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. Louping ill virus (Website 1):
      1. GenBank Taxonomy No.: 11086
      2. Description: The Flaviviridae are a family of over 60 viruses transmitted mainly by mosquito or tick vectors and causing many diseases of man and animals including yellow fever, Japanese encephalitis, dengue, louping ill and tick-borne encephalitis (TBE). Louping ill virus (LIV), which is endemic in upland areas of the UK and Ireland, causes non-suppurative meningoencephalomyelitis in sheep, cattle, horses, pigs, dogs, deer, red grouse, other wildlife species and occasionally man. The natural vector is the tick, Ixodes ricinus. Transmission of LIV to laboratory workers by aerosol infection has been reported(Sheahan et al., 2002). The name 'louping ill' is derived from the old Scots language describing the effect of encephalitis in sheep causing them to 'loup' or spring into the air(Davidson et al., 1991).
      3. Variant(s):
         • Louping ill virus (strain 31) (Website 2):
           • GenBank Taxonomy No.: 36386
           • Parents: Louping ill virus
           • Description: Isolated from an ovine in the Scottish borders in 1931(McGuire et al., 1998).
         • Louping ill virus (strain K) (Website 3):
           • GenBank Taxonomy No.: 36387
           • Parents: Louping ill virus
           • Description: Isolated from a grouse in Grantown, Scotland, in 1980(McGuire et al., 1998).
         • Louping ill virus (strain Negishi 3248/49/P10) (Website 4):
           • GenBank Taxonomy No.: 36388
           • Parents: Louping ill virus
           • Description: Negishi virus (NEG) was originally isolated from a fatal case of encephalitis during and epidemic of Japanese encephalitis in Japan in 1948 and at this time no other tick-borne flaviviruses were being used in the laboratory. NEG virus is serologically related to Russian spring summer encephalitis (RSSE) virus. Subsequent antigenic studies confirmed the close relationship of NEG virus with LI virus and the central European subtype of TBE virus. This relationship has now been investigated more precisely using monoclonal antibodies and nucleotide sequencing. Results are presented suggesting that NEG virus is a strain of LI virus(Venugopal et al., 1992).
         • Louping ill virus (strain Norway) (Website 5):
           • GenBank Taxonomy No.: 36389
           • Parents: Louping ill virus
           • Description: Isolated from an ovine in Norway in 1984(McGuire et al., 1998).
         • Louping ill virus (strain SB 526) (Website 6):
           • GenBank Taxonomy No.: 31640
           • Parents: Louping ill virus
           • Description: Isolated from an ovine in Oban, Scotland, in 1968(McGuire et al., 1998).

1. Genome of Louping ill virus
   1. Description: The genome of LI virus and other flaviviruses comprises a single open reading frame (ORF) approximately 11 kb in length. This ORF encodes a polyprotein consisting of three structural (capsid, premembrane and envelope) and seven nonstructural proteins. The envelope (E) protein is the major structural protein and plays an important role in membrane binding and inducing a protective immune response following virus infection(McGuire et al., 1998). The genomic RNA of flaviviruses is single-stranded and approximately 11 kilobases in length. The genomic RNA is infectious, and thus of poisitive polarity encoding the viral proteins necessary for RNA replication. Genome-length RNAs appear to be the only virus-specific mRNA molecules in flaivirus-infected cells(Chambers et al., 1990).
   2. Single RNA strand(Website 7)
      1. GenBank Accession Number: NC_001809
      2. Size: 10871 bp ss-RNA(Website 7).
      3. Gene Count: The virion RNA is translated into a polyprotein from which structural and non-structural proteins are processed by cellular and viral proteases(Gritsun et al., 1997).
      4. Description: Sequence analysis of the genomic RNAs of several flaviviruses has revealed that they are organized similarly. The viruses are enveloped particles about 50 nm in diameter containing a single stranded RNA molecule, approximately 11 kb in length, of positive sense coding for three structural proteins, designated capsid (C), membrane (M), envelope (E), and seven non-structural proteins designated NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5. The virion RNA is translated into a polyprotein from which structural and non-structural proteins are processed by cellular and viral proteases(Gritsun et al., 1997).
      5. Picture(s):
         • Louping ill virus, complete genome (Website 8)
           
           
           
           
         • Louping ill virus, complete genome (Website 8)
           
           
           
           

1. Biosafety information for Louping ill virus
   1. Level: 3(Website 9).
   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 10).

1. Monolayer Tissue Culture of Pig Kidney (Williams, 1958):
   1. Description: Virus was passaged serially in culture by transferring pooled undiluted or diluted infected medium from one group of four to six tube-cultures to each of a group of newly prepared ones. Passage virus was harvested on the sixth day of incubation and was titrated immediately in mice. By the fifth passage the original virus inoculum titer of 4 x 10(4) mouse LD(50) doses per ml was diluted 10(7) but yielded 3.9 x 10(7) mouse LD(50) doses per ml. Clearly, the cultures were supporting multiplication of the virus. Microscopic examination of live cultures inoculated with low dilutions of mouse brain virus revealed a cytopathogenic effect which commenced on the second to third day. The affected cells became granular, rounded up, and appeared to agglutinate. As the effect increased in severity, there was almost total cell necrosis and destruction of the monolayers. Fixed coverslip preparations of affected cells stained with Giemsa showed shrinking, pyknosis and disintegration. Uninfected cultures and culture inoculated with comparable dilutions of normal mouse brain remained unchanged(Williams, 1958).
   2. Medium: Earle's saline, 0.5 % lactalbumen hydrolysate (enzymatic) and 0.01 % yeast extract. To this mixture was added 10% normal cattle serum as well as antibiotics(Williams, 1958).
   3. Optimal Temperature: 35 C(Williams, 1958).

1. Outbreak Locations:
   1. Currently no epidemic outbreak information is available.
2. Transmission Information:
   1. From: Ixodes ricinus , To: The catholic host preference in the vector ensures that all vertebrates in endemic areas are likely to encounter infection. Antibody has been detected and/or virus isolated from a number of wild species including shrew (Sorex araneus), wood mouse (Apodemus sylvaticus), blue hare (L. timidus), badger (Meles meles), roe deer (Capreolus capreolus), red deer (C. elaphus), feral goat and red grouse (Lagopus scoticus). In addition, disease caused by louping-ill virus has been described in a variety of domestic species including pig, sheep, cattle, horse, dog, and farmed deer as well as in man. However, the principal disease association is in sheep(Reid, 1984).
      Mechanism: Ticks that find a host attach with their mouth parts and, following an initial period when saliva is injected, remove and concentrate blood over a period of 3-10 days(Reid, 1984). Only nymphs and adults that have become infected by ingesting virus with a previous blood meal can transmit virus(Reid, 1984).
   2. From: Vertebrates , To: Ixodes ricinus
      Mechanism: The infection of ticks with virus is complex. Blood is ingested for several days during which the titre of viraemia in the vertebrate will change, and the tick feeds in two distinct phases. Furthermore, not only does virus have to establish in the tick but it must also find its way to the salivary gland of the succeeding instar. Initial investigations in which larvae were fed on laboratory mice infected with louping ill indicated that although viraemias in excess of 10(4) plaque-forming units (p.f.u.) per 0.2 ml of blood occurred, this was insufficient to establish infection in larval ticks. However, experiments with day-old domestic chicks met with greater success and the incidence of infection in freshly engorged larvae approached 100%, the incidence of infection rapidly declined to 10% and remained at this level throughout the moulting process and for the succeeding 2 months. It was subsequently shown that the virus concentration in the blood during the initial feeding phase was directly related to the proportion of the ticks in which infection became established and that this proportion approached 40% as viraemias of 10(6) p.f.u. per 0.2 ml of blood were attained(Reid, 1984).
   3. From: Vertebrates , To: Vertebrates
      Mechanism: Experimentally, LI virus has been shown to be shed in the milk of goats and ewes following infection with the virus. While titers of a virus in the milk of both species were similar, virus was shed for a longer period in goats. Transmission of virus presumably through the ingestion of infective mild was demonstrated in kids that suckled infected goats. Similar attempts to transmit the virus in sheep were unsuccessful(Timoney, 1992). Louping-ill has been transmitted experimentally to various animal species by several parenteral routes of inoculation and following exposure to infective aerosols. Accidental infection of man has occurred following tick-bite, penetration of the virus through skin wounds or by aerosol(Timoney, 1992).
3. Environmental Reservoir:
   1. Sheep:
      1. Description: Of the mammalian species experimentally infected, only sheep consistently developed viremias of a sufficient intensity to infect the vector and are thus the only species that is likely to have a significant role in the maintenance of louping-ill virus(Reid, 1988).
      2. Survival: In experimental studies, 22 of 33 six month-old sheep inoculated subcutaneously (s.c.) with 10(7) mouse LD(50), or cell culture plaque-forming unit (p.f.u.) doses of virus were moribund within 6 to 11 days (mean 8 days). Ataxia rapidly progressed to complete flaccid paralysis within 3 to 5 hr. Two of eleven which survived were 'chronically debilitated'(Smith and Varma, 1981).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Description: Tick-borne flaviviruses are excreted in the urine and faeces 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(Gritsun et al., 2003). 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., 2003).
      2. Delivery Mechanism: In the con 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., 2003). 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., 2003).

1. Organism Detection Test:
   1. IFAT of cell culture assays or tick squashes (Gaunt et al., 1997):
      1. Time to Perform: 2-to-7-days
      2. Description: Sterile glass coverslips were added aseptically to each well of a 24-well tissue culture plate. Porcine kidney (PS) cells were grown to confluence in each well prior to inoculation with the tick homogenates. Three days p.i. cells were washed in PBS and fixed in situ in a 3:2 solution of methanol:acetone at -20 C for 5 min. Infection of cells was confirmed by indirect immunofluorescence antibody test (IFAT) using a monoclonal antibody Mab 813 that binds to the E protein of all flaviviruses(Gaunt et al., 1997). Comparison of the sensitivity of virus infectivity in cell culture and RT-PCR to detect LI virus in field-trapped ticks showed that there was little if any difference between the two methods. However, in terms of speed, simplicity and reliability, the RT-PCR method proved superior, taking only 8 h to demonstrate a cDNA product and a further 48 h to confirm virus identity by nucleotide and deduced amino acid sequence analysis. In contrast, IFAT relied upon a subjective analysis and in samples containing very small quantities of virus-specific antigen, was less reliable(Gaunt et al., 1997).
   2. Virus Isolation (Timoney, 1992):
      1. Description: In the majority of cases, virus isolation is attempted on the brain and spinal cord of dead animals. While this is frequently successful in sheep, results in cattle have been variable. Fresh tissue is best transported to the laboratory in 50 percent glycerol/normal saline or frozen on dry ice and dispatched in a closed, insulated container using an overnight delivery service. Virus can be isolated in the PK1B/RS2 cell line or by the intracerebral inoculation of suckling or adult mice in which the virus produces a fatal encephalomyelitis. Isolates of LI virus can be preliminarily identified either by means of the complement fixation test using a crude mouse brain antigen or simpler still, in a double immunodiffusion test. Final verification of identity is accomplished by means of a neutralization test in cell culture (plaque-reduction or microneutralization) or in mice(Timoney, 1992).
2. Immunoassay Test:
   1. ELISA as Serological Confirmation of LI Infection (Timoney, 1992):
      1. Description: Serological confirmation of a diagnosis of LI virus infection is based on the demonstration of seroconversion or a significant (4 fold or greater) rise in antibody titer to the virus between acute and convalescent sera. Demonstration of specific IgM antibody in serum is also confirmatory of infection. Hemagglutination-inhibition, neutralization and most recently, the enzyme-linked immunosorbent assay (ELISA) tests have been used for the serological diagnosis of LI virus infection. Whereas HI antibodies appear 5 to 10 days after infection and decline after 6 to 12 months, SN antibodies persist for years. The complement fixation test is of very limited value in the diagnosis of this disease in sheep as these antibodies appear late in the course of infection and are transient in duration. A standardized Tick Borne Encephalitis virus antigen is now commercially available for use in an ELISA test for this disease, obviating the need to prepare in-house antigen reagents(Timoney, 1992).
   2. Haemagglutination as Serological Confirmation of LI Infection (Timoney, 1992):
      1. Description: Serological confirmation of a diagnosis of LI virus infection is based on the demonstration of seroconversion or a significant (4 fold or greater) rise in antibody titer to the virus between acute and convalescent sera. Demonstration of specific 1gM antibody in serum is also confirmatory of infection. Hemagglutination-inhibition, neutralization and most recently, the enzyme-linked immunosorbent assay (ELISA) tests have been used for the serological diagnosis of LI virus infection. Whereas HI antibodies appear 5 to 10 days after infection and decline after 6 to 12 months, SN antibodies persist for years. The complement fixation test is of very limited value in the diagnosis of this disease in sheep as these antibodies appear late in the course of infection and are transient in duration. A standardized Tick Borne Encephalitis virus antigen is now commercially available for use in an ELISA test for this disease, obviating the need to prepare in-house antigen reagents(Timoney, 1992).
   3. Neutralization as Serological Confirmation of LI Infection (Timoney, 1992):
      1. Description: Serological confirmation of a diagnosis of LI virus infection is based on the demonstration of seroconversion or a significant (4 fold or greater) rise in antibody titer to the virus between acute and convalescent sera. Demonstration of specific 1gM antibody in serum is also confirmatory of infection. Hemagglutination-inhibition, neutralization and most recently, the enzyme-linked immunosorbent assay (ELISA) tests have been used for the serological diagnosis of LI virus infection. Whereas HI antibodies appear 5 to 10 days after infection and decline after 6 to 12 months, SN antibodies persist for years. The complement fixation test is of very limited value in the diagnosis of this disease in sheep as these antibodies appear late in the course of infection and are transient in duration. A standardized Tick Borne Encephalitis virus antigen is now commercially available for use in an ELISA test for this disease, obviating the need to prepare in-house antigen reagents(Timoney, 1992).
   4. Microneutralization Test in PK(15) Cells (Timoney et al., 1984):
      1. Time to Perform: 2-to-7-days
      2. Description: A microneutralization test in PK(15) cells was developed to measure the neutralizing antibody response of a group of ponies experimentally challenged with louping ill virus. Viral cytopathic effect was maximal after 6 days of incubation, at which point titration endpoints were clear-cut and readily determinable. The assay compared favorably with the mouse neutralization test for accuracy and ease of performance(Timoney et al., 1984). The microneutralization test for LI virus was simple and easy to perform and offered significant advantage over other currently available cell culture assay procedures in that the pig kidney line used was not contaminated with hog cholera virus. The CPE associated with LI virus infection of PK(15) cells was clearly discernible after incubation of cultures for 4 to 6 days and very similar to that previously reported in pig kidney secondary monolayers. Titration endpoints were clear-cut and could be readily determined. In addition to easy reading of test results, the microneutralization assay provides a rapid and relatively inexpensive means of detection and estimation of SN antibody levels to LI virus with satisfactory accuracy. It has considerable advantages over the mouse neutralization test and is less laborious than alternative cell culture procedures(Timoney et al., 1984).
   5. Avidin-Biotin-complex Immunoperoxidase Technique (Krueger and Reid, 1994):
      1. Time to Perform: 2-to-7-days
      2. Description: An immunohistochemical method for the detection of louping ill virus antigen in formalin-fixed, paraffin wax-embedded tissues by an avidin-biotin-complex (ABC) immunoperoxidase technique was established. The tissues examined were from the brains of 10 mice, five sheep and one pig. The mice were experimentally infected with louping ill virus whereas the sheep and the pig were field cases of louping ill confirmed by clinical examination, and by histological and serological methods(Krueger and Reid, 1994). Viral antigen was detected in all 10 experimentally infected mice, in two of the five sheep, and in the pig(Krueger and Reid, 1994).
      3. False Negative: As only two of the five presumptive cases of louping ill in sheep were shown to be positive by the technique it should not at present be the only means of diagnosis(Krueger and Reid, 1994).
      4. Antibody:
         • • Antibody Source: Louping ill virus
3. Nucleic Acid Detection Test: