Tick-borne Encephalitis Virus Complex Research Articles

Selection Pressure Profile Suggests Species Criteria among Tick-Borne Orthoflaviviruses.

Orthoflaviviruses are arthropod-borne viruses that are transmitted by mosquitoes or ticks and cause a range of significant human diseases. Among the most important tick-borne orthoflaviviruses (TBFVs) is tick-borne encephalitis virus (TBEV), which is endemic in Eurasia, and Powassan virus, which is endemic in Asia and North America. There is a significant controversy regarding species assignment in the tick-borne encephalitis virus complex due to the complex phylogenetic, serological, ecological, and pathogenetic properties of viruses. Comparing the rate of non-synonymous to synonymous substitutions (dN/dS) over the course of tick-borne orthoflavivirus diversification suggests that there is a very strong stabilizing selection (Nei-Gojobori dN/dS < 0.1) among tick-borne orthoflaviviruses that differ by less than 13.5% amino acid/21.4% nucleotide sequences, and discretely more rapid accumulation of non-synonymous substitutions (dN/dS > 0.13) among more divergent viruses that belong to distinct species. This pattern was similarly observed in genome regions encoding structural (E) and non-structural (NS3) proteins. Below this distance threshold, viruses appear fit and strongly tied to their ecological niche, whereas above the threshold, a greater degree of adaptation appears necessary. This species criterion suggests that all subtypes of TBEV, all related ovine/caprine encephalomyelitis viruses, and Omsk hemorrhagic fever virus (OHFV) together correspond to a single species. Within this species, viruses make up 11 subtypes that are reliably segregated by a 10% nucleotide distance cut-off suggested earlier for TBEV. The same 10% subtype cut-off suggests that Powassan virus includes two subtypes, Powassan and Deer Tick virus.

Tick-Borne Flavivirus Inhibits Sphingomyelinase (IsSMase), a Venomous Spider Ortholog to Increase Sphingomyelin Lipid Levels for Its Survival in Ixodes scapularis Ticks.

Our previous study showed that cells from medically important arthropods, such as ticks, secrete extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Understanding the molecular determinants and mechanism(s) of arthropod-borne flavivirus transmission via exosome biogenesis is very important. In this current study, we showed that in the presence of tick-borne Langat Virus (LGTV; a member of tick-borne encephalitis virus complex), the expression of arthropod IsSMase, a sphingomyelinase D (SMase D) that catalyzes the hydrolytic cleavage of substrates like sphingomyelin (SM) lipids, was significantly reduced in both Ixodes scapularis ticks (in vivo) and in tick cells (in vitro). The IsSMase reduced levels correlated with down-regulation of its activity upon LGTV replication in tick cells. Our data show that LGTV-mediated suppression of IsSMase allowed accumulation of SM lipid levels that supported membrane-associated viral replication and exosome biogenesis. Inhibition of viral loads and SM lipid built up upon GW4869 inhibitor treatment reversed the IsSMase levels and restored its activity. Our results suggest an important role for this spider venomous ortholog IsSMase in regulating viral replication associated with membrane-bound SM lipids in ticks. In summary, our study not only suggests a novel role for arthropod IsSMase in tick-LGTV interactions but also provides new insights into its important function in vector defense mechanism(s) against tick-borne virus infection and in anti-viral pathway(s).

Geographic distribution of Tick-borne encephalitis virus complex.

A comprehensive understanding of the geographic distribution of the tick-borne encephalitis virus (TBEV) complex is necessary due to increasing transboundary movement and cross-reactivity of serological tests. This review was conducted to identify the geographic distribution of the TBEV complex, including TBE virus, Alkhurma haemorrhagic fever virus, Kyasanur forest disease virus, louping-ill virus, Omsk haemorrhagic fever virus, and Powassan virus. Published reports were identified using PubMed, EMBASE, and the Cochrane library. In addition to TBEV complex case-related studies, seroprevalence studies were also retrieved to assess the risk of TBEV complex infection. Among 1406 search results, 314 articles met the inclusion criteria. The following countries, which are known to TBEV epidemic region, had conducted national surveillance studies: Austria, China, Czech, Denmark, Estonia, Finland, Germany, Hungary, Italy, Latvia, Norway, Poland, Romania, Russia, Switzerland, Sweden, Slovenia, and Slovakia. There were also studies/reports on human TBEV infection from Belarus, Bulgaria, Croatia, France, Japan, Kyrgyzstan, Netherland, and Turkey. Seroprevalence studies were found in some areas far from the TBEV belt, specifically Malaysia, Comoros, Djibouti, and Kenya. Kyasanur forest disease virus was reported in southwestern India and Yunnan of China, the Powassan virus in the United States, Canada, and east Siberia, Alkhurma haemorrhagic fever virus in Saudi Arabia and east Egypt, and Louping-ill virus in the United Kingdom, Ireland, and east Siberia. In some areas, the distribution of the TBEV complex overlaps with that of other viruses, and caution is recommended during serologic diagnosis. The geographic distribution of the TBEV complex appears to be wide and overlap of the TBE virus complex with other viruses was observed in some areas. Knowledge of the geographical distribution of the TBEV complex could help avoid cross-reactivity during the serologic diagnosis of these viruses. Surveillance studies can implement effective control measures according to the distribution pattern of these viruses.

Cervids as sentinel-species for tick-borne encephalitis virus in Norway - A serological study.

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE). TBEV is one of the most important neurological pathogens transmitted by tick bites in Europe. The objectives of this study were to investigate the seroprevalence of TBE antibodies in cervids in Norway and the possible emergence of new foci, and furthermore to evaluate if cervids can function as sentinel animals for the distribution of TBEV in the country. Serum samples from 286 moose, 148 roe deer, 140 red deer and 83 reindeer from all over Norway were collected and screened for TBE immunoglobulin G (IgG) antibodies with a modified commercial enzyme-linked immunosorbent assay (ELISA) and confirmed by TBEV serum neutralisation test (SNT). The overall seroprevalence against the TBEV complex in the cervid specimens from Norway was 4.6%. The highest number of seropositive cervids was found in south-eastern Norway, but seropositive cervids were also detected in southern- and central Norway. Antibodies against TBEV detected by SNT were present in 9.4% of the moose samples, 1.4% in red deer, 0.7% in roe deer, and nil in reindeer. The majority of the positive samples in our study originated from areas where human cases of TBE have been reported in Norway. The study is the first comprehensive screening of cervid species in Norway for antibodies to TBEV, and shows that cervids are useful sentinel animals to indicate TBEV occurrence, as supplement to studies in ticks. Furthermore, the results indicate that TBEV might be spreading northwards in Norway. This information may be of relevance for public health considerations and supports previous findings of TBEV in ticks in Norway.

Characterizing the cellular attachment receptor for Langat virus.

Tick-borne encephalitis infections have increased the last 30 years. The mortality associated to this viral infection is 0.5 to 30% with a risk of permanent neurological sequelae, however, no therapeutic is currently available. The first steps of virus-cell interaction, such as attachment and entry, are of importance to understand pathogenesis and tropism. Several molecules have been shown to interact with tick-borne encephalitis virus (TBEV) at the plasma membrane surface, yet, no studies have proven that these are specific entry receptors. In this study, we set out to characterize the cellular attachment receptor(s) for TBEV using the naturally attenuated member of the TBEV complex, Langat virus (LGTV), as a model. Inhibiting or cleaving different molecules from the surface of A549 cells, combined with inhibition assays using peptide extracts from high LGTV binding cells, revealed that LGTV attachment to host cells is dependent on plasma membrane proteins, but not on glycans or glycolipids, and suggested that LGTV might use different cellular attachment factors on different cell types. Based on this, we developed a transcriptomic approach to generate a list of candidate attachment and entry receptors. Our findings shed light on the first step of the flavivirus life-cycle and provide candidate receptors that might serve as a starting point for future functional studies to identify the specific attachment and/or entry receptor for LGTV and TBEV.

Detection of a novel tick-borne flavivirus and its serological surveillance

Tick-borne encephalitis virus (TBEV), a flavivirus that causes severe neurological symptoms in humans, has been found in Hokkaido, Japan. In the present study, we detected sequences from a novel tick-borne flavivirus, designated Yamaguchi virus (YGV), in liver and serum samples obtained from a wild boar in the Yamaguchi prefecture, Japan. Phylogenetic analysis revealed that YGV belongs to the TBEV complex and is closely related to Langat virus (LGTV). YGV was also detected by specific RT-PCR from 20 in 378 pools of ticks (2923 ticks) collected in Yamaguchi and Wakayama prefectures and from seven in 46 wild boar captured in Wakayama. The major ticks infected with YGV belong to the genus Haemaphysalis. Unfortunately, YGV could not be isolated from any samples from the RT-PCR positive wild boar or ticks. Therefore, ELISA for detection of antibodies against YGV was established using LGTV, and surveillance was performed among wild boar in 10 different prefectures on Honshu Island, the main island of Japan. The results showed that the seroprevalence of tick-borne flavivirus infection in the Wakayama and Hyogo prefectures of western Japan was significantly higher than that in the other prefectures, while antibodies against tick-borne flavivirus were not detected in any wild boar in the Tochigi prefecture in the eastern part of Japan. In addition, wild raccoons or masked palm civets in the Hyogo prefecture did not possess detectable antibodies against tick-borne flaviviruses. In conclusion, YGV appears to be maintained primarily among wild boar and ticks in the western part of Japan. YGV is the second flavivirus (after Japanese encephalitis virus) shown to be circulating on Honshu Island in Japan.

Continued expansion of tick-borne pathogens: Tick-borne encephalitis virus complex and Anaplasma phagocytophilum in Denmark

Tick-borne encephalitis virus (TBEV) is a tick-transmitted flavivirus within the tick-borne encephalitis (TBE) complex. The TBE complex is represented by both TBEV and louping ill virus (LIV) in Denmark. Anaplasma phagocytophilum is also transmitted by ticks and is believed to play an essential role in facilitating and aggravating LIV infection in sheep.This study aimed to describe the distribution of TBE complex viruses in Denmark, to establish the possible emergence of new foci and their association with the distribution of A. phagocytophilum. We performed a nationwide seroprevalence study of TBE complex viruses using roe deer (Capreolus capreolus) as sentinels and determined the prevalence of A. phagocytophilum in roe deer. Danish hunters obtained blood samples from roe deer during the hunting season of 2013-14. The samples were examined for TBEV-specific antibodies by virus neutralization tests (NT). A. phagocytophilum infection was assessed by specific real-time-PCR.The overall seroprevalence of the TBE complex viruses in roe deer was 6.9% (51/736). The positive samples were primarily obtained from a known TBE endemic foci and risk areas identified in previous sentinel studies. However, new TBE complex risk areas were also identified. The overall prevalence of A. phagocytophilum was 94.0% (173 PCR-positive of 184 roe deer), which is twice the rate observed ten years ago.These results point to an expansion of these tick-borne diseases geographically and within reservoir populations and, therefore, rationalize the use of sentinel models to monitor changes in transmission of tick-borne diseases and development of new risk areas. We found no association between TBE complex-positive roe deer and the prevalence of A. phagocytophilum, as almost all roe deer were infected. Based on our findings we encourage health care providers to be attentive to tick-borne illnesses such as TBE when treating patients with compatible symptoms.

Kyasanur forest disease virus: viremia and challenge studies in monkeys with evidence of cross-protection by Langat virus infection

Kyasanur Forest Disease Virus (KFDV), discovered in 1957, is a member of the tick-borne encephalitis virus (TBEV) complex. Diseases caused by members of the TBEV complex occur in many parts of the world. KFDV produces a hemorrhagic fever in humans in South India and fatal illnesses in both species of monkeys in the area, the black faced langur (Presbytis entellus) and the bonnet macaque (Macaca radiata). Experimental infection of the langur and the bonnet macaque with early mouse passage KFDV strain P9605 resulted in a viremia of up to 11 days duration, peak viremia titers as high as 10 9 , and death in 82 = 100% of the animals. Prolonged passage of the KFDV strain P9605 in monkey kidney tissue culture resulted in a markedly reduced virulence of the virus for both species; peak viremia titers in monkeys decreased by 2.5 to 4.0 log LD 50 (p= 0.001), and the mortality decreased to 10% (p= 0.001). In challenge experiments, monkeys previously infected with tissue-culture-adapted KFDV, or with the related Langat virus from Malaysia, were fully protected against virulent KFDV. These studies in non-human primates lend support to the idea that a live virus vaccine from a member of the TBEV complex may be broadly protective against infections by other members of the TBEV complex.

Kyasanur forest disease virus: viremia and challenge studies in monkeys with evidence of cross-protection by Langat virus infection

Kyasanur Forest Disease Virus (KFDV), discovered in 1957, is a member of the tick-borne encephalitis virus (TBEV) complex. Diseases caused by members of the TBEV complex occur in many parts of the world. KFDV produces a hemorrhagic fever in humans in South India and fatal illnesses in both species of monkeys in the area, the black faced langur (Presbytis entellus) and the bonnet macaque (Macaca radiata). Experimental infection of the langur and the bonnet macaque with early mouse passage KFDV strain P9605 resulted in a viremia of up to 11 days duration, peak viremia titers as high as 10 9, and death in 82 = 100% of the animals. Prolonged passage of the KFDV strain P9605 in monkey kidney tissue culture resulted in a markedly reduced virulence of the virus for both species; peak viremia titers in monkeys decreased by 2.5 to 4.0 log LD 50 (p= 0.001), and the mortality decreased to 10% (p= 0.001). In challenge experiments, monkeys previously infected with tissue-culture-adapted KFDV, or with the related Langat virus from Malaysia, were fully protected against virulent KFDV. These studies in non-human primates lend support to the idea that a live virus vaccine from a member of the TBEV complex may be broadly protective against infections by other members of the TBEV complex.

SiRNA Inhibits Replication of Langat Virus, a Member of the Tick-Borne Encephalitis Virus Complex in Organotypic Rat Brain Slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000–3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3′ untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.

Evaluation of the Langat/dengue 4 chimeric virus as a live attenuated tick-borne encephalitis vaccine for safety and immunogenicity in healthy adult volunteers

With the steady rise in tick-borne encephalitis virus (TBEV) infections in Europe, development of a live attenuated vaccine that will generate long-lasting immunity would be of considerable benefit. A chimeric flavivirus, designated LGT/DEN4, was previously constructed to have a genome containing the prM and E protein genes of Langat virus (LGT), a naturally attenuated member of the TBEV complex, and the remaining genetic sequences derived from dengue 4 virus (DEN4). LGT/DEN4 was highly attenuated in rodents and non-human primates, and clinical trials in humans were initiated. Twenty-eight healthy seronegative adult volunteers were randomly assigned in a 4:1 ratio to receive 10(3) plaque-forming units (PFU) of LGT/DEN4 or placebo. Volunteers were closely monitored for clinical responses and for blood chemistry and hematological changes, and the level of viremia and the magnitude and duration of the neutralizing antibody response were determined. The LGT/DEN4 vaccine was safe and viremia was seen in only one vaccinee. Infection induced a neutralizing antibody response to wild-type LGT in 80% of volunteers with a geometric mean titer (GMT) of 1:63 present on day 42 post-immunization; however the antibody response against TBEV was both much less frequent (35%) and lower in magnitude (GMT=1:9). To assess the response to a booster dose, 21 of the original 28 volunteers were re-randomized to receive a second dose of either 10(3) PFU of vaccine or placebo given 6-18 months after the first dose. The immunogenicity against either LGT or TBEV was not significantly enhanced after the second dose of vaccine. Thus, chimerization of LGT with DEN4 yielded a vaccine virus that was highly attenuated yet infectious in humans. The level of replication was sufficiently restricted to induce only a weak cross-reactive antibody response to TBEV. To provide a sufficient level of immunity to widely prevalent, highly neurovirulent strains of TBEV in humans, vaccine candidates will likely need to be based on the TBEV structural protein genes.

Experimental Transmission of Karshi and Langat (Tick-Borne Encephalitis Virus Complex) Viruses byOrnithodorosTicks (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.

Tick-Borne Langat/Mosquito-Borne Dengue Flavivirus Chimera, a Candidate Live Attenuated Vaccine for Protection against Disease Caused by Members of the Tick-Borne Encephalitis Virus Complex: Evaluation in Rhesus Monkeys and in Mosquitoes

Langat virus (LGT), strain TP21, a naturally avirulent tick-borne flavivirus, was used to construct a chimeric candidate virus vaccine which contained LGT genes for premembrane (preM) and envelope (E) glycoprotein and all other sequences derived from dengue type 4 virus (DEN4). The live virus vaccine was developed to provide resistance to the highly virulent, closely related tick-borne flaviviruses that share protective E epitopes among themselves and with LGT. Toward that end the chimera, initially recovered in mosquito cells, was adapted to grow to high titer in qualified simian Vero cells. When inoculated intraperitoneally (i.p.), the Vero cell-adapted LGT TP21/DEN4 chimera remained completely attenuated for SCID mice. Significantly, the chimera protected immunocompetent mice against the most virulent tick-borne encephalitis virus (TBEV). Subsequently, rhesus monkeys were immunized in groups of 4 with 10(5) or 10(7) PFU of LGT strain TP21, with 10(5) PFU of DEN4, or with 10(3), 10(5), or 10(7) PFU of the chimera. Each of the monkeys inoculated with DEN4 or LGT TP21 became viremic, and the duration of viremia ranged from 1 to 5 days. In contrast, viremia was detected in only 1 of 12 monkeys inoculated with the LGT TP21/DEN4 chimera; in this instance the level of viremia was at the limit of detection. All monkeys immunized with the chimera or LGT TP21 virus developed a moderate to high level of neutralizing antibodies against LGT TP21 as well as TBEV and were completely protected against subsequent LGT TP21 challenge, whereas monkeys previously immunized with DEN4 virus became viremic when challenged with LGT TP21. These observations suggest that the chimera is attenuated, immunogenic, and able to induce a protective immune response. Furthermore, passive transfer of serum from monkeys immunized with chimera conferred significant protection to mice subsequently challenged with 100 i.p. 50% lethal doses of the highly virulent TBEV. The issue of transmissibility of the chimera by mosquitoes was addressed by inoculating a nonhematophagous mosquito, Toxorhynchites splendens, intrathoracically with the chimera or its DEN4 or LGT parent. Neither the LGT TP21/DEN4 vaccine candidate nor the wild-type LGT TP21 virus was able to infect this mosquito species, which is highly permissive for dengue viruses. Certain properties of the chimera, notably its attenuation for monkeys, its immunogenicity, and its failure to infect a highly permissive mosquito host, make it a promising vaccine candidate for use in immunization against severe disease caused by many tick-borne flaviviruses.

Experimental Transmission of Langat (Tick-Borne Encephalitis Virus Complex) Virus by the Soft Tick Ornithodoros sonrai (Acari: Argasidae)

Laboratory studies determined that the soft tick Ornithodoros sonrai Sautet & Witkowski is a competent vector of Langat (tick-borne encephalitis virus complex) virus. When ticks fed on suckling mice having a mean viremia of 10(7.2) plaque-forming units per ml, 52% (n = 208) became infected, and 84% (n = 87) of the infected ticks transmitted virus by bite when fed individually on suckling mice > or = 27 d after the infectious blood meal. Overall, 79 of 184 (43%) of ticks exposed to the original viremic mice individually transmitted virus by bite when tested up to 351 d after the infectious blood meal. In addition, ticks transmitted virus both transstadially and transovarially. Some ticks that transmitted virus during their first transmission attempt were retested. These ticks transmitted virus during 81 (99%) of 82 refeeding attempts, including ticks that transmitted virus 512 d after the initial infectious meal. Therefore, Ornithodoros spp. should be considered as potential vectors of Langat and other tick-borne encephalitis viruses.

Rapid detection of viruses of the tick-borne encephalitis virus complex by RT-PCR of viral RNA

Studies were performed to identify a pair of primers, specific for the tick-borne encephalitis (TBE) virus complex of the Flaviviridae, with which to develop a rapid and specific identification system based on reverse transcription and the polymerase chain reaction (RT-PCR). The specificity of a putative primer pair was examined by RT-PCR of representative viruses from other antigenic complexes of the Flaviviridae and by computer sequence homology checks. All viruses of the TBE complex tested, with a single exception, were identified by RT-PCR using the identified primer pair. Accumulated data suggest that one of the putative primers identified in these studies may have flavivirus group specificity. The advantages of such a primer in the development of identification systems for all virus complexes of the Flaviviridae is discussed.

Inefficient Mechanical Transmission of Langat (Tick-Borne Encephalitis Virus Complex) Virus by Blood-Feeding Mites (Acari) to Laboratory Mice

One day after feeding on a viremic mouse, tropical rat mites, Ornithonyssus bacoti (Hirst), transmitted Langat (tick-borne encephalitis virus complex) virus to a naive suckling mouse in one of four trials. However, no transmissions to naive mice by O. bacoti were recorded either immediately after the viremic blood meal (0/4 trials) or on days 4-18 (0/20 trials). After feeding on a viremic mouse, chicken mites, Dermanyssus gallinae (De Geer), failed to transmit Langat virus to naive suckling mice in any trials (0/24). Although virus failed to replicate in either species of mite, it was detectable in 20% (2/10) of O. bacoti individuals 1 d after a viremic blood meal, but only immediately after the viremic blood meal in 20% (2/10) of D. gallinae mites. Neither mite appears to be an efficient vector of Langat virus.

RAPID TYPING OF DENGUE VIRUSES BY THE MICRO-PRECIPITIN AGAR-GEL DIFFUSION TECHNIQUE.

THE agar-gel precipitin technique has been used to differentiate members of the tick-borne encephalitis virus complex of group B arboviruses1. This report describes the application of this technique in the rapid typing of dengue virus types 1, 2, 3 and 4.