Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Powassan virus (Website 1):
      1. GenBank Taxonomy No.: 11083
      2. Description: 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(Gritsun et al., 2003). Powassan virus is a flavivirus and a member of the tick-borne encephalitis (TBE) antigenic complex(Kuno et al., 2001).
      3. Variant(s):
         • Tick-borne powassan virus (strain lb) (Website 2):
           • GenBank Taxonomy No.: 39008
           • Parents: Powassan virus
           • Description: The LB strain was isolated in 1958 in Ontario, Canada from a human brain(Kuno et al., 2001). 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(Artsob, 1989).

1. 1. Description: 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(Burke and Monath, 2001).

1. Genome of Powassan virus
   1. Description: 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(Chambers et al., 1990).
   2. Single RNA strand(Website 3)
      1. GenBank Accession Number: NC_003687
      2. Size: 10839 bp ss-RNA(Website 3).
      3. Gene Count: 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(Mandl et al., 1993).
      4. Description: 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(Mandl et al., 1993).
      5. Picture(s):
         • Powassan virus (Website 4)
           
           
           
           Description: Powassan virus, complete genome(Website 4).
         • Powassan virus (Website 4)
           
           
           
           Description: Powassan virus, complete genome(Website 4).

1. Biosafety information for Powassan virus
   1. Level: 3(Website 52).
   2. Precautions: Biosafety Level 3 practices, safety equipment, and facilities are recommended for activities using potentially infectious clinical materials and infected tissue cultures, animals, or arthropods(Website 53).

1. Cytopathic effects :
   1. Description: 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(Artsob, 1989). Other tissue cultures used to test for POW N antibodies have included BHK-21 and WI-38(Artsob, 1989).

1. Outbreak Locations:
   1. During September 1999 - July 2001, four Maine and Vermont residents with encephalitis were found to be infected with POW virus(MMWR, 2001).
2. Transmission Information:
   1. From: Vertebrate, Powassan virus , To: invertebrate, Powassan virus , With Destination:invertebrate, Powassan virus
      Mechanism: 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(Ebel et al., 2004).
   2. From: invertebrate, Powassan virus , To: Vertebrate, Powassan virus , With Destination:Vertebrate, Powassan virus, Mammals
      Mechanism: Infectious POW virus is present in tick salivary secretions inoculated during the earliest stages of feeding, and may be immediately inoculated(Ebel et al., 2004). 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(Artsob, 1989).
   3. From: Vertebrate, Powassan virus , To: Vertebrate, Powassan virus , With Destination:Vertebrate, Powassan virus
      Mechanism: 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(Woodall and Roz, 1977).
3. Environmental Reservoir:
   1. Mammals:
      1. Description: 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(Calisher, 1994). 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(Artsob, 1989). 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(Artsob, 1989).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Description: 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(Gritsun et al., 2003b).
      2. Delivery Mechanism: 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(Gritsun et al., 2003b). 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(Gritsun et al., 2003b).

1. Immunoassay Test:
   1. Hemagglutination inhibition assay :
      1. Time to Perform: unknown
      2. Description: 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(Ralph, 1999). 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(McLean et al., 1961).
   2. EIA-IC :
      1. Description: 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(Xiao et al., 1986).
   3. Neutralization tests for PV :
      1. Time to Perform: unknown
      2. Description: 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(McLean et al., 1961).
   4. Complement Fixation test :
      1. Time to Perform: unknown
      2. Description: 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(McLean et al., 1964).
2. Nucleic Acid Detection Test: