Powassan virus Powassan virus (POWV) circulates in South Dakota, the Eastern and Western United States, Canada, and Far Eastern Russia and is readily differentiated from the other tick-transmitted virus species by serologic tests. It was first isolated in 1958 from the brain of a 5-year old boy who died from encephalitis in Powassan, Ontario, Canada. In North America, POWV causes severe encephalitis in humans with a high incidence of neurologic sequelae and up to 60% case-fatality rate. Powassan virus is a flavivirus and a member of the tick-borne encephalitis (TBE) antigenic complex. Tick-borne powassan virus (strain lb) The LB strain was isolated in 1958 in Ontario, Canada from a human brain. Shestopalova et al. undertook a comparative electron microscopic study of two POW strains: the prototype LB strain from Canada and strain AN-750 isolated from a pool of An. hyrcanus collected in the USSR. They studied virus in the cerebral and cerebellar cortex of newborn and adult white mice that had been inoculated I.S. Both strains exhibited identical morphological characteristics by electron microscopy. In North America, the virus is transmitted in a cycle involving small mammals (principally squirrels and ground hogs) and Ixodes species ticks, including Ixodes marxi and Ixodes cookei in the east, and Ixodes spinipalpis in the west. The genome RNA of flaviviruses is single-stranded and approximately 11 kilobases in length. The genomic RNA is infectious, and thus of positive polarity encoding the viral proteins necessary for RNA replication. Genome-length RNAs appear to be the only virus-specific mRNA molecules in flavivirus-infected cells. http://staff.vbi.vt.edu/pathport/pathinfo_images/Powassan_Virus/Genome.jpg Powassan virus, complete genome 10839 bp ss-RNA The genome of POW virus is 10839 nucleotides long and contains a single long open reading frame which codes for a 3415 amino acid-long polyprotein. The genome of POW virus is 10839 nucleotides long and contains a single long open reading frame which codes for a 3415 amino acid-long polyprotein. The computer analysis of this protein sequence and its comparison with TBE virus revealed the presence of homologous potential protease cleavage sites, internal signal sequences, stop transfer, and transmembrane sequences, suggesting that the POW virus polyprotein is co- and post-translationally cleaved into the mature viral protein (structural proteins C, (pr) M, and E; nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) in the same manner as other flaviviruses. Powassan virus infection appears to be one of the least common causes of arbovirus encephalitis reported in cases from the United States and Canada, ranking behind LaCrosse, St. Louis and eastern and western equine encephalitis. However, Powassan virus and eastern equine encephalitis have the dubious distinction of having the highest case-fatality rates and are associated with a very high incidence of neurologic sequelae. Humans are accidental victims when they enter into areas where the virus, the arthropod vector (an ixodid tick) and the vertebrate natural hosts coexist. Among the most commonly implicated natural hosts are the woodchuck and snowshoe hare. However, other animals that humans come into contact with including coyotes, foxes, raccoons and skunks have shown serological evidence of infection. Moreover, the scope of transmission of the virus may be broadened by domestic cats and dogs, which can act as harbingers of infected ticks and thereby expose humans. Cases of arbovirus encephalitis have been reported from Ontario, Quebec and New Brunswick in Canada and from New York, Pennsylvania and Massachusetts in the United States. Surveillance serologic studies have been positive in up to 3% of the population in certain northern Ontario communities, suggesting that infection without encephalitis can occur in humans. During September 1999 - July 2001, four Maine and Vermont residents with encephalitis were found to be infected with POW virus. Infected deer ticks (Ixodes scapularis) were allowed to attach to naive mice for variable lengths of time to determine the duration of tick attachment required for Powassan (POW) virus transmission to occur. Viral load in engorged larvae detaching from viremic mice and in resulting nymphs was also monitored. Ninety percent of larval ticks acquired POW virus from mice that had been intraperitoneally inoculated with 10(5) plaque-forming units (PFU). Engorged larvae contained approximately 10 PFU. Transstadial transmission efficiency was 22%, resulting in approximately 20% infection in nymphs that had fed as larvae on viremic mice. Titer increased approximately 100-fold during molting. Nymphal deer ticks efficiently transmitted POW virus to naive mice after as few as 15 minutes of attachment, suggesting that unlike Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum, no grace period exists between tick attachment and POW virus transmission. Infectious POW virus is present in tick salivary secretions inoculated during the earliest stages of feeding, and may be immediately inoculated. D. andersoni were fed on viremic New Zealand white rabbits and POW virus multiplication demonstrated in various tick organs including salivary glands, Gene's organ glands, and accessory glands. POW virus was transmitted to rabbits, hamsters, and guinea pigs by the bites of infected ticks. A lactating goat with a 74-day-old kid was inoculated with 10(3) mouse 50% lethal dose (LD50) of Powassan virus. No ensuing viremia could be detected, but virus was secreted in the milk on postinoculation days 7 through 15, with a titer of 10(5) LD50/ml on day 12. Neutralizing antibody was found in the serum on days 22 through 36 and in the milk on day 36. The offspring was not inoculated but was allowed to continue feeding on its mother's milk. It developed neutralizing antibody by day 22. Since the kid was past the age when it could resorb antibody from the milk, its serum antibody was evidence of active infection. Neither animal showed any clinical sign of illness. A serum survey of 499 goats in New York State showed that 9 had neutralizing antibodies to Powassan virus. These immune goats came from widely scattered localities, including counties where human cases have been confirmed. The findings suggest the possibility of milk-borne transmission of Powassan virus from goat to man. Neutralizing antibodies to Powassan virus have been found in numerous small mammals. Powassan virus has been isolated from marxi ticks collected from one red squirrel, T. hudsonicus, and from the blood of another squirrel. Isolations of Powassan virus, as well as high antibody prevalence in squirrels and ground hogs in the North Bay-Powassan region of Ontario, have led to the consideration of these animals as important reservoirs in the maintenance cycle of Powassan virus. The catholic host preference of POW vectors ensures that many vertebrates in endemic areas likely to encounter virus infection, including domestic animals. The viremia in many POW infected vertebrates has tended to be relatively low, i.e. 10(2.5) LD(50) or less. However, the long feeding periods of most ticks and their enormous meals may allow sufficient virus intake to infect ticks. Thus, numerous mammals, primarily of the orders Rodentia and Carnivora, likely can contribute to the POW amplification cycle. Vertebrate behavioral patterns, including the nesting and social interaction of potential reservoir hosts, are important contributing factors to the probability of animals serving as tick hosts. The use of a single den by several individuals, such as a female woodchuck and her offspring or males seeking females, provides opportunities for exchanges of POW-infected ticks. The population dynamics of the vertebrate hosts are of relevance. Many of the Rodentia and Carnivora that serve as reservoir hosts of POW virus have brief life spans (usually less than 2 or 3 years) and high reproductive rates (two to ten progeny per year). Thus, many new susceptibles are introduced into the population each year ensuring the ready availability of POW amplifying hosts in endemic areas. Main presented the following three postulated enzootic cycles for POW virus in North America: 1. Arboreal squirrels and Ix. marxi in the East. 2. Medium-sized rodents and carnivores and Ix. cookei in the East and the Midwest. 3. Small and medium-sized mammals and Ix. spinipalpus in the Northwest. Tick-borne flaviviruses are excreted in the urine and feces of experimentally infected animals but it is unlikely that this form of virus would provide an efficient route of infection for humans. Perhaps their greatest weakness as biological weapons is the fact that they are normally transmitted to vertebrate hosts via the bite of an infected tick, and the natural habitat of ticks is the forest or moist thick grassy vegetation as found on uplands. Under most circumstances this means that humans and even most animals would be a dead-end for virus transmission because few humans are exposed to the bite of a tick. Another important factor is that these viruses are all antigenically closely related. Therefore, immunity against one strain is likely to produce cross-immunity against the others. Moreover, in endemic regions there is a reasonably high level of immunity amongst the indigenous viruses. One can ask the question whether or not it is feasible to spread the virus by infecting large numbers of ticks with the virus. This would not be a logical approach for the following reasons: (a) very large numbers of infected ticks would be required and logistically this would be technically extremely difficult; (b) ticks only feed three times, at very critical stages of their life cycle and it would be extremely difficult to arrange for them to be infected and ready to feed when delivered as weapons; (c) the production of a sufficiently large number of ticks to pose a threat to human or animal populations would also be a difficult technical exercise. In summary, these viruses are unlikely to be the most effective front line weapons in biological warfare but they might be capable of causing significant problems on a small scale. In the context of bioterrorism, we have shown that the tick-borne flaviviruses are pathogenic for humans and some animals. Some strains are more virulent than others but even the most virulent viruses are unlikely to produce high fatality rates. These viruses can infect via the alimentary tract and also when inoculated intranasally into experimental animals. Presumably, therefore concentrated aerosols would be infectious or high virus concentrations delivered as a powder contaminating food might infect a significant proportion of people eating the food. One can ask the question whether or not it is feasible to spread the virus by infecting large numbers of ticks with the virus. This would not be a logical approach for the following reasons: (a) very large numbers of infected ticks would be required and logistically this would be technically extremely difficult; (b) ticks only feed three times, at very critical stages of their life cycle and it would be extremely difficult to arrange for them to be infected and ready to feed when delivered as weapons; (c) the production of a sufficiently large number of ticks to pose a threat to human or animal populations would also be a difficult technical exercise. Human Homo sapiens The disease occurs in Russia, Canada, and the United States. In North America, Powassan encephalitis has been reported in Ontario, New York, and Pennsylvania, with a total of 20 cases, 18 in people younger than 20 years of age, and one death. Most cases have been in males, probably reflecting increased outdoor activity and exposure to ticks. Human infections are rare; antibody surveys in the United States and Canada have generally shown prevalence rates of 0.5% to 4%. The virus has a much wider geographic distribution than indicated by case reports, and Powassan infection should therefore be suspected in cases of encephalitis throughout the United States and Canada. The prevalence of exposure to POW virus among human residents of deer tick-infested areas, and the relationship between viral inoculum and POW viral pathogenesis remains poorly described. It may be that a large viral inoculum, delivered over several hours or days, is required to produce illness in humans. Because there is no vaccine or specific therapy for POW encephalitis, the best means of prevention is protection from tick bite. This includes using insect repellents, wearing light-colored clothing with long sleeves and pants tucked into socks or boots, avoiding or clearing brushy areas, and removing ticks before they attach or as soon after attachment as possible. Checking family pets also can prevent ticks from entering the home. Because Ix. cookei are often found on woodchucks and skunks and may be the primary vector of POW virus, environmental controls reducing human contact with small and medium-sized mammals should reduce risk for exposure to POW virus-infected ticks. Persons should keep areas adjacent to their home clear of brush, weeds, trash, and other elements that could support small and medium-sized mammals. When removing rodent nests, avoid direct contact with nesting materials and use sealed plastic bags for disposal to prevent direct contact with ticks. There is currently no vaccine available for Powassan virus. Education is the best possible defense; people should be aware of tick-borne diseases and learn to avoid any contact with suspected vectors. Human protection is mainly achieved through wearing adequate clothing to minimize exposed skin, treating clothes with insecticides and avoiding or clearing brushy areas. The use of tick repellents and insecticides should be encouraged, and an effort should be made to control ticks in domestic and farm animals and in buildings that they frequent. Woodall and Roz demonstrated the possibility of POW virus transmission through goat's milk. Thus, avoidance of unpasteurized milk is desirable. Because so few cases of clinically apparent POW virus infection have been recorded, few definitive and generally applicable statements can be made about infection with this virus. In the few human infections described, onset is sudden, with headache and fever to 40 C and convulsions. Prodromal symptoms include sore throat, sleepiness, headache, and disorientation. Encephalitic cases are characterized by vomiting, prolonged fever or fever of variable length, respiratory distress, lethargy, and other nonspecific symptoms throughout the acute phase. Patients may become semicomatose with some paralytic manifestations, but general neurologic signs of meningeal irritation presage encephalitis, which is often severe. Most diagnosed cases display evidence of focal lesions, but one patient had major involvement of the right temporal lobe, more typical of herpes encephalitis. Russian workers, analyzing 14 cases of POW encephalitis, reported seven patients (one death) with signs of meningoencephalitic lesions, two with meningeal manifestations, and five with unapparent or uncomplicated febrile infections. POW encephalitis was characterized by signs of cerebellovestibular lesions, which differ from signs of tick-borne encephalitis. Patients may become semicomatose with some paralytic manifestations, but general neurologic signs of meningeal irritation presage encephalitis, which is often severe. The incubation period is at least one week after being fed on by an infected tick. The reported incubation periods for Powassan virus range from 8 to 34 days. POW encephalitis is associated with significant long-term morbidity and has a case-fatality rate of 10% to 15%. In Canada and the USA, POWV causes severe encephalitis in humans with a high incidence of neurological sequelae and up to 60% case fatality rate. In Far Eastern Russia POWV infections were described as milder than those produced by TBEV (tick-borne encephalitis virus). Over half (11/20) of the patients who survived had sequelae, and this rate may actually be higher because follow-up information was not available in some cases. Powassan encephalitis is the name applied to the human disease. Clinical disease has not been demonstrated in lower animals infected with Powassan virus in nature. Twenty-seven symptomatic cases of Powassan virus encephalitis have been reported in North America between 1958 and 1998; 23 are published, and 4 are unpublished. Of note, 11 of the 23 cases were acquired in Canada, 7 of those in Ontario, and the majority (10/12) of cases reported in the United States originated in New York state. Sore throat Prodromal symptoms include sore throat, sleepiness, headache, and disorientation. Sleepiness Prodromal symptoms include sore throat, sleepiness, headache, and disorientation. Headache In the few human infections described, onset is sudden, with headache and fever to 40 C and convulsions. Disorientation Prodromal symptoms include sore throat, sleepiness, headache, and disorientation. Vomiting Encephalitic cases are characterized by vomiting, prolonged fever or fever of variable length, respiratory distress, lethargy, and other nonspecific symptoms throughout the acute phase. Respiratory distress Encephalitic cases are characterized by vomiting, prolonged fever or fever of variable length, respiratory distress, lethargy, and other nonspecific symptoms throughout the acute phase. Convulsions In the few human infections described, onset is sudden, with headache and fever to 40 C and convulsions. Fever In the few human infections described, onset is sudden, with headache and fever to 40 C and convulsions. Encephalitic cases are characterized by vomiting, prolonged fever or fever of variable length, respiratory distress, lethargy, and other nonspecific symptoms throughout the acute phase. Hemiplegia The most common indication of neurologic damage was hemiplegia, but recurrent severe headaches and damage to the upper cervical cord resulting in paralysis and atrophy of shoulder muscles, a common feature of Russian spring-summer and Central European encephalitides, also have been reported. Minor memory impairment Recurrent severe headaches, minor memory impairment, and damage to the upper cervical cord resulting in paralysis and the wasting of right shoulder muscles were also reported. Paralysis and wasting of right shoulder muscles The most common indication of neurologic damage was hemiplegia, but recurrent severe headaches and damage to the upper cervical cord resulting in paralysis and atrophy of shoulder muscles, a common feature of Russian spring-summer and Central European encephalitides, also have been reported. Lethargy Encephalitic cases are characterized by vomiting, prolonged fever or fever of variable length, respiratory distress, lethargy, and other nonspecific symptoms throughout the acute phase. Hypotonia Neuromuscular manifestations, specifically hypotonia, have been documented in a middle-aged male patient from Ontario, Canada Spasticity Spasticity can persist for weeks after the initial illness in patients who eventually improve. Ophthalmoplegia Powassan infection was diagnosed from serum drawn 19 days after onset that was positive for Powassan-specific IgM and a neutralizing antibody titer of 1:640. PCR testing is not a requisite for diagnosis. Over the succeeding months, all nonocular signs improved. Seven months after onset, she had normal visual acuity, color vision, visual fields, lids, pupils, anterior segments, vitreous, and fundi. The right eye was slightly exotropic. She appeared unable to execute any eye movements on command, look to an eccentric target, or follow a target. However, with prolonged effort, after a 10- to 20-second delay, both eyes would drift conjugately 30 up or down and 20 left or right. The eyes would return slowly to the primary position. Doll's eye maneuvers elicited nearly full, but slow and delayed, conjugate excursions. Three months later, she could promptly initiate up-gaze, but the movements remained slow. No improvement occurred thereafter. Treatment is symptomatic. Mus musculus Powassan virus Simultaneous inoculation of mice with tick-borne and Powassan viruses was shown, depending on experimental conditions, to result either in stimulation of infection or its unchanged course as compared with monoinfection and inoculation with the viruses at 2--3-week intervals in cross protection of mice against the superinfecting virus. Simultaneous inoculation of mice with the two viruses was accompanied by their multiplication in the blood and brains of mice and formation of antihemagglutinating antibodies to each of them. In the virus population in the brains of mice there was either formation of a mixture of two viruses or their phenotypic mixing. In cross protection, multiplication of the superinfecting virus in the blood and brain of mice was slightly inhibited, the antihemagglutinating antibody to a second virus either did not form or appeared in low titers. Macaca mulatta, Rhesus monkey Powassan virus We have carried out a comparative study of the experimental infection of monkeys with the P-40 strain of the Powassan virus, isolated in the Primor'e Territory of the USSR, and with the Canadian prototype LB strain. The Powassan virus was found to be pathogenic for Macaca rhesus. The clinical and pathomorphological picture of the experimental encephalitis was studied, and the full identity of the infection produced in the monkeys by the P-40 strain and the Canadian LB strain of the Powassan virus was demonstrated. On electron microscopic examination of the central nervous system the virus was detected in the neurons, glial cells, and intercellular spaces. The virions of the strains studied have identical morphological parameters, being 37-45 nm in diameter and of spherical shape. The data obtained indicated a marked neurotropism of the virus. They will contribute to the elucidation of the role of the virus in the infection pathology of humans, i.e., in the differentiation of encephalitis cases not associated etiologically with the virus of the spring-summer tickborne encephalitis. Oryctolagus cuniculus, rabbit Powassan virus strain M794 Powassan virus strain M794, a member of the Flavivirus genus known to infect man and animals in Canada, was inoculated intracerebrally into rabbits and horses. No clinical signs were observed in rabbits, but widespread encephalitis resulted, characterized by lymphoid perivascular cuffing, lymphocytic meningitis, and lymphocytic choroiditis. The virus could not be re-isolated from the rabbit or horse brains. Equus caballus Powassan virus strain M794 Powassan virus strain M794, a member of the Flavivirus genus known to infect man and animals in Canada, was inoculated intracerebrally into rabbits and horses. In horses, eight days after inoculation, prominent neurological signs occurred and lesions were those of non-suppurative encephalomyelitis, neuronal necrosis, and focal parenchymal necrosis. The virus could not be re-isolated from the rabbit or horse brains. Cricetinae Powassan virus Transmission experiments were performed with Ixodes scapularis ticks from an uninfected laboratory colony. Immature and adult ticks were exposed to Powassan (POW) viremic hamsters and rabbits, respectively. Oral infection rates for engorged larvae, nymphs and females fed on POW-infected hosts were 10%, 40%, and 57%, respectively. Transstadial transmission rates for nymphs exposed to POW virus as larvae, adults exposed as larvae, and adults exposed as nymphs, were 9.5%, 10%, and 54%, respectively. Evidence of transovarial transmission occurred when two uninfected hamsters, exposed to F2 larvae and nymphs originally exposed to POW virus in the F1 nymphal stage, seroconverted to POW virus with hemagglutination inhibition titers of 80 and 5,120, respectively; the transovarial transmission rate was 16.6%. Capra hircus Powassan virus Latent infection and milk-borne transmission of POWV in goats has been demonstrated experimentally. Dermacentor andersoni POW virus has been isolated from four species in North America ticks including Ixodes cookei (22 reported isolates), Ix. marxi (1 isolate), Ix. spinipalpis (1 isolate), and D. andersoni (2 isolates). Dermacentor silvarum In Russia, POWV has been isolated from other tick species, namely I. persulcatus, H. neumanni, H. consinna and D. silvarum and replication in different tick species is believed to be a selection factor for different POWV strains. In the environment POWV was isolated mainly from D. silvarum possibly because these ticks have a shorter life cycle feeding twice annually as larvae and nymphs, giving them a potential advantage for virus transmission. Dermacentor variabilis In Canada, POWV has been isolated from Ixodes cookei that feeds mainly on groundhogs (Marmota monax), Ixodes augustus which often bite humans and cats and Ixodes scapularis and Dermacentor variabilis which frequently bite humans and dogs. Ixodes angustus In Canada, POWV has been isolated from Ixodes cookei that feeds mainly on groundhogs (Marmota monax), Ixodes augustus which often bite humans and cats and Ixodes scapularis and Dermacentor variabilis which frequently bite humans and dogs. Ixodes cookei In Canada, POWV has been isolated from Ixodes cookei that feeds mainly on groundhogs (Marmota monax), Ixodes augustus which often bite humans and cats and Ixodes scapularis and Dermacentor variabilis which frequently bite humans and dogs. Ixodes marxi POW virus has been isolated from four species in North America ticks including Ixodes cookei (22 reported isolates), Ix. marxi (1 isolate), Ix. spinipalpis (1 isolate), and D. andersoni (2 isolates). Taiga tick Ixodes persulcatus In Russia, POWV has been isolated from other tick species, namely I. persulcatus, H. neumanni, H. consinna and D. silvarum and replication in different tick species is believed to be a selection factor for different POWV strains. Black-legged tick Ixodes scapularis Powassan (POW) virus (Flaviviridae, Flavivirus) strains belonging to Lineage II (also referred to as tick virus), appear to circulate in nature in an enzootic cycle with deer ticks (Ixodes scapularis) serving as the main vector. In Canada, POWV has been isolated from Ixodes cookei that feeds mainly on groundhogs (Marmota monax), Ixodes augustus which often bite humans and cats and Ixodes scapularis and Dermacentor variabilis which frequently bite humans and dogs. Ixodes spinipalpis POW virus has been isolated from four species in North America ticks including Ixodes cookei (22 reported isolates), Ix. marxi (1 isolate), Ix. spinipalpis (1 isolate), and D. andersoni (2 isolates). Haemaphysalis spp. In Russia, POWV has been isolated from other tick species, namely Ixodes persulcatus, Haemaphysalis neumanni, H. consinna and Dermacentor silvarum and replication in different tick species is believed to be a selection factor for different POWV strains. Ochlerotatus togoi, Aedes togoi Phylogenetic analysis shows that POWV emerged as the most ancestral lineage of the mammalian tick-borne viruses. Interestingly, the virus has also been isolated from mosquitoes, Anopheles hyrcanus and Aedes togoi. Anopheles hyrcanus Phylogenetic analysis shows that POWV emerged as the most ancestral lineage of the mammalian tick-borne viruses. Interestingly, the virus has also been isolated from mosquitoes, Anopheles hyrcanus and Aedes togoi. Ammospermophilus 24 out of 214 Ammospermophilus nelsoni were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. Heermann's kangaroo rat Dipodomys heermanni 13 of 88 Dipodomys heermanni were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. Kangaroo rat Dipodomys nitratoides 37 of 1,004 Dipodomys nitratoides were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. North American porcupine Erethizon dorsatum Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson's ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. Yellow-pine chipmunk Tamias amoenus, Neotamias amoenus Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson's ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. Yellow-bellied marmot Marmota flaviventris Neutralization tests revealed POW antibodies in two M. flaviventris. Woodchuck Marmota monax Many small mammals serve as hosts for ixodid ticks. Those implicated in the natural cycle of POW virus include marmots (woodchuck [Marmota monax]) and snowshoe hares, which assist in amplification of both tick and virus populations. Numerous isolates and/or high antibody prevalences have been documented in the woodchuck, Marmota monax. Meadow vole Microtus pennsylvanicus 4 out of 59 Microtus pennsylvanicus were found to have antibodies to Powassan virus by hemagglutination-inhibition assay, and 1 out of 49 M. pennsylvanicus tested positive for Powassan virus by a neutralization test. Meadow mouse Microtus sp Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson's ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. House mouse Mus musculus 8 out of 97 Mus musculus were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. 15 out of 20 Mus musculus were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. Woodland jumping mouse Napaeozapus insignis Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson's ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. Southern grasshopper mouse Onychomys torridus 1 out of 34 Onychomys torridus was found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Chaetodipus californicus 2 out of 8 Chaetodipus (Perognathas) californicus were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. White-footed mouse Peromyscus leucopus Other isolates of POW virus have been obtained from a red squirrel (Tamiasciurus hudsonicus), a white-footed mouse (Peromyscus leucopus), and a spotted skunk (Spilogale putorius). Their significance in the natural cycle of the virus is unknown; these isolates may reflect only the catholic feeding habits of the tick vectors. Three P. leucopus had monotypic HI titers of 1:40 to Powassan virus. Deer mouse Peromyscus maniculatus Powassan antibody was detected in sera from 107 snowshoe hares (Lepus americanus) or 79 other mammals including 1 deer, 4 flying squirrels (Glaucomys sabrinus), 10 jumping mice (Napaeozapus sp.) 22 deer mice (Peromyscus sp.), 1 mole, 5 shrews, 35 voles or 1 weasel. Norway rat Rattus norvegicus I out of 3 Rattus norvegicus was found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Two Norway rats were found to have HI titers of 1:40 to Powassan virus. Another Norway rat had a HI titer of 1:160 to Powassan virus. Western harvest mouse Reithrodontomys megalotis 1 of 14 Reithrodontomys megalotis was found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Gray squirrel Sciurus carolinensis Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson's ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. California ground squirrel Spermophilus beecheyi 16 of 67 Spermophilus (Citellus) beecheyi were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. Columbian ground squirrel Spermophilus columbianus Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson's ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. 4 out of 60 Citellus columbianus were found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Golden-mantled ground squirrel Spermophilus lateralis Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson's ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. Richardson's ground squirrel Spermophilus richardsonii Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. Eastern chipmunk Tamias striatus Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. American red squirrel Tamiasciurus hudsonicus Other Rodentia from which POW isolates and/or neutralizing antibodies have been obtained include Columbian ground squirrel, golden mantled ground squirrel, Richardson ground squirrel, porcupine, chipmunk, meadow mouse, woodland jumping mouse, deer mouse, gray squirrel, eastern chipmunk, and red squirrel. Coyote Canis latrans Serological studies for arboviruses were conducted on 725 animal sera collected in 22 Ontario townships between 1975 and 1980 including 44 coyote (Canis latrans), 277 red fox (Vulpes vulpes), 192 raccoon (Procyon lotor) and 212 striped skunk (Mephitis mephitis). Hemagglutination inhibition antibodies to two flaviviruses, namely St. Louis encephalitis and Powassan were found in 50% of coyote, 47% of skunk, 26% of fox and 10% of raccoon sera. Striped skunk Mephitis mephitis Serological studies for arboviruses were conducted on 725 animal sera collected in 22 Ontario townships between 1975 and 1980 including 44 coyote (Canis latrans), 277 red fox (Vulpes vulpes), 192 raccoon (Procyon lotor) and 212 striped skunk (Mephitis mephitis). Hemagglutination inhibition antibodies to two flaviviruses, namely St. Louis encephalitis and Powassan were found in 50% of coyote, 47% of skunk, 26% of fox and 10% of raccoon sera. In addition, neutralizing antibodies to POW virus have been demonstrated in striped skunk, short- and long-tailed weasel, and raccoon of the order Carnivora and snowshoe hare of the order Lagomorpha. Ermine Mustela erminea In addition, neutralizing antibodies to POW virus have been demonstrated in striped skunk, short- and long-tailed weasel, and raccoon of the order Carnivora and snowshoe hare of the order Lagomorpha. Long-tailed weasel Mustela frenata In addition, neutralizing antibodies to POW virus have been demonstrated in striped skunk, short- and long-tailed weasel, and raccoon of the order Carnivora and snowshoe hare of the order Lagomorpha. Raccoon Procyon lotor Serological studies for arboviruses were conducted on 725 animal sera collected in 22 Ontario townships between 1975 and 1980 including 44 coyote (Canis latrans), 277 red fox (Vulpes vulpes), 192 raccoon (Procyon lotor) and 212 striped skunk (Mephitis mephitis). Hemagglutination inhibition antibodies to two flaviviruses, namely St. Louis encephalitis and Powassan were found in 50% of coyote, 47% of skunk, 26% of fox and 10% of raccoon sera. In addition, neutralizing antibodies to POW virus have been demonstrated in striped skunk, short- and long-tailed weasel, and raccoon of the order Carnivora and snowshoe hare of the order Lagomorpha. Eastern spotted skunk Spilogale putorius Other isolates of POW virus have been obtained from a red squirrel (Tamiasciurus hudsonicus), a white-footed mouse (Peromyscus leucopus), and a spotted skunk (Spilogale putorius). Their significance in the natural cycle of the virus is unknown; these isolates may reflect only the catholic feeding habits of the tick vectors. Gray fox Urocyon cinereoargenteus Urocyon cinereoargenteus has been found to be infected with Powassan virus in New York. An additional strain of POW virus was isolated from the brain of a gray fox. The brain was received by the Rabies Group of the Laboratories for Veterinary Science of this Division on July 1. The animal had been found with choreiform movements in Broome County 48 hours before it had died. The brain suspension was inoculated intracerebrally into 10- to 12-g mice July 1, and an infectious agent was isolated which was submitted in its 4th passage to use for identification. In suckling mice the titer was 10(8.5) LD(50) per 0.03 ml smb suspension. Two immune rabbit sera, one prepared with POW and the other with strain 64-7062, protected all of 16 mice inoculated with 40 LD(50) of the fox strain. Red fox Vulpes vulpes Serological studies for arboviruses were conducted on 725 animal sera collected in 22 Ontario townships between 1975 and 1980 including 44 coyote (Canis latrans), 277 red fox (Vulpes vulpes), 192 raccoon (Procyon lotor) and 212 striped skunk (Mephitis mephitis). Hemagglutination inhibition antibodies to two flaviviruses, namely St. Louis encephalitis and Powassan were found in 50% of coyote, 47% of skunk, 26% of fox and 10% of raccoon sera. Snowshoe hare Lepus americanus Many small mammals serve as hosts for ixodid ticks. Those implicated in the natural cycle of POW virus include marmots (woodchuck [Marmota monax]) and snowshoe hares, which assist in amplification of both tick and virus populations. In addition, neutralizing antibodies to POW virus have been demonstrated in striped skunk, short- and long-tailed weasel, and raccoon of the order Carnivora and snowshoe hare of the order Lagomorpha. Black-tailed jackrabbit Lepus californicus 4 out of 100 Lepus californicus were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. Desert cottontail Sylvilagus auduboni 3 of 50 Sylvilagus auduboni were found to have antibodies to Powassan virus by hemagglutination-inhibition assays. Southern opossum Didelphis marsupialis One Didelphis marsupialis was found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Dog Canis familiaris Serologic surveys and virus isolations have shown infections in wild mammals, including rodents, hares, dogs, skunks, and foxes. Other research provided evidence of latent infection with POWV in humans, domestic pets (cats, dogs, goats) and local rodents. Birds Aves Isolations of POW virus have been reported in several species of birds in the U.S.S.R. including mallard, common teal, pintail, and masked bunting. Kilenko et al. tested sera from 190 U.S.S.R. birds by HI and found antibodies in four birds, all of different species. No significant evidence exists for POW virus infection of North American birds. Natrix Eight snakes were collected throughout the year. the following three species were represented: 6 Thamnops sirtalis (garter snake), I Thamnopsis sauriti sauritus (ribbon snake), and 1 Natrix sipedo (red bellied water snake). The water snake had a HI titer of 1:20 to Powassan virus. There were no virus isolations from either the liver or brain of any of the snakes listed above. Red-bellied snake Storeria occipitomaculata A single Storeria occipitomaculata was found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Eastern garter snake Thamnophis sirtalis 3 out of 6 Thamnophis sirtalis were found to have antibodies to Powassan virus by hemagglutination-inhibition assay. 1 out of 6 Thamnophis sirtalis was found to have antibodies to Powassan virus by neutralization test. Eastern painted turtle Chrysemys picta 2 out of 43 Chrysemys picta were found to have antibodies to Powassan virus by hemagglutination-inhibition assay. 1 out of 43 Chrysemys picta was found to have antibodies to Powassan virus by neutralization test. American toad Bufo americanus Three Bufo americanus were found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Green frog Rana clamitans 4 out of 13 Rana clamitans were found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Eight amphibians were captured, with two species represented: four Rana catesbeiana (bull frog) and four Rana clamitans (green frog). One green frog had a HI titer of 1:20 to Powassan virus. Tissues from these amphibians were negative in isolation attempts. Pickerel frog Rana palustris 3 out of 4 Rana palustris were found to have antibodies to Powassan virus by hemagglutination-inhibition assay. Northern leopard frog Rana pipiens 7 out of 15 >Rana pipiens were found to have antibodies to Powassan virus by hemagglutination-inhibition assay. 3 Biosafety Level 3 practices, safety equipment, and facilities are recommended for activities using potentially infectious clinical materials and infected tissue cultures, animals, or arthropods. POW virus produced cytopathic effects and/or plaques in several vertebrate cell lines including baby hamster kidney, BHK-21, rhesus monkey kidney, LLC-MK2, African green monkey kidney, VERO primary swine kidney, and human embryo lung, WI-38 (Mayflick) cells. Growth of POW virus without cytopathic effect occurred in primary chick embryo, rhesus monkey kidney, and primary cells from the ticks, Hyalomma dromedarii and D. silvarum. POW virus did not propagate in mosquito, Culex tarsalis and Ae. aegypti nor in the emperor gum worm, Antherae eucalypti cell culture. Isolation procedures for POW virus have employed i.c. inoculation of suckling mice almost exclusively; however, McLean et al. used primary swine kidney cells to reisolate and titrate POW virus in tick pools and clotted blood from which virus had originally been isolated using suckling mice. Other tissue cultures used to test for POW N antibodies have included BHK-21 and WI-38. Currently in Ontario the Provincial Health Laboratory performs a hemagglutination inhibition assay on acute and convalescent sera for Powassan virus antibody. Although the test will cross-react with antibodies of other flaviviruses such as dengue, St. Louis encephalitis and yellow fever, an epidemiologic history of the patient should help distinguish among them. The major drawback is that the detection of seroconversion may require a week or more, delaying diagnosis. Hemagglutination-inhibition tests were performed in plastic plates. Unheated serums were absorbed with kaolin to remove inhibitors of hemagglutination, and naturally occurring agglutinins were removed by absorption with chick cells. Antigens were prepared by extraction of infected suckling mouse brains with borate saline, pH 9.3, centrifugation at 10,000 rpm for 1 hour, and treatment of the resultant supernatant with 2.5 mg per ml protamine. Serum-virus mixtures diluted in borate saline, pH 9, containing 0.4% bovine serum albumin were held at 4 C overnight before addition of a 0.25% suspension of erythrocytes from newly hatched chicks. Erythrocytes were diluted in virus-adjusting diluent to give a final pH of 6.4 for eastern equine encephalomyelitis antigen and 6.7 for Powassan antigen. Comparative titrations of alpha-, flavi- and Bunyamwera viruses were made by EIA-IC and according to cytopathic effect (CPE). Specific enzymatic reactions appeared earlier and in higher titres than CPE. The titres of dengue type 1, Mayaro, Powassan and Langat viruses measured by EIA-IC were comparable to those measured by intracerebral inoculation of mice. The cross-reactivity testing of EIA-IC among alphaviruses (Chikungunya, Sindbis and Mayaro), flaviviruses (Japanese encephalitis, Murray valley encephalitis, Kunjin, West Nile, yellow fever and louping ill, Powassan, Langat) and Bunyamwera arboviruses using polyclonal immune ascitic fluids confirmed the high specificity of EIA-IC. Homologous reactions mostly showed higher titres than heterologous ones. No cross-reactivity was seen between alpha-, flavi- and bunyaviruses, among the three alphaviruses, between mosquito-borne and tick-borne flaviviruses, or between JE complex and YF viruses. However, a cross-reactivity to different extent was observed among the four JE complex viruses and among louping ill, Powassan and Langat viruses. The results of EIA-IC cross tests showed that this method can distinguish togavirus group- or species-specific antigens, more precisely than conventional ELISA. Neutralization tests were performed by intracerebral inoculation of serum-virus mixtures into 3-week-old mice. Mixtures containing 0.1 ml aliquots of unheated test serum, unheated normal ox serum ("accessory factor"), and Powassan virus diluted in 10% ox serum saline to give a final concentration of 50 to 100 mouse LD(50) were held at room temperature (22 C) for 1 hour. Groups of 5 mice were inoculated intracerebrally with 0.03 ml aliquots of each serum-virus mixture. Serums which neutralized at least 50 LD(50) of virus were considered positive. Whenever sufficient serum was available, complement-fixation tests were performed against the antigens of six arboviruses: Powassan, Silverwater, California, Colorado tick fever, Tensaw and eastern equine encephalomyelitis (EEE), prepared by extraction of infected suckling mouse brains with borate-saline solution of pH 9.3. Using a universal primer set designed to match the sequence of the NS1 gene of flaviviruses, the virus RNA of dengue (DEN), Japanese encephalitis (JEV), powassan and langat of Flaviviridae were successfully amplified by polymerase chain reaction (PCR) via cDNA; and with different internal primers, the serotypes of the dengue viruses were identified. Of the 78 clinically diagnosed dengue fever patients, 18 patients were positive for DEN 1, 48 patients for DEN 2 and 8 patients concurrently infected with DEN 4. Of the 52 patients admitted with Japanese encephalitis (JE), 45 were determined to be JEV infections. By nested PCR, we completed the identification of flaviviruses within 2 days. The results show that seven primers have a potential value for rapid clinical diagnosis of flavivirus infections. DJS(+) GACATGGGGTATTGGAT DJA(-) TCCATCCCATACCTGCA Viral RNA was detected using a quantitative real-time reverse transcriptase-polymerase reaction (RT-PCR) using primers (forward: 5' - gtgactggctatttgagcacctt-3', reverse: 5'-tggatctaaccttcgctatgaattc-3') designed to amplify an 83-base pair region of the POW virus nonstructural protein 5 coding region, and a probe (5'-6FAM-cgagccagggtga-MGBNFQ-3') designed to specifically bind all known linage II POW virus strains. Forward: 5' - gtgactggctatttgagcacctt-3 Reverse: 5'- tggatctaaccttcgctatgaattc-3' 5'-6FAM-cgagccagggtga-MGBNFQ-3' Woodall JP Roz A Am J Trop Med Hyg. 1977 21 6 190 192 McLean DM Crawford MA Ladyman SR Peers RR, Purvin-Good KW.. Am J Epidemiol. 1970 92 4 266 272 McLean DM Bergman SK Gooddard EJ Graham EA, Purvin-Good KW. Can J Public Health 1971 62 2 120 124 Whitney E Jamnback H Means RG Watthews TH. Am J Trop Med Hyg 1968 17 4 645 650 Hardy JL Reeves WC Scrivani RP Roberts DR. Am J Trop Med Hyg 1974 23 6 1165 1177 Bast TF Whitney E Benach JL Am J Trop Med Hyg 1973 22 1 109 115 Whitney E Jamnback H Proc Soc Exp Biol Med 1965 119 432 435 McLean DM de Vos A Quantz EJ Am J Trop Med Hyg 1964 13 4-6 747 753 McLean DM Walker SJ MacPherson LW Scholten TH, Ronald K, Wyllie JC, McQueen EJ. J Infect Dis 1961 109 19 23 Meiyu F Huosheng C Cuihua C Xiaodong T, Lianhua J, Yifei P, Weijun C, Huiyu G. Microbiol Immunol 1997 41 3 209 213 Lessell S Collins TE Neurology 2003 60 10 1726 1727 Xiao ZS Jia LL Qu XS Zhang YH. Acta Virol. 1986 30 6 487 493 Costero A Grayson MA Am J Trop Med Hyg 1996 55 5 536 546 Khozinskaia GA Pogodina VV Vopr Virusol 1982 27 4 :491 :495 Little PB Thorsen J Moore W Weninger N. Vet Pathol 1985 22 5 500 507 Frolova MP Isachkova LM Shestopalova NM Pogodina VV. Neurosci Behav Physiol 1985 15 1 62 69 Gholam BIA Puksa S Provias JP CMAJ 1999 161 11 1419 1422 MMWR Morb Mortal Wkly Rep 2001 50 35 761 764 Calisher CH Clin Microbiol Rev 1994 7 1 89 116 Artsob H Spence L Th'ng C Lampotang V, Johnston D, MacInnes C, Matejka F, Voigt D, Watt I. Can J Vet Res 1986 50 1 42 46 Kuno G Artsob H Karabatsos N Tsuchiya KR, Chang GJ. Am J Trop Med Hyg 2001 65 5 671 676 Ebel GD Kramer LD Am J Trop Med Hyg 2004 71 3 268 271 Mandl CW Holzmann H Kunz C Heinz FX. Virology 1993 194 11 173 184 Gritsun TS Lashkevich VA Gould EA Antiviral Res 2003 57 1-2 129 146 Ralph ED CMAJ 1999 161 11 1416 1417 Chambers TJ Hahn CS Galler R Rice CM. Annu Rev Microbiol 1990 44 649 688 Gritsun TS Nuttall PA Gould EA Adv Virus Res 2003 61 317 371 Artsob H Powassan Encephalitis Steele JH. CRC Handbook Series in Zoonoses. Section B: Viral ZoonosesVolume I. 1981 156 158 CRC Press, Inc. Boca Raton, Florida Burke DS Monath TP Flaviviurses Knipe DM Howley PM Fields Virology. 2001 1043 1125 Lippincott Williams and Wilkins Philadelphia Pa Artsob H Powassan encephalitis Monath TP The Arboviruses: Epidemiology and Ecology. Volume IV. 1989 29 49 CRC Press Boca Raton, Florida http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=11083&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=39008&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=20260779 http://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=16176 http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34620&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34621&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=35564&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34615&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=35565&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=6945&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34614&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=55967&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=138534&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34622&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10018&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34844&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=64679&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=93162&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9995&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10058&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10090&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=101671&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=38674&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=145408&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10041&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10042&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10116&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=44234&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=30640&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=45474&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=34862&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=76772&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=50862&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=37591&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10009&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=160400&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9614&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=30548&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=36723&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=55048&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9654&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=30552&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=55040&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9627&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=48086&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=48087&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=30581&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9268&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10053&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9606&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=9615&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s74.htm http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4s7h.htm http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=45485&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10016&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8782&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8782&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8583&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&:id=46282&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=35019&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8479&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8389&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=145282&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=298395&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=8404&lvl=3&lin=f&keep=1&srchmode=1&unlock http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=145282&lvl=3&lin=f&keep=1&srchmode=1&unlock Wattam Rebecca wattam@vbi.vt.edu 09/26/04 0.83 pathinfo@vbi.vt.edu