimeric vaccines. There are a couple ways to help keep you and your family from infection. The best thing to do is talk to your primary care physician if you believe you are at risk and stay up to date on your immunizations. The Center of Disease Control's protocol for disease outbreak starts with analyzing the risk probability of an area and assessing the surrounding resources. Also to remember to keep the community involved with current updates, proper training of all emergency staff and conduct exercise to help prepare the community.
This blogger provides a source of information to better inform the public of Japanese Encephalitis virus. Increasing awareness of JE is our main priority.