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= Rio Negro Virus =

Background and History
The Rio Negro Virus is an Alphavirus (one of 31 other species) that was isolated in Argentina in 1980. The virus was first called Ag80-663, but was renamed to Rio Negro Virus in 2005. The virus is a subtype of the Venezuelan Equine Encephalitis complex, specifically type VI. "The Venezuelan equine encephalitis (VEE) complex comprises a broadly distributed group of alphaviruses in the Americas that have the potential to emerge and cause severe disease". Rio Negro isn't the only subtype of VEE to be given it's own name. There is also subtype IIIA named Mucambo Virus and subtype II; Everglades Virus.

Viral Structure
The Rio Negro Virus is an spherical, enveloped virus. This means that after the virion (complete infectious form of a virus outside of a host cell) leaves the host cell, it carries part of the cell membrane with it. This gives it easy access to other cells using the surface proteins. There are 80 'spikes' embedded in the stolen membrane, which are arranged in a T=4 icosahedral. In all, the Rio Negro virion is ~70 nm in diameter with 3 structural proteins making up the capsid. There is 2 open reading frames on the genome. The proteins (E1, E2, and C) are encoded in the C-terminal ORF near the 3' end while the non-structural proteins (nsPs 1-4) are encoded in the N-terminal ORF the 5' end. The E1 and the E2 proteins are used to make up the base of the protrusions that interact between the capsid and the membrane.

Viral Genome
The virus has a +ssRNA genome that is 11.5 kilobases long. Other viruses with the same genome are SARS, MERS, Hepatitis C, Poliovirus, West Nile Virus, and the Norovirus. To be able to use host machinery, the genome has to be translated from positive strand to a negative strand, or mRNA. To do this, Rio Negro is a Bring-Your-Own-Enzyme virus. It needs to encode for RNA Dependent RNA Polymerase. The Rio Negro genome is a non segmented genome, meaning that to make copies or proteins, the whole genome must be translated to get a specific part. These are then broken up into sub-genomic proteins, which encode the structural proteins. The genome and the sub-genome have 5' caps and poly(A) tails.

Transmission
The main vector of transmission for Rio Negro is mosquitoes and rodents. This makes the virus an 'arbovirus'. This means that it uses mosquitoes are its main mode of transportation. There are 7 mosquitoes that have been scientifically shown to be carriers of the Rio Negro virus. The species with the most evidence were the Culex (Culex) interfor, Culex quin-quefasciatus, Aedes  albifasciatus, and the Culex (Melanoconion) taeniopus . There is 4 criteria that have to be met for a virus to be considered an arbovirus.


 * 1) The arthropod must have significant contact with a known pathogen host.
 * 2) There is an association between contact with the suspected vector and infection.
 * 3) There is repeated demonstration of the natural infection due to suspected vector happening in the wild and
 * 4) Sufficient experiments run to conclusively theorize that the suspected arthropod is the vector for the virus.



Entry into the Cell
Rio Negro Virus, like all Alphaviruses, has glycoprotein receptors, called E proteins, on it's envelope, which recognize cellular receptors in order to perform membrane fusion. On the viral envelope, there are originally two of these glycoprotein receptors, P62 and E1, which form a dimer. P62 is eventually cleaved into E2 and E3 proteins, forming a trimer, and this prepares these proteins to be reactive to acidic conditions. Membrane fusion is initiated by receptor recognition, followed by clatherin-mendiated endocytosis, and then, in response to the low pH of the endosome, an irreversible change in the conformation of the glycoprotein trimer occurs. The E2 protein's cytoplasmic domain interacts with the nucleocapsid of the virus, while its ectodomain binds to receptors on the surface of the host membrane. When the E2 protein binds to host receptors, the virus is engulfed into the host via endocytosis. Once the virus is in a cellular endosome, with low pH, the E1 and E2 proteins disassociate. This conformational change exposes the viruses' fusion peptides, which then fuse the membrane of the virus and the cellular endosome, which transports the nucleocapsid of the virus into the cytoplasm of the host cell.

Replication and Transcription
After membrane fusion, the genome of Rio Negro Virus enters the host's cytoplasm, and this is where replication and transcription occur. The viral genomic (+)ssRNA is used both to translate proteins and to transcribe (+)ssRNA copies of the viral genome. The viral genome has two open reading frames (ORFs) which generate the nonstructural and structural polyproteins. There are five structural proteins - C, E3, E2, 6K, and E1 - as well as the nonstructural polyprotein - nsP1-4. Rio Negro Virus, as a type of Alphavirus, encodes four nonstructural proteins (nsP1-4) in it's genome, which are used in RNA synthesis. These are initially produced as a polyprotein, but are later cleaved by viral or host proteases, to form separate polyproteins. The first cleavage, produces polyprotein P123 and nsP4, and these form a (-)ssRNA template strand to be used to replicate the viral genome. Then, the P123 polyprotein is further cleaved to form nsP1, nsP2, and nsP3 proteins, in addition to nsP4. These produce (+)ssRNA copies of the viral genome, using (-)ssRNA strand as a template, that will later be distributed to the virions which will be released after assembly. Alphavirus polymerase is capable of de novo RNA synthesis.

Assembly and Release
Alpha virus nucleocapsids are assembled in the cytoplasm from the capsid proteins produced in translation of the viral genome. Alpha virus virions are composed of the lipid envelope in which the E2 and E1 glycoproteins are located, and the nucleocapsid, composed of the capsid protein, which surrounds the genome. The capsid proteins have two domains: the C-terminal protease domain and the N-terminal domain, which has a strong positive charge. The protease function of the C-terminal serves to cleave the capsid protein from the polyprotein in which it was produced, so that it can separate to form the capsid.

The viral genome contains conserved regions that serve as packaging signals, which increase the efficiency of viral packaging. One of these packaging signals is in the nsP1 coding sequence in the Venezuelan, Eastern, and Western equine sncephalitis viruses. This area of the genome sooms to form eight stem loops. Each of these loops contain triplet guanine (GGG) nucleotides at the stem tip. In other species of Alphaviruses, the packaging sequences When the nucleocapsid of the virus is assembled, encapsulating the newly produced viral genome, it exits the cell by budding through the plasma membrane. This is also where virus-encoded surface glycoprotein E1 and E2 are assimilated onto the virion.

Host Interactions
The Rio Negro virus tricks the cell into believing that it's not an invader. It does this by shutting down the host cell's gene expression. So even if the cell did detect the virus, there would be no proteins to fight it. The virus does this by disrupting the creation of the transcription factor: STAT1. The virus capsid and de novo viral gene expression is needed to shut down STAT1 creation. Not only that, research shows that VEEC viruses don't seem to be affected by some of the cell's defences, mainly type I and II IFN.

Associated Diseases
As stated many times above, the Rio Negro virus is a sub-virus of the Venezuelan Equine Encephalitis Complex. Humans can get infected, but this usually only results in acute fevers. The complex has caused reoccurring outbreaks in Central and South Americas with at least one outbreak in Mexico that resulted in the complex entering the United States.

All Viruses in the Venezuelan Equine Encephalitis Complex:

 * Rio Negro virus
 * Cabassou virus
 * Everglades virus
 * Mosso das Pedras virus
 * Mucambo virus
 * Paramana virus
 * Pixuna virus
 * Trocara vrius
 * Subtype: Bijou Bridge virus
 * Venezuelan equine encephalitis virus

Tropism
Infection in humans vs horses are different. In horses, the virus attacks the central nervous system, causing paralysis and eventual death. When the virus infects humans, symptoms manifest as fever, chills, headaches, nausea, vomiting, and muscle/back pain. Humans tend to recover in a few weeks with only the first couple of days having sever symptoms. Fatality rate for human adults is only 1%, but it is much more sever for children, rising to 20% while horses have a 10% chance of dying from infection. Like most virus infections, there is the normal plethera of 'flu-like' symptoms from the immune system fighting back. There is a 4-14% chance of neurological complications developing from infection. Death is usually caused by encephalitis and brain/lung/intestinal bleeding.