Thursday, October 12, 2006

W N V

West Nile Virus

Authored by Jennifer Quick & reviewed by JP Saleeby, MD





History/Demographics

Protecting ourselves against those pesky mosquitoes could save you not only from an annoying bug bite, but also from transmission of a nasty virus introduced into the United States fairly recently. West Nile Virus (WNV) was first isolated from a female patient presenting with fever like symptoms in Uganda in 1937. It is, however, a relatively new disease to the Western Hemisphere, presenting itself in 1999 when the first cases in America were documented in New York City. The outbreak resulted in 62 infections causing serious illness and seven deaths. Since then, the virus has spread to multiple areas including the mid-west, southern and western states, resulting in a steady increase in the number of infections annually. According to the Center for Disease Control, as of August 22, 2006 there were already 581 reported cases of West Nile Virus infection, with 18 of those cases resulting in the death of the victim. Not only are humans targeted by this virus, WNV infects birds as well as other mammals. Researchers continue to study the pathways of infection, trends in immunity, and signs and symptoms of the illnesses in order to more effectively combat this spreading pathogen.

Bird Infection

Although WNV can be a fatal infection in humans, it is primarily a detrimental disease in birds. The virus is transmitted to other birds through different species of mosquitoes including Aedes, Culex, or Anopheles. These mosquitoes carry and amplify the virus in their salivary glands and subsequently, during a blood meal, the virus is transfered to the bird. The transmission cycle continues as the birds transmit the virus to other feeding mosquitoes and those mosquitoes go on to infect more birds or other mammals. As of September 2000, WNV has been isolated in at least 70 different species of dead birds found throughout the United States.

There are some differences in WNV infection among various species of birds. Dr. Richard Bowen, a researcher at the Animal Reproduction and Biotechnology Laboratory at Colorado State University, has explored some of these differences in various birds such as crows, chickens, and pigeons. He comments that, “There are a lot of differences among birds. Very few American crows survive WNV and typically circulate at 10^8 to 10^9 pfu's of virus/ml of blood. Fish crows, on the other hand, have only a 25-50% mortality rate.” When studying chickens, Dr. Bowen found that, “Chickens greater than 1 week of age that become infected have low viremia levels and usually never get sick. On the other hand, baby chicks infected at less then one week of age do get higher viremias causing illness and death.” Additionally, Dr. Bowen has studied the effects of WNV immunity and immunosupression in chickens. His findings conclude that when immune hens (female chicken) give birth, they transfer their antibody to the chick, thereby protecting the chick with this maternal antibody for approximately one month. After that time, the chicks become susceptible to viremia but do not show signs of illness. Also, it was found that immunosuppression of the chickens increased the magnitude of viremia, but did not make them susceptible to illness. Pigeons, on the other-hand, are “sort of between non-susceptible chickens and highly susceptible American crows.” They show moderate viremia levels with very low mortality rates. As for future research, Dr. Bowen is working on trying to understand what is so lethal about the NY99 strain of West Nile Virus. He is also working on determining whether immunity to WNV makes birds immune to other related viruses such as Japanese encephalitis.

Human Infection

The virus affects not only birds but humans as well. Twenty percent of infected individuals will develop symptoms and one out of 150 of those infections results in encephalitis or meningitis. Additionally, the mortality rate from severe illness is 3-15% depending on who is collecting the data. The severity of infection depends on the degree of central nervous system (CNS) invasion, exposure to multiple bites, and age of the victim. Morbidity and mortality increase with ages over 50 and prove to be especially significant in people over the age of seventy-six. High-risk areas include the Midwest, accounting for 55% of cases, as well as the Southern and Western states. Symptoms in patients usually appear in June and taper off in November.




2006 West Nile Virus Activity in the United States(Reported to CDC as of August 22, 2006*)
http://www.cdc.gov/ncidod/dvbid/westnile/Mapsactivity/surv&control06Maps.htm




Infectious Agent

The West Nile Virus is a member of the family Flaviviridae. It is an enveloped, spherical, positive sense single stranded RNA virus that contains one open reading frame. The (+) ssRNA serves as the mRNA template that is then translated into one polypeptide and subsequently processed by cellular and viral proteases producing various proteins. These proteins include a capsid protein (C), an envelope protein (E), a premembrane protein (preM), as well as seven non-structural proteins. The E and M proteins are thought to give the mature virion it’s rough appearance.

In order to gain entry into host cells, WNV attaches to a yet unknown receptor on the host cell and undergoes clathrin-mediated endocytosis. More research must be done to verify the actual mechanism of entry, but it has been observed that there is an interaction of the virus with toll-like receptors as well as an increase in tumor necrosis factor alpha (TNF-a) before penetration of the virus into the CNS. Toll-like receptors (TLR's) are transmembrane proteins that were first identified in the fruit fly and later found to be present in various types of mammals and even in plants. There are various types of TLR's and when activated, these proteins begin a cascade of events that alert the immune system to begin combating a pathogen such as a virus. In order to better understand the entry of WNV after supposed interaction with TLR's, it is imperative to know a bit about it's entrance into host cells via clathrin- mediated endocytosis. This process starts when the virus binds to its receptor which then causes a clathrin coat to build up inside the membrane below this binding. The clathrin forms around the pit that is to eventually be endocytosed into the cell (It is thought that clathrin drives the process of endocytosis as well as stabilizes the whole process). Once endocytosed, the vesicle containing the virus loses its coat. Then, vesicles join other vesicles uniting to become what is known as an early endosome. After a series of steps in which the early endosome transforms into a late endosome, the late endosome is then fused with a lysosome. A drop in pH within this lysosome causes a conformational change in the E surface protein thus causing the hydrophobic domain of that protein to insert itself into the lysosome membrane. This fusion allows the nucleocapsid of the virus to be inserted into the cytoplasm of the host cell and begin its replication. This ends the whole process of entry into the host. At this point, host cell ribosomes translate the (+) ssRNA into the mRNA template going on to the Endoplasmic reticulum (ER) to be translated into various proteins. These proteins are cleaved by viral and host proteases and directed to their corresponding places within the cell by host direction. For instance, the E and preM proteins are synthesized in the membrane of the ER and are translocated by host interaction into the lumen of the ER. Similarly, host cell machinery directs the C protein as well as additional proteins such as N2A through N5 into the cytoplasm of the cell.

In order to make more virions it is imperative that more (+) ssRNA be made. Synthesis of this vital component is accomplished in part by the protein N5 which is thought to be the viruses' RNA dependent RNA polymerase. Along with the help of N3 it is able to synthesize (-) RNA from the (+)RNA . This allows more (+)RNA to be made which can then function as an additional template for mRNA synthesis or go on to be packaged in virions that will exit the cell and go on to infect other cells.

Once enough protein and (+)RNA has been made, the virus is ready to package these items and export them out of the cell in the form of new infectious particles. A nucleocapsid is formed from multiple copies of the C protein. (The C protein is one of the three structural proteins encoded by the (+)RNA.) Shortly thereafter, all components are gathered. As the virion is exiting the host cell, the E and preM proteins complex, rendering E ineffective. This critical step is important so membrane fusion does not occur and the virion is able to exit the cell without reentry. Exit of the virion continues with budding through the ER membrane and final departure via the secretory pathway. On exiting the cell, host proteases cleave the preM protein that was once in complex with the E protein, thereby rendering the E protein effective so that the new virus is capable of entry into new cells.

Pathogenesis / Symptoms


After infection by a mosquito vector, the virus incubates in mammals for around 5-15 days. In the 20% of individuals that show signs of infection, symptoms will usually only last for about 3-6 days. Those showing symptoms are classified as either having WNV encephalitis, in which neurological signs of disease are seen, or classified as having WNV fever, ill victims but showing no signs of CNS malfunction. Neurological signs of disease include muscle weakness, flaccid paralysis, photophobia, seizures, mental status changes, respiratory symptoms, inflammation of the brain and spinal cord. WNV fever symptoms include fever, nausea, anorexia, malaise, myalgia, headache, rash, eye pain, and vomiting. Those infected who never show symptoms may never know they were infected unless tested for antibody to the virus.


Diagnosis

The most effective way to diagnose West Nile Virus infection involves serologic testing to detect IgM antibodies specific for WNV. This is done with the use of the IgM antibody capture Enzyme-Linked ImmunoSorbent Assay (MAC-ELISA). This test can, however, provide false positives due to the close relation of WNV to other Flaviviruses and should therefore be confirmed with a plaque reduction neutralization test (PRNT). Also helping in diagnosis is a detailed history from the patient including information about visits to high exposure areas as well as information on exposure to outdoors during peak times of mosquito prevalence. Information about histories involving organ transplants, breast feeding in mothers, as well as blood transfusions is also imperative as contraction the virus has been observed via these routes as well.

Prevention / Deterrence

There is no known medical treatment (pharmaceutical or vaccine) for West Nile Virus infection at this time aside from supportive care. Avoiding mosquitoes is thus the primary way of preventing this disease. This can be done with the use of insect repellents containing N,N-diethyl-meta-toluamide (DEET, 10-30% is considered effective) and ethyl hexandiol, using clothing to cover exposed areas of the body, and staying indoors from dusk till dawn can help in reducing the risk of infection. There is even a clothing manufacturer with the trade name Buzz Off that uses a permethrin compound incorporated in the clothing material.

An alternative to DEET is Picaridin, also known as KBR 3023, an ingredient found in many mosquito repellents used in Europe, Australia, Latin America and Asia for some time. Evidence indicates that it works very well, often comparable with DEET products. Still another “natural” alternative is Oil of lemon eucalyptus (also known as p-menthane 3,8-diol or PMD) is a plant-based mosquito repellent that provided protection time similar to low concentration DEET products in two recent studies.

Also, reporting dead birds to the proper health officials is important in early detection of this virus in your area. Other deterrence mechanisms include picking up garbage and draining standing water from vacant areas and parks in order to hinder breeding grounds for mosquitoes. Researchers on intently studying ways to develop a human vaccine and antiviral drugs to treat WNV for the years to come.


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Reference:

Childs, Gwen V. “Receptor Mediated Endocytosis.” Text copyright 1996.
http://www.cytochemistry.net/Cell-biology/recend.htm. Accessed September 15, 2006.


Hayes, Edward B. et al. “Virology, Pathology, and Clinical Manifestations of West Nile Virus Disease.” Emerging Infectious Diseases. National Center for Infectious diseases, Centers for Disease Control and Prevention. August 2005.
http://findarticles.com/p/articles/mi_m0GVK/is_8_11/ai_n14920182. Accessed August 27, 2006.

Microbiology and Bacteriology. “The West Nile Virus.”
Timothy Paustian. 1999-2006. http://www.bact.wisc.edu/Microtextbook/index.php?name=Sections&req=viewarticle&artid=206&page=1. Accessed August 25, 2006.

“New York State West Nile Virus Response Plan – Guidance Document.” May 2001.
http://www.health.state.ny.us/nysdoh/westnile/2001/responseplan/2001wnv_responseplan.pdf
Accessed August 26, 2006.

Salinas, Jess D. et al. West Nile Virus. w
ww.emedicine.com/pmr/topic236.htm. Accessed August 26, 2006.

“Statistics, Surveillance, and Control.” Center for Disease Control.
http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount06_detailed.htm.
Accessed August 26, 2006.

“West Nile Virus Transmission Cycle.”
http://environmentalrisk.cornell.edu/WNV/WNVEducDocs/WNVTransCycle.pdf.
Accessed August 26, 2006.

http://www.ces.ncsu.edu/depts/ent/notes/Urban/repel.htm, Accessed October 2, 2006.

http://www.buzzoff.com/ Accessed October 2, 2006

Jennifer Quick interview with
Dr. Richard Bowen, DVM, PhD, Professor & Researcher Animal Reproduction and Biotechnology Laboratory, Colorado State University, 3801 W. Rampart Road
Fort Collins, CO 80523 USA
, ARBL@colostate.edu.
http://www.cvmbs.colostate.edu/bms/ARBL/index.htm


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Jennifer Quick received her BS degree (2006) in Microbiology from Colorado State University. Also obtaining a minor in Biomedical Sciences (Anatomy and Neurobiology). She has experience as a research assistant in a West Nile Virus lab (2003-2005) as well as an HIV lab (2005-2006); Jen is acknowledged in a publication titled "Derivation of phenotypically and functionally normal macrophages from lentiviral vector transduced human embryonic stem (hES) cells for HIV-1 gene therapy.”

JP Saleeby, MD is assistant medical director of the Emergency Department at Liberty Regional Medical Center in Hinesville, GA. He held a faculty position at Georgia Southern University in the department of nursing. He is a medical and health writer for several online and print journals. He has authored a book entitled “Wonder Herbs: A Guide to Three Adaptogens”, published in 2006.

© 2006

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