Rapid spread of the disease has inspired accelerated research
Since first making an appearance in New York City in 1999, West Nile Virus (WNV) has claimed more than 300 lives and infected thousands more. The fastest-moving vector-borne disease ever encountered within North America, WNV has firmly established itself within the United States. The virus has been detected within 45 states as well as Canada and Mexico, and experts predict it will be found in all 48 contiguous states this year. The speed with which the disease has spread has surprised scientists, the medical community, and the government.

A cure and control remain elusive. Clinically effective agents have yet to be identified; treatment is symptomatic and/or supportive, depending upon the severity of the infection. As a result, there has been much focus on prevention and diagnosis, and the government has taken steps to minimize the risk of contracting WNV with mosquito control programs, public education, research grants, and industry collaboration.

Yet the case count, as reported to ArboNet, the national electronic surveillance system established by the Centers for Disease Control and Prevention (CDC) to assist states in tracking WNV and other mosquito-borne viruses, showed a large jump after the first week of August. On August 1, 69 cases and three deaths had been reported in 2003. By August 8, the case count had risen to 182 cases and five deaths, with additional cases and deaths awaiting confirmation. At the same time last year, 112 cases had been reported. In 2002, there were 4,156 cases and 284 deaths reported.

The main period of risk for contracting WNV is from July through September, though cases have been reported as early as May and as late as December. In Florida, where the weather is warm and humid year-round, risk is constant.

Classification: Flavivirus
WNV was first isolated in the West Nile district of Uganda in 1937—hence its name. It has since been recorded in Africa, Europe, the Middle East, west and central Asia, Oceania, and, more recently, North America. In addition to the US outbreak, epidemics have occurred recently in Israel (2000) and Russia (1999).

Classified as a Flaviviridae (genus Flavivirus), the WNV family includes Japanese encephalitis, Kunjin, Murray Valley encephalitis, and St Louis encephalitis. According to the CDC’s Division of Vector-Borne Infectious Diseases, flaviviruses share a common size (40–60 nm), symmetry (enveloped, icosahedral nucleocapsid), nucleic acid (positive-sense, single-stranded RNA approximately 10,000–11,000 bases), and appearance in the electron microscope.

  An electron micrograph of WNV. A typical member of the flaviviruses, the virus is roughly 50 nm in diameter. Source: Public Health Image Library, CDC/Cynthia Goldsmith

Typical of the group, the WNV particle is approximately 50 nm in diameter and consists of a host-derived lipid bilayer membrane surrounding a nucleocapsid core containing a single-stranded, positive-sense RNA genome of approximately 11,000 nucleotides. The virus can be divided genetically into two lineages, only one of which has been definitely associated with human disease. The strain introduced in New York in 1999 has been traced to the Middle East, but it is not yet known how it made the trip.

Nature’s natural reservoir for WNV has been identified as birds. The virus is amplified and sustained through a mosquito-bird-mosquito transmission cycle. When a mosquito bites, it injects into the skin of the bitten person or animal a small amount of blood, which may carry diseases such as WNV or human immunodeficiency virus (HIV). In this way, mosquitoes have passed the infection to humans and other animals, including horses, cats, and dogs. These mammals are considered “dead-end” or incidental hosts; the level of virus in their blood does not become sufficient enough for them to be reservoirs and contribute to the transmission cycle. Though no human-to-human transmission has been recorded, the CDC confirmed transmission by transfusion last year, and a study published in the May issue of The New England Journal of Medicine (“Transmission of West Nile Virus from an Organ Donor to Four Transplant Recipients”) documents the transmission of WNV by organ transplantation into four recipients.

WNV Statistics
Such news concerns public health officials because of the increased risk factors associated with transfusion and transplant patients. These groups frequently have weakened immune systems, making them more vulnerable to infections. Indeed, of the four persons infected from the one organ donor, three of the cases progressed to encephalitis and one developed febrile illness.

Though 80% of WNV-infected persons will never realize they have been infected and remain asypmtomatic, 20% will develop a mild illness, and one in 150 will suffer severe neurological disease, with encephalitis reported more frequently than meningitis. Victims of advanced age, particularly those more than 70 years old, and those with weakened immune systems are at greater risk for progression of the disease to WN encephalitis.

The incubation period for the virus is thought to range from 3 to 14 days. Those with WN fever experience flu-like symptoms for about 3 to 6 days, including malaise, anorexia, nausea, vomiting, eye pain, headache, myalgia, rash, and lymphadenopathy. Victims of WN encephalitis suffer more severe symptoms, such as fever, weakness, gastrointestinal distress, and changes in mental status. Overall fatality rates range from 4% to 14%; during the 1999 outbreak in New York, 12% of patients died.

According to the CDC, the laboratory findings in victims of recent outbreaks show:

  • Total leukocyte counts in peripheral blood mostly normal or elevated, with lymphocytopenia and anemia also occurring;
  • Hyponatremia sometimes present, particularly among patients with encephalitis;
  • Cerebral spinal fluid (CSF) examinations indicating pleocytosis, usually a predominance of lymphocytes;
  • Protein universally elevated; and
  • Glucose normal.

In addition, computed tomographic scans of the brain primarily did not show evidence of acute disease, but in roughly one third of patients, magnetic resonance imaging (MRI) showed enhancement of the leptomeninges, the periventricular areas, or both.

Effective Diagnostics: Antibody Testing
Brain imaging, using either CT or MRI, as well as electroencephalography (EEG), may be used in developing a diagnosis. However, health care providers typically rely on two familiar methods: virus and/or antibody detection.

Serology tests identifying antibodies IgM and IgG have been developed, with IgM often the preferred choice. Wayne Hogrefe, PhD, vice president of Marketing, Focus Technologies in Herndon, Va, explains: “IgG is indicative of long-term immunity and will be present in anyone who has been exposed to the virus in the past but who is no longer infective. IgG lasts a lifetime.” IgM, however, is more indicative of a recent infection, though Hogrefe notes that research has shown it to remain in the blood for up to 6 months. Some studies have shown its presence more than a year later.

 Gen-Probe’s Procleix, distributed by Chiron, uses a nucleic acid amplification technique called transcription-mediated amplification (TMA) to detect WNV RNA in donated blood.

Still, according to the CDC, “the most efficient diagnostic method is detection of IgM antibody to WNV in serum collected within 8 to 14 days of illness onset or CSF collected within 8 days of illness onset using the IgM antibody capture enzyme-linked immunoabsorbent assay (MAC-ELISA). Since IgM antibody does not cross the blood-brain barrier, IgM antibody in CSF strongly suggests central nervous system (CNS) infection.”

The first test for use as an aid in the clinical laboratory diagnosis of WNV employs this technique and was cleared by the US Food and Drug Administration (FDA) in July—PanBio’s West Nile Virus IgM Capture ELISA requires confirmation of positive results with an additional test. Focus Technologies completed clinical trials on its West Nile Virus IgG and IgM ELISA diagnostic kits and has filed a 510(k) application with the FDA; its test also requires confirmation.

Confirmation eliminates false positives resulting from recent vaccinations for or infections from related flaviviruses. To prevent this, the test battery should include other flaviviral antigens for comparison, and the diagnosis should be confirmed with the plaque reduction neutralization test (PRNT), which can also determine if the infection is past or acute. In addition, PRNT can be used to confirm results of indirect immunoflourescence and hemagglutination inhibition tests, which some health care providers still use, though not as frequently.

The IgM antibody test may also show a false negative if performed too early in the infection cycle, before the body has been able to manufacture any antibodies. Among persons infected in New York City in 1999 and 2000, 90% of serum samples obtained within 8 days of symptom onset were positive for IgM antibody, and 70% to 80% of patients had detectable levels of IgM by the 8th day of illness.

Effective Blood Screening: Nucleic Acid Testing
At this point, the level of virus in the blood has typically decreased. “The viremic stage is very short. By the time a patient feels ill, he has usually begun clearing the virus,” says Hogrefe. Because a WNV-infected person may not realize he or she is ill, screening donated blood has become a top priority for government health care agencies, blood banks, and health care providers. The organ donor in The New England Journal of Medicine study mentioned earlier received the WNV infection from a blood transfusion; subsequent testing found that one of the 63 possible blood donors tested seropositive for WNV IgM antibodies during the 2 months following donation. The study concluded that this person was the original source of infection, confirming transmission by both transfusion and transplant.

Before the degree of risk could even be determined, the FDA took immediate action. Blood establishments have been ordered to defer donors not in good health; those with recent headaches or fevers are to be turned away. Additional screening questions to identify these symptoms (ie, asking about headaches) have been added. If a probable or proven transmission case is discovered, the product will be quarantined, removed from the blood supply, and destroyed.

The FDA called a public scientific meeting to discuss accelerated development of commercial tests to screen blood donors and prevent WNV-contaminated blood from entering the blood supply. The meeting was widely attended by the blood industry, and the FDA challenged its manufacturers to develop a product in time for the 2003 WNV season, with a deadline to begin screening on July 1.

The industry met the challenge. According to the CDC, every blood bank in the United States has been screening donated blood for WNV since July 14.

Some centers began screening even earlier. An FDA spokesperson notes that both Roche Diagnostics and Gen-Probe Inc filed Investigational New Drugs (INDs) with the agency ahead of the July 1 deadline. Both tests detect viral levels rather than antibodies, which are typically not yet being produced in the asymptomatic individual.

On May 16, the FDA accepted the protocol for Gen-Probe’s nucleic acid blood screening test for the detection of WNV, which will be distributed by Chiron Corporation. The Procleix test is undergoing clinical trials, after which the company will submit a Biologics License Application (BLA) to the FDA to permit commercial sales of the product. The assay uses a nucleic acid amplification technique called transcription-mediated amplification (TMA), which detects and amplifies viral nucleic acids in the test tube. The test is run on the same test platform as an HIV-1/HCV assay and, as a result, requires little additional operator training. Kim Weissenburger, a Chiron spokesperson, says, “The test runs on the same instrumentation as the current screen of tests. The WNV product requires an upgrade to the software to pull results, so operator training is minimal. The first results are seen within 5 hours.”

Roche also seeks to minimize operator training and error, having produced an automated test that also screens for other flaviviruses, including Japanese encephalitis, Kunjin, and St Louis encephalitis. According to James Gallardo, director of Blood Screening Development at Roche, the TaqScreen West Nile Virus test utilizes polymerase chain reaction (PCR) technology, which is “the gold standard of testing worldwide for detection of the virus and detection in the preseroconversion period.”

Roche’s test was also available early, with its screening products June 16 at the South Bend Medical Foundation, one of 11 clinical trial sites evaluating the new system. By July 3, the test had already identified a unit of blood carrying WNV; the infected blood was intercepted at the Gulf Coast Regional Blood Center in Houston, Tex, another of the trial sites.

Both Gen-Probe and Roche completed development within 9 months, a remarkable amount of time according to spokespeople at both companies.

Vaccines in Development

While the FDA has been focused on the nation’s blood supply and preventing spread of the WNV through transfusion, other government agencies, such as the National Institute of Allergy and Infectious Diseases (NIAID), have focused on other prevention methods, including vaccines and drug treatments.

NIAID states, “Drugs may be effective against West Nile Virus because the infection is typically not chronic and antiviral drugs have been identified to be effective in the laboratory against other flaviviruses … NIH [National Institutes of Health] has funded investigators to establish a system to screen chemical compounds for possible antiviral activity against WNV. … More than 550 drugs have been screened and approximately 3% have shown promise for additional testing in animals.”

Other research has found ribavirin in high doses, and interferon alpha-2b has some activity against WNV in vitro, but controlled studies have yet to be completed.

Vaccine research has shown more potential more quickly. In 1999, NIAID funded a fast-track project of its own. Working with biotechnology firm Acambis, NIAID found that the research has resulted in a prototype vaccine that will, according to company spokesperson Lyndsay Wright, begin Phase I human clinical trials this year. The trials will address human safety and efficacy of the vaccine candidate, which has been successfully tested in hamsters, mice, monkeys, and horses.

“The candidate vaccine uses an attenuated yellow fever backbone and substitutes the genes encoding the coat proteins with those of the target—ie, West Nile—so that it expresses a West Nile coat but has the safety/replication profile of a yellow fever vaccine,” says Wright. The technology, ChimeriVax, has already been applied to a Japanese encephalitis vaccine, which is in Phase II clinical trials, and a dengue vaccine, in Phase I.

NIAID intramural scientists have also used this chimeric technology to develop a WNV vaccine candidate that uses dengue virus as the backbone. Expected to begin Phase I human trials in late 2003, it has already been tested in monkeys with promising results.

In addition, the NIAID Vaccine Research Center (NRC) is researching WNV vaccines, focusing on preclinical studies of DNA constructs that express WNV proteins and production of clinical-grade plasmid DNA for future Phase I trials.

Independent research is also under way. David Watumull, president and CEO of Hawaii Biotech Inc, states that his company is conducting animal studies of a genetically engineered WNV vaccine. “Genetically engineered vaccines have an advantage over live, attenuated vaccines in that they do not carry any risk of infection from the actual virus. Different vaccines will have different rates of infection, but every vaccine must be evaluated in terms of risk of infection versus benefits of protection.”

Noninfectious vaccines may be preferable for the elderly and immune-compromised who would be the logical targets of a vaccine program. Other volunteer targets would include those who experience high exposure to mosquitoes, whether through work or leisure.

A study reported in Emerging Infectious Diseases (“Efficacy of Killed Virus Vaccine, Live, Attenuated Chimeric Virus Vaccine, and Passive Immunization for Prevention of West Nile Virus Encephalitis in Hamster Model”) evaluated the efficacy of a killed virus vaccine; a live, attenuated chimeric virus vaccine; and passive immunization in the hamster model and found that all three protected the animals from clinical illness and death. However, “the live, attenuated chimeric virus vaccine candidate induced the highest humoral antibody responses,” which led researchers to conclude that it “provides the longest lasting immunity.”

Mosquito Control as Prevention

Until a vaccine is approved, however, the public will have to rely on mosquito control—both personal and public health protective measures—to avoid contracting the infection at all. Within the United States, the Culex species is thought to play a large role in disease transmission, though other species have also been identified as carriers.

Personal mosquito protective measures include reducing time outdoors, particularly during peak insect activity at dusk and dawn; wearing long pants and long-sleeved shirts, with light-colored items providing greater contrast for spotting mosquitoes; applying mosquito repellant to clothing and exposed skin areas; keeping screens and windows in good repair; and eliminating standing water around the home, which provides a breeding ground for mosquitoes.

Public health measures also target water, spraying pesticides in these habitats to eliminate either mosquito larvae or adults, as well as surveillance systems. The United States Geological Survey (USGS) is working with the CDC on a 3-year study to learn the current extent of WNV; to understand how it moves between birds, mosquitoes, and humans; and to predict future movements of the virus. Active wild bird surveillance along the Atlantic Flyway and simultaneous collection of mosquitoes are used to detect the presence of WNV. Graphical displays and animated programs compile the data to determine the virus’s pattern and spread.

Additional government-funded research is occurring at Cornell University’s Northeast Regional Climate Center (NRCC) and the Department of Entomology. Its goal is to develop a Web-based, degree-day calculator that will warn public health officials when, where, and under which conditions infectious mosquitoes may thrive or die.

A habitat’s temperature may also determine the mosquito species that reside there. “Adults of different mosquito species may prefer different habitats,” says Allen James, president of Responsible Industry for a Sound Environment (RISE), “but all species have a larval stage that thrives in similar breeding grounds.”

James advocates an integrated approach to control. Larvicide and adulticide, the targeted pesticide killing of larvae and adult mosquitoes, respectively, each play a role in mosquito control. “Areas where standing water cannot be eliminated should be sprayed with a larvicide. This should be later followed with an adulticide to eliminate those mosquitoes that escaped the initial spraying,” says James.

Unfortunately, adulticides tend to kill their victims on contact only, meaning that they do not spread through the mosquito population as easily as WNV has spread through the North American population and are therefore not as effective as their intended victims. Given enough time, however, prevention methods will take hold—and since many of these methods are on the fast track, they are in a race with the virus, a race public health officials are determined to win.

Renee DiIulio is a contributing writer for Clinical Lab Products.

1. Lyndsay Wright, Director of Communications, Acambis, 44.0.1223.275.316, fax: 44.0.1223.416.300, [email protected]
2. Kim Weissenburger, Chiron Corporation, 510.923.5706, [email protected]
3. Paul Richards, FDA Spokesperson/Trade Press Liaison, Center for Biologics, 800.835.4709
4. Wayne Hogrefe, PhD, Focus Technologies, 800.445.0185, ext. 2034
5. David Watumull, Hawaii Biotech Inc, 808.486.5333, [email protected]
6. Allen James, Responsible Industry for a Sound Environment, 202.872.3862
7. James Gallardo, PhD, Roche Diagnostics; PR contact: Melinda Baker, 925.730.8379, [email protected]
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