Continuing education for physical therapists,
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Continuing education for physical therapists,
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West Nile Virus This course is free through September 30, 2008. Occupational therapy courses are accredited by AOTA and are accepted by the NBCOT Certification Renewal program. For information specific to this course, click here. Physical therapists—please click here for accreditation information.
West Nile virus (WNV) is a mosquito-borne virus that was recently introduced into the temperate regions of North America and Europe. While it is not known how long the virus has been in the United States, CDC officials believe it has been in the eastern United States since at least summer 1999 (CDC, 2004f). Experts believe WNV is established as a seasonal epidemic in North America that flares up in the summer and continues into the fall (CDC, 2006f). West Nile virus is an arbovirus, meaning it is spread by an arthropod—in this case the Culex mosquito. Birds infected by a WNV-carrying mosquito inadvertently spread the virus when mosquitoes take a blood meal from the bird (Figure 1). Although most birds infected with WNV will not die, the occurrence of large numbers of bird deaths is a sentinel event and signals the presence of WNV in a particular geographic area. 
Figure 1 Mosquito feeding. Culex pipiens mosquitoes carry West Nile virus. Photo courtesy of CDC. People and other animals, particularly horses, can be infected by a mosquito that has fed on an infected bird. Because the virus must enter the bloodstream to cause an infection, it is not likely for an individual to become infected by having casual contact with infected birds, animals, or people. There is no evidence that birds can directly infect other birds or animals. There are no reported cases of animal-to-human infection other than mosquitoes. There are a number of alternative modes of transmission to humans of WNV; however, it should be noted that these represent a very small proportion of cases. These alternative modes are: organ transplants, blood transfusions, breast milk, trans-placental (mother-to-child), and occupational exposure (CDC, 2007a). West Nile virus can cause mild or severe symptoms. Most people who become infected with WNV develop no clinical illness or symptoms. In previous outbreaks in the Northern Hemisphere, an estimated 80% of people who became infected never developed symptoms attributable to the infection. Of the approximately 20% of infected people who do develop symptoms, most develop what has been termed West Nile fever. The incubation period for WNV infection is thought to range from about 3 to 14 days from the time of the mosquito bite, although longer incubation periods have been documented in immunosuppressed persons (CDC, 2007a, 2004a). The most severe response to being infected with West Nile virus is sometimes called "neuro-invasive disease," because it affects a person's nervous system. Specific types of neuro-invasive disease include:
Serious illness can occur in people of any age, however people over age 50 and some who are immuno-compromised (eg, transplant patients) are at the highest risk for becoming severely ill when infected with WNV. Less than 1% of people who become infected with West Nile virus will develop severe illness—most people who get infected do not develop any disease at all (CDC, 2006a). GEOGRAPHIC DISTRIBUTIONWest Nile virus is present in Africa, Europe, the Middle East, west and central Asia, Oceania, and North America. In the United States from 1999 through 2007 WNV has been documented in every area except Alaska and Hawaii (see Figure 2, below). The discovery of virus-infected, overwintering mosquitoes during the winter of 1999–2000 in New York focused on identifying and documenting WNV infections in birds, mosquitoes and horses as sentinel animals that could predict the occurrence of human disease. By the end of the 2000 transmission season, WNV activity had been identified in a twelve-state area from Vermont and New Hampshire in the north to North Carolina in the south (CDC, 2003a). The 2002 WNV epidemic was the largest recognized arboviral meningoencephalitis epidemic in the Western Hemisphere and the largest WNV meningoencephalitis epidemic ever recorded. Significant human disease activity was recorded in Canada for the first time, and WNV activity was also documented in the Caribbean basin and Mexico (CDC, 2003a). By 2004 WNV had spread to the west coast of the United States; in early 2004 the first confirmed case of WNV was reported in California, including one death. By the end of 2007, WNV had spread to all areas of the country except Alaska and Hawaii (Figure 2) (CDC, 2008). 
Figure 2 2007 West Nile Virus activity in the United States. This map reflects surveillance findings occurring from January 1, 2007 through December 31, 2007 as reported to CDC's ArboNET system for public distribution by state and local health departments. Map shows the distribution of avian, animal, or mosquito infection occurring during 2007 with the number of human cases, if any, by state. If West Nile virus infection is reported to CDC from any area of a state, that entire state is shaded. EPIDEMIOLOGYWest Nile virus is a member of the family Flaviridae (genus Flavivirus), part of the Japanese encephalitis virus complex that includes St. Louis encephalitis (SLE), Japanese encephalitis, dengue, tick-borne encephalitis, yellow fever, Kunjin, and Murray Valley encephalitis viruses, as well as others. For unknown reasons, deaths among birds from WNV infection have occurred only in the United States, Israel, Canada, and Mexico. Since 1999, very few genetic changes have occurred in the WNV strains circulating in the United States (CDC, 2004b). West Nile virus (WNV) was first isolated and identified in 1937 in a febrile person in the West Nile district of Uganda. Prior to 1999, the virus was found only in the Eastern Hemisphere, with wide distribution in Africa, Asia, the Middle East, and Europe. There were infrequent reports of human outbreaks, mainly associated with mild febrile illnesses, in Israel and Africa. These were mostly in groups of soldiers, children, and healthy adults. One notable outbreak in Israeli nursing homes in 1957 was associated with severe neurologic disease and death (CDC, 2004c). Since the mid-1990s, the frequency and apparent clinical severity of WNV outbreaks have increased. Outbreaks in Romania (1996), Russia (1999), and Israel (2000) involved hundreds of persons with severe neurologic disease. It is unclear if this apparent change in disease severity and frequency is due to differences in the circulating virus's virulence or to changes in the age structure, background immunity, or prevalence of other predisposing chronic conditions in the affected populations (CDC, 2004c). The first appearance of WNV in North America was in 1999, with encephalitis reported in humans and horses, and the subsequent spread in the United States may be an important milestone in the evolving history of this virus (CDC, 2003a). The spread of WNV in North America has been rapid. From 1999 through April 8, 2008 there were more than 27,000 confirmed cases of WNV human illness in the United States reported to CDC and 1086 fatalities (Table 1).
Among all reported cases in humans the median age is 55; 54% of cases are in men and 46% are in women. In 2002 the epidemic peaked during the week ending August 17 in the southern states and the week ending August 24 in the northern states. During 2002, Illinois, Michigan, and Ohio had the highest caseloads (CDC, 2003a). Table 2 shows the activity of West Nile virus for the year 2007.
ENTOMOLOGYMosquito Life CycleMosquitoes are insects. They go through four life stages (complete metamorphosis) and look completely different at each stage: egg, larva, pupa, and adult. The first three life stages are spent in water or wet places. Adults emerge from the pupal stage full size and able to fly. Adult females bite to get a blood meal that provides the nutrients they need to form each brood of eggs (CDC, 2004d). 
Figure 3 The mosquito life cycle. Courtesy of CDC. There are about 200 different species of mosquitoes in the United States, all of which live in specific habitats, exhibit unique behaviors, and bite different types of animals. Despite these differences, all mosquitoes share some common traits, such as a four-stage life cycle. After the female mosquito obtains a blood meal (male mosquitoes do not bite), she lays her eggs directly on the surface of stagnant water, in a depression, or on the edge of a container where rainwater may collect and flood the eggs (CDC, 2004d). 
Figure 4 A female mosquito laying her eggs in a stagnant body of water. Courtesy of CDC. The eggs hatch and a mosquito larva or "wriggler" emerges. The larva lives in the water, feeds and develops into a pupa or "tumbler." The pupa also lives in the water, but no longer feeds. Finally, the mosquito emerges from the pupal stage and the water as a fully developed adult, ready to bite (CDC, 2004d). Culex MosquitoesAlthough over 60 species of mosquitoes have tested positive for the West Nile virus, Culex pipiens (Northern house mosquitoes) are the species of mosquitoes most closely associated with transmitting WNV in the Northeast United States. These mosquitoes "prefer" to bite birds, but if breeding sites are available near homes and domestic animal enclosures, Culex pipiens may bite people and domestic animals. Culex pipiens are most active between dawn and dusk. Culex quinquefasciatus (Southern house mosquitoes) fill this niche in the southern United States (CDC, 2003a). Some other types of mosquitoes have been found to be WNV-positive in the United States. These include Culex salinarius and Aedes vexans, which are of potential concern because they feed more readily on mammals, including humans, than do other mosquito species associated with WNV. (Cx. Pipiens, Cx. restuans, and Culiseta melanura all prefer to feed on birds). Some of these species bite during the daytime. In all, 62 species of mosquitoes are known to be infected with WNV (CDC, 2007b, 2003a). Transmission CycleIn 1999 WNV was transmitted principally by Culex species mosquitoes. In 2000 a total of fourteen WNV-infected mosquito species were identified, although 89% of positive mosquito pools were Culex. In contrast to Culex, many of these other species are daytime feeders and mammal feeders. The effect that this widened spectrum of WNV-infected mosquito species will have on WNV ecology in the United States is not known (CDC, 2003a). 
Figure 5 West Nile virus transmission cycle. Courtesy of CDC. Infectious mosquitoes carry virus particles in their salivary glands and infect susceptible bird species during blood-meal feeding. Birds will sustain an infectious viremia (virus circulating in the bloodstream) for 1 to 4 days after exposure, after which these hosts develop lifelong immunity. People, horses, and most other mammals are not known to develop infectious-level viremias very often, and thus are probably "dead-end" or incidental-hosts (CDC, 2007c). BirdsMortality in a wide variety of bird species has been a hallmark of WNV in the United States. The reasons for this are not known; however, public health officials were able to use bird mortality (particularly birds from the family Corvidae such as crows, blue jays, and ravens) to effectively track WNV expansion in 2000. Although WNV infection is fatal in a large percentage of Corvids, many other bird species survive WNV infection. It is not known if affected birds develop immunity or if they become chronically infected and susceptible to related illness during times of stress. Supportive treatment with antibiotics and nonsteroidal anti-inflammatories early in the illness appears to help. Migrating birds likely contribute to natural transmission cycles and dispersal of the virus (Cornell University, 2005a). Through April 2007 WNV had been detected in at least 317 bird species. The following bird species have been reported to CDC's West Nile Virus avian mortality database from 1999 to 2007 (CDC, 2007d).
There is no evidence that an individual can get WNV from handling live or dead infected birds. But people should avoid bare-handed contact when handling any dead animals, and use gloves or double plastic bags to place the bird carcass in a garbage bag or contact their local health department for guidance (CDC, 2007c). Dogs and CatsWest Nile virus does not appear to cause extensive illness in dogs or cats. There is a single published report of WNV isolated from a dog in southern Africa (Botswana) in 1982. West Nile virus was isolated from a single dead cat in 1999. A sero-survey in New York City of dogs in the 1999 epidemic area indicated that dogs are frequently infected. Nonetheless, disease from WNV infection in dogs has yet to be documented (CDC, 2007c). There is no documented evidence of person-to-person or animal-to-person transmission of WNV. Because WNV is transmitted by infectious mosquitoes, dogs or cats could be exposed to the virus in the same way humans become infected. Veterinarians should take normal infection-control precautions when caring for an animal suspected to have WNV infection (CDC, 2007c). It is possible that dogs and cats could become infected by eating dead infected animals such as birds, but this is undocumented. There is no reason to destroy an animal just because it has been infected with WNV. Full recovery from the infection is likely. Treatment would be supportive and consistent with standard veterinary practices for animals infected with a viral agent (CDC, 2007c). HorsesCases of WNV disease in horses have been documented, either by virus isolation or by detection of WN virus-neutralizing antibodies in 1999, 2000, and 2001 (Figure 6). Approximately 40% of equine WN virus cases results in the death of the horse. Horses most likely become infected with WNV in the same way humans become infected, by the bite of infectious mosquitoes (CDC, 2007c). 
Figure 6 Equine cases of West Nile virus during 2007, state by state. Total cases: 468. Courtesy of USDA, 2008. In locations where WNV is circulating, horses should be protected from mosquito bites as much as possible. Horses vaccinated against Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), and Venezuelan equine encephalitis (VEE) are not protected against WNV infection (CDC, 2007c). According to the American Association of Equine Practitioners (AAEP), 1086 cases of equine WNV encephalitis were predicted in the United States; however, as of October 2007, only 250 cases had been reported. The presumption is that the decline is due to both vaccination programs and natural immunity (AAEP, 2008). The mortality rate for horses exhibiting clinical signs of West Nile virus infection is approximately 33%. Data has supported that 40% of horses that survive the acute illness caused by WNV still exhibit residual effects, such as gait and behavioral abnormalities that were attributed to the illness by owners 6 months post diagnosis (AAEP, 2008). There is no reason to destroy a horse just because it has been infected with WNV. Data suggest that most horses recover from the infection. Treatment would be supportive and consistent with standard veterinary practices for animals infected with a viral agent (CDC, 2007c). Signs of severe WNV illness in horses may include ataxia (lack of coordination, stumbling, staggering), difficulty walking, knuckling over, head tilt, muscle twitches or tremors, inability to stand, circling, weakness or paralysis of limbs, apparent blindness, lip droop, grinding teeth, and death. However, these symptoms could also be caused by other diseases including rabies, eastern and western equine encephalitis virus infections, and equine herpes virus-1 infection (USDA, 2004). HORSE VACCINEThere are currently three licensed equine vaccines available: inactivated whole virus vaccine, recombinant vector vaccine, and modified live chimera vaccine. Efficacy of all three vaccines appears to be approximately 95%. Schedules and procedures for each vaccine vary and more detailed information is available at http://www.aaep.org/wnv.htm. In all cases horses should be vaccinated prior to mosquito season and booster shots will be necessary (AAEP, 2008). Use of these vaccines is no longer restricted to licensed veterinarians (USDA, 2004). Other VertebratesThe CDC has also received a small number of reports of WNV infection in bats, a chipmunk, a skunk, a squirrel, and a domestic rabbit (Cornell University, 2005a). CLINICAL FINDINGSSigns and SymptomsMost people who become infected with WNV do not get sick. If infection does occur, it may take 3 to 14 days to become ill. Older people are more likely to become very ill from WNV. It is estimated that about 20% of the people who become infected will develop West Nile fever: the symptoms include fever, headache, and fatigue. Occasionally symptoms may include a skin rash on the trunk of the body, swollen lymph glands, or eye pain (CDC, 2004a). Most WNV infections are mild and often clinically unapparent:
Severe infection (West Nile encephalitis or meningitis) is sometimes referred to as "neuro-invasive disease." It is estimated that approximately 1 in 150 persons infected with the WNV will develop a more severe form of disease (CDC, 2006a, 2004a). The most significant risk factor for developing severe neurologic disease is advanced age. Encephalitis is more commonly reported than meningitis. Neurologic presentations included:
A minority of patients with severe disease developed a maculopapular or morbilliform rash involving the neck, trunk, arms, or legs. Several patients experienced severe muscle weakness and flaccid paralysis (CDC, March 2004). Although not observed in recent outbreaks, myocarditis, pancreatitis, and fulminant hepatitis have been described (CDC, 2004a). In 1999 in New York, approximately 40% of laboratory-positive humans with encephalitis or meningitis had severe muscle weakness; 10% developed flaccid paralysis with electromyographic findings consistent with axonal neuropathy. Healthcare and Laboratory WorkersPeople working outdoors when mosquitoes are actively biting are at risk of infection and should be educated about this occupational health issue and available recommendations. Although WNV is most often transmitted by the bite of infected mosquitoes, the virus can also be transmitted through contact with infected animals, their blood, or other tissues. Thus laboratory, field, and clinical workers who handle tissues or fluids infected with WNV or who perform necropsies are at risk of WNV exposure (CDC NIOSH, 2006). These workers include laboratory diagnosticians and technicians, pathologists, researchers, veterinarians and their staff, wildlife rehabilitators, entomologists, ornithologists, wildlife biologists, zoo and aviary curators, health care workers, emergency response and public safety personnel, public health workers, and others in related occupations (CDC NIOSH, 2006). WNV may be present in blood, serum, tissues and CSF of infected humans, birds, mammals and reptiles. The virus has been found in the oral fluids and feces of birds. Parenteral inoculation with contaminated materials poses the greatest hazard for laboratory workers and contact exposure of broken skin is a possible risk. Sharps precautions should be strictly adhered to when handling potentially infectious materials. Workers performing necropsies on infected animals may be at high risk of infection (CDC, 2003a). TESTING AND TREATMENTWest Nile virus (WNV) infection can be suspected in a person based on clinical symptoms and patient history. Laboratory testing is required for a confirmed diagnosis. The most commonly used WNV laboratory test measures antibodies that are produced very early in the infected person. These antibodies, called IgM antibodies, can be measured in blood or cerebrospinal fluid (CSF), which is the fluid surrounding the brain and spinal cord. This blood test may not be positive when symptoms first occur; however, the test is positive in most infected people within 8 days of onset of symptoms. A test for WNV IgM-antibody is used by CDC, state and local public health labs, and, increasingly, at private laboratories. When testing is conducted at private laboratories, the health department or CDC will often confirm results in their own laboratories before officially reporting WNV cases. In some instances, health departments may conduct or request additional testing from CDC before officially reporting a case to CDC's ArboNET Surveillance System. The state or CDC reference laboratory may repeat the initial IgM-antibody testing. A state may also perform or ask CDC to perform an additional, different test on a specimen (CDC, 2007e). There is no specific treatment for WNV infection, and care is supportive in nature. In severe cases, supportive care often involves hospitalization, intravenous fluids, respiratory support, and prevention of secondary infections. (CDC, 2007e, 2004e). Clinical trials involving both drugs and potential vaccines are ongoing. In severe cases, when WNV meningitis, encephalitis, or other neurologic symptoms are present, intravenous hydration and mechanical ventilation may be needed. The American College of Physicians (ACP) recommends the following non-drug therapy in severe cases:
Drug therapy includes:
PREVENTIONExposure to WNV can be prevented in two ways: (1) personal protective measures to reduce contact with mosquitoes, and (2) public health measures to reduce the population of infected mosquitoes in the environment. Personal protection measures include reducing time outdoors, particularly in early evening hours, wearing long pants and long-sleeved shirts, and applying mosquito repellent to exposed skin areas (CDC, 2003a). Public health measures include elimination of larval habitats or spraying of insecticides to kill juvenile (larvae) and adult mosquitoes. In emergency situations, wide-area aerial spraying is used to quickly reduce the number of adult mosquitoes. Precautions should continue until there have been two hard frosts (CDC, 2003a). A critical component of any prevention and control program for vector-borne diseases is public education about these diseases, how they are transmitted, and how to prevent or reduce risk of exposure (CDC, 2003a). As stated earlier, mosquitoes breed in wet areas, and Culex are found particularly where there is decaying organic matter (eg, leaves, grass clippings, animal wastes). There does not have to be much water and the water does not have to be left standing for very long. Thus, they can reproduce throughout the mosquito breeding season in your area (and especially after each rainstorm, drizzle, watering of the garden, or washing of the car). Follow these guidelines for eliminating potential mosquito breeding habitat:
RepellentsRepellents are an important tool to assist people in protecting themselves from mosquito-borne diseases. A wide variety of insect repellent products are available. The CDC recommends the use of products containing active ingredients that have been registered with the U.S. Environmental Protection Agency (EPA) for use as repellents applied to skin and clothing. EPA registration of repellent active ingredients indicates the materials have been reviewed and approved for efficacy and human safety when applied according to the instructions on the label (CDC, 2007f, 2006e). Of the active ingredients registered with the EPA, two have demonstrated a higher degree of efficacy in the peer-reviewed literature. Products containing these active ingredients typically provide longer-lasting protection than others:
Repellents containing oil of lemon eucalyptus (p-menthane 3, 8-diol [PMD]), a plant-derived active ingredient, are also registered with EPA. In two recent scientific publications, when repellents containing oil of lemon eucalyptus were tested against mosquitoes found in the United States they provided protection similar to repellents with low concentrations of DEET. This recommendation refers to EPA-registered repellents containing the active ingredient oil of lemon eucalyptus (PMD). "Pure" oil of lemon eucalyptus (eg, essential oil) has not received similar, validated testing for safety and efficacy, is not registered with EPA as an insect repellent, and is not covered by this CDC recommendation (CDC, 2007f, 2006e). In addition, certain products that contain permethrin are recommended for use on clothing, shoes, bed nets, and camping gear, and are registered with EPA for this use. Permethrin is highly effective as an insecticide and as a repellent. Permethrin-treated clothing repels and kills ticks, mosquitoes, and other arthropods and retains this effect after repeated laundering. The permethrin insecticide should be reapplied following the label instructions. Some commercial products are available pretreated with permethrin (CDC, 2007f, 2006e). Permethrin is not to be used directly on skin (CDC, 2007f). Length of protection from mosquito bites varies with the amount of active ingredient, ambient temperature, amount of physical activity/perspiration, any water exposure, abrasive removal, and other factors. For long duration protection use a long-lasting (micro-encapsulated) formula and re-apply as necessary according to label instructions (CDC, 2007f, 2006e). VaccinesNo human vaccine against WNV is currently available. Clinical trials continue and promising results have been obtained in Phase II studies conducted by the biotechnology company Acambis on its ChimeriVax-West Nile vaccine; however, at this time no human vaccine is available. The best defense continues to reside in personal protective measures, public health measures, and surveillance and awareness. SURVEILLANCE AND CONTROLSurveillance is the organized monitoring of levels of virus activity, vector populations, infections in vertebrate hosts, human cases, weather, and other factors to detect or predict changes in the transmission dynamics of arboviruses. Optimal environmental conditions allow rapid increase of vectors and virus amplification in vertebrate hosts. It is urgent, therefore, that a well-organized surveillance program be in place well in advance of the virus transmission season (Moore et al, 1993). Surveillance is a high priority for states that are affected or that are at higher risk for being affected by WNV because of bird migration patterns and virus spread. Depending on the geographic location of the state, active surveillance should be implemented in the spring and continued until the late fall (for states where mosquito activity will cease because of cold weather) or through the winter months (for southern states where mosquito activity may be continuous throughout the year). In all states that face potential WNV activity, the following surveillance activities should be emphasized (CDC, 2003a):
Appropriate and timely response to surveillance data is the key to preventing human and animal disease associated with WNV and other arboviruses. If increasing levels of virus activity are detected in the birds or mosquitoes, the response must be effective mosquito control without delay. (For more information see Guidelines for Arbovirus Surveillance in the United States, available at http://www.cdc.gov/ncidod/dvbid/arbor/arboguid.htm.) CONCLUSIONIndividuals and healthcare workers need to remain vigilant about WNV, to protect themselves and to counter potential public health threats. West Nile cases have been identified in all 48 contiguous states, with most states presenting both avian/animal and human cases. Most people do not develop any symptoms after exposure to WNV but a small group will develop WNV fever and an even smaller group will develop more severe illness. Medical practitioners should remain vigilant about WNV, especially if they are in mosquito areas, and they should be prepared to follow their applicable state reporting requirements. Posted July 1, 2008 Expires July 1, 2010 Copyright © 2008 Wild Iris Medical Education. All rights reserved. REFERENCESAmerican Association of Equine Practitioners (AAEP). (2008). West Nile Virus. Retrieved April 26, 2008 from http://www.aaep.org/wnv.htm. American Association of Equine Practitioners (AAEP). (2005). West Nile Virus Vaccination Guidelines. Retrieved April 26, 2008 from http://www.aaep.org/pdfs/AAEP_WNV_Guidelines_2005.pdf. American College of Physicians (ACP). (2006). Physicians' Information and Education Resource. West Nile Virus Disease: Non-Drug Therapy. Retrieved May 29, 2006 from http://pier.acponline.org/physicians/public/d951/ CDC, Division of Vector-Borne Infectious Diseases. (2008). West Nile Virus: Statistics, Surveillance, and Control. Maps and Data. 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Retrieved April 20, 2008 from http://www.aphis.usda.gov/vs/nahss/equine/wnv/ada_wnv_2007.pdf. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
