Experimental transmission of Karshi and Langat (tick-borne encephalitis virus complex) viruses by Ornithodoros ticks (Acari: Argasidae)

Selected species of mosquitoes and Ornithodoros ticks were evaluated for their potential to transmit Karshi and Langat (tick-borne encephalitis virus complex) viruses in the laboratory. Although there was no evidence of replication of Karshi virus in either of the two mosquito species tested [Ochlerotatus taeniorhynchus (Wiedemann) or Culex pipiens (L.)], Karshi virus replicated in and was transmitted by all three species of Ornithodoros ticks tested (Ornithodoros parkeri Cooley, Ornithodoros sonrai Sautet & Witkowski, and Ornithodoros tartakovskyi Olenev). When inoculated with Karshi virus, 90% of Ornithodoros ticks (44/49) transmitted this virus by bite to suckling mice, and transmission continued to occur for at least 1 yr, the longest extrinsic incubation tested. After feeding on a suckling mouse with a viremia of approximately 10(5) suckling mouse subcutaneous lethal dose. units of Karshi virus per milliliter of blood, all three species of Ornithodoros tested became infected with and transmitted Karshi virus both trans-stadially and horizontally by bite to suckling mice. In addition, female O. tartakovskyi transmitted Karshi virus vertically to their progeny. In a continuation of a previous study, O. sonrai, orally exposed to Langat virus, were able to transmit this virus after >3 yr, the longest interval tested. Therefore, Ornithodoros spp. should be considered as potential vectors and as possible long-term maintenance hosts for Karshi virus and other members of the tick-borne encephalitis virus complex.     

The differentiation of viruses of the tick-borne encephalitis complex by means of RNA-DNA hybridization]

Nucleic acid spot hybridization with cloned cDNA of tick-borne encephalitis (TBE) virus, strain Sofjin, was used to differentiate strains of TBE and other flaviviruses. The cDNA probe reacted with strains of TBE and flaviviruses of TBE subgroup with the exception of Powassan virus. The probe did not react with viruses of Japanese encephalitis and Gendue subgroups. The viruses of TBE subgroup and some strains of TBE virus were differentiated from TBE strain Sofjin by thermal stability of RNA-DNA hybrids. Negishi and Louping ill viruses were found to be most closely related to TBE strain Sofjin among viruses of the TBE subgroup.     

To the antigenic classification of some viruses from the tick-borne encephalitis complex by monoclonal antibodies

Two monoclonal antibodies (MoAb) to the Skalica virus from the tick-borne encephalitis (TBE) complex were used to compare Karshi and Royal Farm viruses with the Russian spring-summer encephalitis, Central European encephalitis (Hypr and Kumlinge strains) Skalica, Langat and Powassan viruses. The first MoAb was prepared by fusion of P3NS1 cells with BALB/c mouse spleen cells, immunized with the Skalica virus; it was of IgM class and reacted in haemagglutination-inhibition (HI) test (MoAb type 1). The second MoAb was of IgG class and reacted in complement-fixation (CF) test (MoAb type 2). MoAb type 1 reacted in the HI test with Russian spring-summer encephalitis (RSSE), Central European encephalitis (CEE) virus strains, Skalica and Langat viruses. No reaction was observed with Powassan, Karshi, and Royal Farm viruses. MoAb type 2 reacted in the CF test with all members of tick-borne encephalitis complex except the Powassan, Karshi, and Royal Farm viruses.     

Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis

Tick-borne encephalitis virus (TBEV) is an important human pathogen that causes severe neurological illness in large areas of Europe and Asia. The neuropathogenesis of this disease agent is determined by its capacity to enter the central nervous system (CNS) after peripheral inoculation ("neuroinvasiveness") and its ability to replicate and cause damage within the CNS ("neurovirulence"). TBEV is a small, enveloped flavivirus with an unsegmented, positive-stranded RNA genome. Mutations affecting various steps of its natural replication cycle were shown to influence its neuropathogenic properties. This review describes experimental approaches and summarizes results on molecular determinants of neurovirulence and neuroinvasiveness that have been identified for this virus. It focuses on molecular mechanisms of three particular steps of the viral life cycle that have been studied in some detail for TBEV and two closely related tick-borne flaviviruses (Louping ill virus (LIV) and Langat virus (LGTV)), namely (i) the envelope protein E and its role in viral attachment to the cell surface, (ii) the 3'-noncoding region of the genome and its importance for viral RNA replication, and (iii) the capsid protein C and its role in the assembly process of infectious virus particles. Mutations affecting each of these three molecular targets significantly influence neuropathogenesis of TBEV, particularly its neuroinvasiveness. The understanding of molecular determinants of TBEV neuropathogenesis is relevant for vaccine development, also against other flaviviruses.     

Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure

The glycosylation pattern of several flavivirus E proteins as well as the role of carbohydrate in biological functions and the antigenic structure of tick-borne encephalitis (TBE) virus were investigated by the use of specific endoglycosidases. Endoglycosidase F digestion revealed the presence of a single asparagine-linked oligosaccharide side chain in TBE virus (Western and Far Eastern subtype), Louping III virus, Murray Valley encephalitis virus, and Rocio virus. Consistent with published sequence data, the E protein of West Nile virus apparently is not glycosylated at all. Evidence derived from digestion experiments using endoglycosidase H indicates that the tick-borne viruses contain high-mannose type N-linked oligosaccharide side chains, whereas that of the mosquito-borne Murray Valley encephalitis virus and Rocio virus is endoglycosidase H resistant. Complete deglycosylation of TBE virus by endoglycosidase F did not impair infectivity and HA activity. Carbohydrate does not seem to play a major role in the antigenic structure of the TBE virus glycoprotein since the reactivity of the native virus and the deglycosylated virus was identical when analyzed with monoclonal as well as polyclonal immune sera.     

Antigenic relationships among viruses of the tick-borne encephalitis complex as studied by monoclonal antibodies

Tick-borne encephalitis (TBE) monoclonal antibodies showed haemagglutination-inhibiting (HI) activity against viruses belonging to the TBE complex except of Powassan virus. The HI titre with Kyasanur forest disease virus was lower than with tick-borne encephalitis virus, when monoclonal antibodies were incubated with the antigen at +4 degrees C for 30 min. Langat virus could be distinguished from other viruses of the TBE complex when the antigen was incubated with monoclonal antibodies at +4 degrees C for 18 hr. A close antigenic relationship was demonstrated between tick-borne encephalitis, louping-ill, Negishi and Omsk haemorrhagic fever viruses.     

Analysis of the complete genome of the tick-borne flavivirus Omsk hemorrhagic fever virus

Omsk hemorrhagic fever virus (OHF) is a tick-borne flavivirus endemic to Western Siberia. This virus is the only known tick-borne flavivirus to cause hemorrhagic disease in humans in the absence of encephalitis. OHF virus circulates within a small, defined niche in which other tick-borne complex flaviviruses are also present. The objectives of this study were to genetically classify OHF virus based on its complete genome and to identify genetic determinants that might be involved in tissue tropism and viral replication leading to the disease state caused by this virus. The OHF virus genome was sequenced and phylogenetic analysis demonstrated that OHF virus falls within the tick-borne encephalitis serocomplex of flaviviruses, yet is distinct from other members of the complex, including those closely associated geographically. OHF is also distinct from Alkhurma (ALK) and Kyasanur forest disease (KFD) viruses, both of which cause disease that includes hemorrhagic and encephalitic manifestations. Several amino acid residues were found to be distinct among OHF, KFD, and ALK viruses; these residues include E-76, which is closely associated with the viral envelope protein fusion peptide. In addition, variation between the viral 5'-untranslated region of OHF and other tick-borne flaviviruses suggests potential variability in viral replication. These data demonstrate that OHF is a unique virus among the tick-borne flaviviruses and also provide insight to viral biodiversity and tropism.     

Genomic sequence of the structural proteins of louping ill virus: comparative analysis with tick-borne encephalitis virus.

The genomic RNA of louping ill virus coding for capsid, premembrane, membrane, and envelope proteins was cloned and sequenced. Hydrophilicity profiles of the deduced amino acid sequence shared homologous functional domains with other flaviviruses. The premembrane and envelope proteins contain N-glycosylation sites and conserved cysteine residues which are important for maintaining the secondary structures of the proteins. Sequence comparisons of louping ill envelope protein showed greater homology with tick-borne than mosquito-borne flaviviruses and greater homology with the western than the far eastern subtype of tick-borne encephalitis virus. With the capsid and membrane proteins, the degree of homology between louping ill and the western subtype was greater than that between the two subtypes, indicating very close evolutionary relationships between louping ill and the western subtype of tick-borne encephalitis. Thus, louping ill and tick-borne encephalitis may be varieties of a common tick-borne ancestral virus. The average amino acid sequence diversity between members of the tick-borne serogroup was significantly lower than that of mosquito-borne serogroups, suggesting that tick-borne flaviviruses have been subjected to different evolutionary immune selection pressure from the mosquito-borne viruses. Using the published model of tick-borne encephalitis envelope protein and our sequence data on louping ill virus, we have identified three discontinuous peptides (amino acids 81-88, 207-212, and 230-234) which may represent critical molecular determinants within the receptor binding site of tick-borne flaviviruses and may provide a specific genetic marker for these viruses.     

Flavivirus encephalitis: focus on West Nile encephalitis

The genus flavivirus, family Flaviviridae includes a serocomplex of vector-borne neurotropic viruses as in the tick-borne encephalitis virus serocomplex and the Japanese encephalitis virus serocomplex. The tick-borne encephalitis virus serocomplex includes Central European encephalitis virus and Russian spring summer encephalitis virus. West Nile virus (WNV), which in the Western hemisphere was first detected in New York, in the United States in 1999, is one of several mosquito-borne members of the Japanese encephalitis serocomplex, that cause similar encephalitis. The virus has moved to western states of the U.S. since 2002 and spread rapidly to neighboring countries, including Canada, Mexico and Caribbean islands during 2003-2004. Serology has a dominant role in the laboratory diagnosis of WNV and other flaviviruses in humans. This review aims to describe recent developments in the understanding of flavivirus encephalitis to assist in the event of a WNV outbreak in East Asia.     

Tick-borne flaviviruses: dissecting host immune responses and virus countermeasures

The tick-borne encephalitis (TBE) serocomplex of viruses, genus Flavivirus, includes a number of important human pathogens that cause serious neurological illnesses and hemorrhagic fevers. These viruses pose a significant public health problem due to high rates of morbidity and mortality, their emergence to new geographic areas, and the recent rise in the incidence of human infections. The most notable member of the TBE serocomplex is tick-borne encephalitis virus (TBEV), a neurotropic flavivirus that causes debilitating and sometimes fatal encephalitis. Although effective prophylactic anti-TBEV vaccines have been developed, there is currently no specific treatment for infection. To identify new targets for therapeutical intervention, it is imperative to understand interactions between TBEV and the host immune response to infection. Interferon (IFN) has a critical role in controlling flavivirus replication. Dendritic cells (DCs) represent an early target of TBEV infection and are major producers of IFN. Thus, interactions between DCs, IFN responses, and the virus are likely to substantially influence the outcome of infection. Early IFN and DC responses are modulated not only by the virus, but also by the tick vector and immunomodulatory compounds of tick saliva inoculated with virus into the skin. Our laboratory is examining interactions between the triad of virus, tick vector, and mammalian host that contribute to the pathogenesis of tick-borne flaviviruses. This work will provide a more detailed understanding of early events in virus infection and their impact on flavivirus pathogenesis.     

Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses

The genome sequence of tick-borne encephalitis (TBE) virus (Western subtype vaccine strain Neudoerfl) was determined. This extends the previously published sequence of the structural proteins to the nonstructural protein region and noncoding sequences at the 5'- and 3'-termini. The amino-termini of the individual proteins were assigned by comparison with other flavivirus sequences. Amino acid homology calculations between TBE virus and mosquito-borne flaviviruses were performed for all nonstructural proteins. An evolutionary tree based on protein NS1 is presented that reveals the molecular basis of relationships among flaviviruses. Tick-borne and mosquito-borne flaviviruses share a common hydrophilicity profile and also other features of their primary sequences, such as the presumably functional Gly-Asp-Asp sequence element within protein NS5. Other characteristics, such as the potential N-glycosylation sites of protein NS1 and a potential proteolytic cleavage site within protein NS4B, are conserved within the mosquito-borne group, but differ in the TBE virus sequence.     

Nucleotide sequence of the envelope glycoprotein of Negishi virus shows very close homology to louping ill virus.

Negishi virus, a member of the family Flaviviridae, was originally isolated in Japan, during an outbreak of Japanese encephalitis. Antigenically, however, Negishi virus resembles the tick-borne rather than the mosquito-borne flaviviruses. Monoclonal antibodies that bind louping ill virus showed a close antigenic relationship between louping ill and Negishi virus. The genes encoding the envelope glycoprotein of Negishi virus (strain 3248/49/P10) and louping ill virus (strain SB526) were cloned and sequenced. They showed a very close homology at both the nucleotide and deduced amino acid levels. Comparison with the known sequence of another strain of louping ill virus (strain 369/T2) and with other tick-borne flaviviruses showed that Negishi virus was more closely related to louping ill virus than to the other tick-borne viruses. The significance of this observation for virus evolution, virus distribution in the environment, and the potential use of nucleotide sequencing for rapid and precise identification of flaviviruses are discussed.     

Use of recombinant E protein domain III-based enzyme-linked immunosorbent assays for differentiation of tick-borne encephalitis serocomplex flaviviruses from mosquito-borne flaviviruses

The serological diagnosis of infection by flaviviruses is complicated by the presence of flavivirus cross-reactive antibodies that produce false-positive results for flavivirus infections, especially in regions where more than one virus is endemic. Current diagnostic reagents for tick-borne flavivirus infection have been found to cross-react with yellow fever- or dengue virus-positive sera. This study utilized recombinant flavivirus E protein domain 3 (rE-D3) as a diagnostic reagent to differentiate between infection by mosquito- and tick-borne flaviviruses. This study found that the use of rE-D3 in an enzyme-linked immunosorbent assay (ELISA)-based format allowed the differentiation between serum specific for either mosquito- or tick-borne flaviviruses, but not among the members of the tick-borne encephalitis (TBE) serocomplex of flaviviruses. Sera derived against several TBE serocomplex rE-D3 were found to cross-react with heterologous rE-D3 within the TBE serocomplex, but not with those from mosquito-borne flaviviruses, in both Western blots and ELISAs. Mouse hyperimmune sera generated against TBE serocomplex viruses were also found to react specifically with TBE serocomplex rE-D3, but not with rE-D3 from mosquito-borne viruses and vice versa. When a similar test using virus-derived antigen was performed, a loss of both specificity and sensitivity was observed. These results indicate that flavivirus rE-D3 would be a useful reagent for the detection of infection by TBE serocomplex flaviviruses, several of which are potential biothreat agents, but would not provide the ability to differentiate among infections by separate members of the serocomplex.     

A comparative study of tick-borne encephalitis virus RNA synthesis in acutely and persistently infected cells

Summary Rate zonal and buoyant density gradient centrifugation did not reveal any difference between tick-borne encephalitis virus virions released from acutely or persistently infected cells. All three RNA species characteristic for flavivirus replication were found both in acutely or persistently infected cells, but increased levels of intracellular 42S RNA polyadenylation was observed in persistently infected cells.With 2 Figures     

Tick-borne virus diseases of human interest in Europe

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A-C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.     

Heterologous resistance to superinfection by louping ill virus persistently infected cell cultures

Summary Louping ill virus, a tick-borne arbovirus readily established a persistent infection in porcine kidney (PS) cells after initially inducing minor cytopathic changes. Nucleotide sequence analysis of the envelope glycoprotein of the viral RNA recovered from the persistently infected cells showed no changes as compared with the virus used to establish persistent infections. More than 80 per cent of the cells contained virus specific antigen when analysed by indirect immunofluorescence microscopy. This persistently infected cell line resisted superinfection with either homologous or most heterologous flaviviruses. However, the yellow fever French neurotropic virus (YF FNV) multiplied in the persistently infected cells and evidence of dual infections in these cells was obtained using specific monoclonal antibodies in double labelling immunofluorescence tests. The relevance of these observations is discussed in the light of other evidence that tick-borne viruses can survive for long periods in wild animal species.     

Monoclonal antibodies to flavivirus antigens induced by a vaccinal strain of yellow fever

Monoclonal antibodies (MABs) YEL-2 induced by the vaccine FNS Dakar yellow fever (YF) virus were characterized for their capacity to enter into serological reactions and to react with heterologous flaviviruses. YEL-2 MABs belong to the IgG2a class of immunoglobulins, possess the antihemagglutination properties, are active in indirect IF test but do not activate complement and have no neutralizing properties. The inability to enter into CFT in the presence of antihemagglutinating properties suggests that YEL-2 MABs are directed for the structural E glycoprotein. YEL-2 MABs reacted similarly with the vaccine 17D strain and the FNS Dakar strain by which they had been induced. In addition to YF virus, YEL-2 MABs reacted with Tyuleniy, Rosio, Ilhéus, Uganda S, Karshi, and Sokuluk viruses, the reaction with Tyuleniy virus reaching the same titer as with the homologous virus but was of one-way nature. No reaction of YEL-2 MABs was observed with the viruses of the tick-borne encephalitis complex, Japanese encephalitis, West Nile, Dengue 2 and 4 viruses. These results specify the antigenic classification of flaviviruses.     

Detection of flavivirus antibodies in human serum by epitope-blocking immunoassay

Human flavivirus group-reactive, dengue complex-reactive, and encephalitis virus complex-reactive antibodies were detected using epitope-blocking immunoassays in which the binding of selected mouse monoclonal antibodies to flavivirus antigens was blocked by human serum. When late (greater than 6 months after illness) convalescent sera were tested, the epitope-blocking immunoassays were superior to the hemagglutination inhibition test and comparable to the plaque reduction neutralization for identifying subjects immune to dengue, to Japanese encephalitis, or both viruses.     

Comparison of six different commercial IgG-ELISA kits for the detection of TBEV-antibodies

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a pathogenic human flavivirus endemic in some parts of Europe and Asia. Commercial enzyme immunoassays (EIA) for the detection of IgG antibodies are often used in TBEV-seroprevalence studies, as well as for the confirmation of a successful vaccination against TBEV. However, the detection of TBEV-specific antibodies can be biased by the cross-reactivity between different flavivirus genera. OBJECTIVES: To compare different EIA test systems for the detection of TBEV-IgG antibodies. STUDY DESIGN: Six commercial EIA kits for the detection of TBEV-specific antibodies are compared, using serum panels (n=139) of subjects with a documented clinical history (109 sera from TBEV infected patients, 30 sera from people vaccinated against TBEV). For the analysis of possible cross-reactivities, 24 sera from yellow fever vaccinated people and 13 sera positive for Dengue virus-specific antibodies were also included. RESULTS: The sensitivity of the different TBEV test systems ranges from 73 to 99%. However, when testing the yellow fever and Dengue virus positive specimens, problems with the flavivirus cross- reactivity become obvious, resulting in specificities between 14 and 81%. CONCLUSIONS: This study shows the necessity of further improvement of the existing TBEV test systems regarding both sensitivity and specificity.