An antivector vaccine protects against a lethal vector-borne pathogen

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a "dual action" in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: "transmission," number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; "support," number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and "survival," number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.     

Genetic diversity of tick-borne pathogenes in Tomsk and environs

Two urban and two suburban biotopes of Tomsk were studied for tick-transmitted disease prevalence in ticks collected in the wild. Tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Borrelia spp., Rickettsia spp., and Ehrlichia spp. were found in 6.5%, 2.2%, 8%, 2.5%, and 1.7% of tick specimens, respectively. Genetic markers of Powassan virus, Bartonella spp., and Babesia spp. were not found. Analysis of the genetic diversity of revealed pathogens demonstrated that TBEV strains belonged to the Siberian and Far-Eastern subtypes, and the Far-Eastern subtype of TBEV is most frequently found in urban biotopes (up to 43% of urban strains of TBEV). WNV strains belonged to the 1a genotype. Borrelia spp. was classified as B. garinii, Rickettsia spp. was classified as R. tarasevichiae and probably as a new Rickettsia raoultii subspecies, and Ehrlichia spp. was classified as E. muris. The coexistence of several pathogens was found in 5.7% of tick specimens, and TBEV + Borrelia spp. was the most frequent combination.     

Differences in the distant transmission of the tick-borne encephalitis virus by ixodid ticks of 2 subfamilies]

Simultaneous but separate feeding of ticks on nonviremic animal (guinea pig) has shown that Amblyomminae ticks are practically unable to transmit distantly tick-borne encephalitis virus (TBEV) to the specimens of their own subfamily and to Ixodinae as well. Ixodes persulcatus and I. ricinus displayed their ability as donors and recipients of TBEV (adults and nymphs) not only for their own subfamily representatives but also as donors for recipients of Amblyomminae subfamily (nymphs and adults of Dermacentor and Rhipicephalus and nymphs of Haemaphysalis). Experimental and literature data analysis permits the authors to conclude that the very important role of TBEV circulation in nature belongs to the distant virus transmission. The absence of such type of virus exchange among Amblyomminae excludes this group of ticks from active virus circulation in TBEV foci.     

Virus–Pesticide interactions in Ixodes persulcatus and Amblyomma hebraeum ticks (Acarina: Ixodidae)

Sublethal doses of the organophosphate Dursban were tested on non-infected and tick-borne encephalitis virus (TBEV)-infected nymphs of Amblyomma hebraeum and Ixodes persulcatus. It was shown that contact with this pesticide enhanced the survival of non-infected ticks, whereas TBEV infection reduced survival. This effect of TBEV infection was greater in I. persulcatus than in A. hebraeum ticks. This phenomenon may be the result of a virus-specific action on nymphs, whereby the virus may enhance activity and thus food reserves may deplete faster. Our data emphasize that the acaricidal action on non-infected ticks and those infected by different tick-borne pathogens may be different.     

The correlation between tick (Ixodes persulcatus Sch.) questing behaviour and synganglion neuronal responses to odours

We examined the behavioural and electrophysiological responses of taiga ticks (Ixodes persulcatus) to several olfactory stimuli: Osmopherone® (5-a-androst-16-en-3-ol), Osmopherine® (butanoic and 3-methylbutanoic acids), DEET® (N,N-diethyl-meta-toluamide), ethanol (96%), and water (control stimulus). To study individual tick behavioural reactions to these stimuli, we used a Y-shaped glass maze (n = 50). To study the electrophysiological reactions of the ticks’ synganglia to these olfactory stimuli, we recorded the shifts of total potential (TP) of pre-oesophageal neurons in response to odour stimulation of Haller’s organ (n = 25). We found that Osmopherine® attracted ticks and frequently evoked negative shifts of TP, whereas the response to Osmopherone® did not differ from the reaction to water. DEET® and ethanol acted as tick repellents and generally evoked positive shifts of TP. We also tested each tick for its questing height (QH) on a glass rod that was at an incline of 75°, and we tested for the presence of pathogens i.e., DNA of Borrelia burgdorferi sp. s.l. and RNA of tick-borne encephalitis virus (TBEV). The degree of response to Osmopherine® positively correlated with the QH. The ticks with the highest values of QH showed a greater prevalence of the tick-borne pathogen Borrelia sp. s.l. compared with the ticks that did not reach the maximum QH. The present results show a correlation between the electrophysiological reaction of the synganglia of ticks and their behavioural responses to different odours.     

Tick-borne encephalitis virus in northern Italy: molecular analysis, relationships with density and seasonal dynamics of Ixodes ricinus

Ixodes ricinus ticks were collected from dragging vegetation and from shot roe deer in the province of Trento and Belluno in northern Italy. Ticks were pooled for analyses and from 1060 pools of ticks collected in the province of Belluno and 12390 tick samples collected in Trentino, four proved positive by immunofluorescence microscopy using a tick-borne encephalitis (TBE)-specific antiserum. The identity of the virus isolates was determined by RT-PCR cycle sequencing and they were all found to be closely similar (> 98% nucleotide identity) to typical western European TBE complex viruses as found in Austria. The isolates from Trentino differed from the Neudorfl strain of western European TBE virus at eight nucleotide positions but as these nucleotide substitutions were all synonymous, there were no amino acid changes. These results imply that the virus isolates in Trentino have changed slightly from the typical European strains isolated in nearby Austria. The abundance of questing ticks and ticks feeding on roe deer was greater in TBE positive hunting districts than in hunting districts where TBE complex viruses were only probable or believed to be absent. In TBE positive and probable districts synchrony in the seasonal dynamics of larvae and nymphs of L. ricinus was observed. This study provides evidence to suggest that roe deer may have an important role to play in the maintenance of tick density and in the persistence of TBE virus.     

Molecular evolution of the tick-borne encephalitis and Powassan viruses

Issues associated with newly emerging viruses, their genetic diversity, and viral evolution in modern environments are currently attracting growing attention. In this study, a phylogenetic analysis was performed and the evolution rate was evaluated for such pathogenic flaviviruses endemic to Russia as tick-borne encephalitis virus (TBEV) and Powassan virus (PV). The analysis involved 47 nucleotide sequences of the TBEV genome region encoding protein E and 17 sequences of the PV NS5-encoding region. The nucleotide substitution rate was estimated as 1.4 × 10⁻⁴ and 5.4 × 10⁻⁵ substitutions per site per year for the E protein-encoding region of the TBEV genome and for the NS5 genome region of PV, respectively. The ratio of non-synonymous to synonymous nucleotide substitutions (dN/dS) in viral sequences was calculated as 0.049 for TBEV and 0.098 for PV. The highest dN/dS values of 0.201–0.220 were found in the subcluster of Russian and Canadian PV strains, and the lowest value of 0.024 was observed in the cluster of Russian and Chinese strains of the Far Eastern TBEV genotype. Evaluation of time intervals between the events of viral evolution showed that the European subtype of TBEV diverged from the common TBEV ancestor approximately 2750 years ago, while the Siberian and Far Eastern subtypes emerged approximately 2250 years ago. The PV was introduced into its natural foci of the Russian Primorskii krai only approximately 70 years ago; these strains were very close to Canadian PV strains. The pattern of PV evolution in North America was similar to the evolution of the Siberian and Far Eastern TBEV subtypes in Asia. The moments of divergence between major genetic groups of TBEV and PV coincide with historical periods of climate warming and cooling, suggesting that climate change was a key factor in the evolution of flaviviruses in past millennia.     

The basic reproductive number of tick–borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is reciprocally transmitted between Ixodes ricinus ticks and small mammals. Recently, transmission between co-feeding ticks has been postulated as an epidemiological by important mechanism of perpetuating the agent. To empirically examine the question whether the “traditional” mode of transmission is sufficient to maintain enzootic TBEV transmission, the basic reproductive number R₀ of TBEV could be estimated under this model for sites in which TBEV is enzootic. I propose an empirical estimator of R₀ for TBEV which is based on longitudinal stage–specific local tick infestation densities assessed by live trapping of small mammals. A Gibbs sampler–based 95%–credibility interval is presented. When applied to published field data from TBEV enzootic sites sub–critical R₀ estimates are obtained for both sites. I discuss potential shortcomings of this method and possible implications of these findings on the discussion of supplemental mechanisms of transmission. I thank Marc Lipsitch (Harvard School of Public Health, Boston, MA) who helped with an early version of this paper. I also thank Dr. Andrea Pugliese and two anonymous referees for their insightful comments and helpful suggestions.     

Prevalence of tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks from natural and urban environment,Poland

Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus) is an arthropod-borne virus, an etiologic agent of tick-borne encephalitis (TBE), a human infection involving the central nervous system. The disease is endemic in a large region in Eurasia, where it is transmitted mainly by Ixodes ricinus and Ixodes persulcatus ticks. It is known that also Dermacentor reticulatus is involved in circulation of TBEV. However, the current knowledge of D. reticulatus importance in TBE epidemiology is still insufficient. A total of 471 adult D. reticulatus ticks were collected by flagging vegetation in the Bia?owie?a Primeval Forest, Biebrza National Park, Masurian Landscape Park (North-Eastern Poland) and in the city of Warsaw in the years 2007–2010. All collected ticks were examined individually for the presence of RNA of TBEV using nested RT-PCR assay. Positive results were noted in all investigated localities with the infection rate ranging from 0.99 to 12.5 % with a total mean of 2.12 %. The difference in the percentage of infective males and females was not statistically significant.     

Molecular evolution of the tick-borne encephalitis and Powassan viruses

The problem of emerging viruses, their genetic diversity and viral evolution in nature are attracting more attention. The phylogenetic analysis and evaluationary rate estimation were made for pathogenic flaviviruses such as tick-borne encephalitis virus (TBEV) and Powassan (PV) circulated in natural foci in Russia. 47 nucleotide sequences of encoded protein E of the TBEV and 17 sequences of NS5 genome region of the PV have been used. It was found that the rate of accumulation of nucleotide substitutions for E genome region of TBEV was approximately 1.4 x 10(-4) and 5.4 x 10(-5) substitutions per site per year for NS5 genome region of PV. The ratio of non-synonymous nucleotide substitutions to synonymous substitution (dN/dS) for viral sequences were estimated of 0.049 for TBEV and 0.098 for PV. Maximum value dN/dS was 0.201-0.220 for sub-cluster of Russian and Canadian strains of PV and the minimum - 0.024 for cluster of Russian and Chinese strains of Far Eastern genotype TBEV. Evaluation of time intervals of evolutionary events associated with these viruses showed that European subtype TBEV are diverged from all-TBEV ancestor within approximately 2750 years and the Siberian and Far Eastern subtypes are emerged about 2250 years ago. The PV was introduced into natural foci of the Primorsky Krai of Russia only about 70 years ago and PV is a very close to Canadian strains of PV. Evolutionary picture for PV in North America is similar to evolution of Siberian and Far Eastern subtypes TBEV in Asia. The divergence time for main genetic groups of TBEV and PV are correlated with historical periods of warming and cooling. These allow to propose a hypothesis that climate changes were essential to the evolution of the flaviviruses in the past millenniums.     

Ixodes ricinus tick saliva modulates tick-borne encephalitis virus infection of dendritic cells

Tick-borne encephalitis virus is an important human pathogen, naturally delivered into host skin via a tick bite. To examine the effects of the virus on dendritic cell biology, we cultured dendritic cells with two tick-borne encephalitis virus strains of different virulence in the presence of Ixodes ricinus tick saliva. Tick saliva treatment increased proportion of virus-infected cells, led to a decrease in virus-induced TNF-α and IL-6 production and to reduced virus-induced apoptosis. Our data indicate that tick saliva modulate virus-mediated alterations in dendritic cells, thus probably being involved in the early infection process in the host.     

Tick infestation of small mammals in an English woodland

Tick infestations on small mammals were studied from April to November, 2010, in deciduous woodland in southern England in order to determine whether co‐infestations with tick stages occurred on small mammals, a key requirement for endemic transmission of tick‐borne encephalitis virus (TBEV). A total of 217 small mammals was trapped over 1,760 trap nights. Yellow‐necked mice (Apodemus flavicollis) made up the majority (52.5%) of animals, followed by wood mice (A. sylvaticus) 35.5% and bank voles (Myodes glareolus) 12%. A total of 970 ticks was collected from 169 infested animals; 96% of ticks were Ixodes ricinus and 3% I. trianguliceps. Over 98% of ticks were larval stages. Mean infestation intensities of I. ricinus were significantly higher on A. flavicollis (6.53 ± 0.67) than on A. sylvaticus (4.96 ± 0.92) and M. glareolus (3.25 ± 0.53). Infestations with I. ricinus were significantly higher in August than in any other month. Co‐infestations with I. ricinus nymphs and larvae were observed on six (3.6%) infested individuals, and fifteen small mammals (8.9%) supported I. ricinus – I. trianguliceps co‐infestations. This work contributes further to our understanding of European small mammal hosts that maintain tick populations and their associated pathogens, and indicates that co‐infestation of larvae and nymph ticks does occur in lowland UK. The possible implications for transmission of tick‐borne encephalitis virus between UK ticks and small mammals are discussed.     

Epidemiology of a tick‐borne viral infection: theoretical insights and practical implications for public health

The morbidity of tick‐borne encephalitis (TBE) varies yearly by as much as 10‐fold among the people of Western Siberia. This long‐term variation is dependent on many factors such as the density of the tick populations, the prevalence of TBE virus (TBEV) among sub‐adult ticks, the yearly virulence of the TBEV, and prophylactic measures. Here we highlight the role of small mammal hosts in the circulation of TBEV through the ecosystem. Refining classical models of non‐viremic horizontal transmission, we emphasize the recently understood fact that the physiological and immunological status of the small mammal hosts affects the tick and virus‐host interactions. In addition to its theoretical interest, our approach may lead to some practical improvements in the precision of epidemiological forecasts and perhaps in forestalling the severity of outbreaks of TBE, or, at least, in forewarning medical authorities and the general public of impending TBE outbreaks.     

Altitudinal patterns of tick and host abundance: a potential role for climate change in regulating tick-borne diseases?

The impact of climate change on vector-borne infectious diseases is currently controversial. In Europe the primary arthropod vectors of zoonotic diseases are ticks, which transmit Borrelia burgdorferi sensu lato (the agent of Lyme disease), tick-borne encephalitis virus and louping ill virus between humans, livestock and wildlife. Ixodes ricinus ticks and reported tick-borne disease cases are currently increasing in the UK. Theories for this include climate change and increasing host abundance. This study aimed to test how I. ricinus tick abundance might be influenced by climate change in Scotland by using altitudinal gradients as a proxy, while also taking into account the effects of hosts, vegetation and weather effects. It was predicted that tick abundance would be higher at lower altitudes (i.e. warmer climates) and increase with host abundance. Surveys were conducted on nine hills in Scotland, all of open moorland habitat. Tick abundance was positively associated with deer abundance, but even after taking this into account, there was a strong negative association of ticks with altitude. This was probably a real climate effect, with temperature (and humidity, i.e. saturation deficit) most likely playing an important role. It could be inferred that ticks may become more abundant at higher altitudes in response to climate warming. This has potential implications for pathogen prevalence such as louping ill virus if tick numbers increase at elevations where competent transmission hosts (red grouse Lagopus lagopus scoticus and mountain hares Lepus timidus) occur in higher numbers.     

First detection of TBE virus sequences in Ixodes ricinus from Friuli Venezia Giulia (Italy)

We report, for the first time, the presence of tick-borne encephalitis (TBE) virus in the tick Ixodes ricinus collected in the Friuli Venezia Giulia region of north-eastern Italy. Using molecular methods, we demonstrate that the TBE virus carried by ticks from FVG is a western European strain. Sequence analysis of the 5' NCR showed 98.4% identity to the Neudoerfl strain.     

Tick-borne encephalitis virus prevalence in Ixodes ricinus ticks collected in high risk habitats of the south-Bohemian region of the Czech Republic

A survey for tick-borne encephalitis virus in Ixodes ricinus ticks was conducted in May 2000 in two districts of the South-Bohemian region of the Czech Republic with a high occurrence of tick-borne encephalitis (TBE) in humans. Homogenized ticks were tested on PS cells, which were examined for any cytopathic effect, plaque assay and indirect fluorescent antibody test (IFT). The IFT has proved to be the most sensitive and most rapid test to demonstrate the virus in ticks. TBE virus was found in 17 of 187 pooled samples, consisting of a total of 2,968 ticks. The mean minimum infection rate was 0.6% for all tick stages combined. Infection rates in nymphs collected indifferent locations varied between 0.2 and 1.3% and between 5.9 and 11.1% in adult ticks. The observed TBE prevalence in ticks was compared with data obtained elsewhere in the Czech Republic. It is concluded that screening of ticks for TBE virus prevalence using IFT is a valuable indicator for the degree of risk to contract TBE in as particular habitat. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Three Subtypes of Tick-Borne Encephalitis Virus Induce Encephalitis in a Natural Host,the Bank Vole (Myodes glareolus)

Tick-borne encephalitis virus (TBEV) infects bank voles (Myodes glareolus) in nature, but the relevance of rodents for TBEV transmission and maintenance is unclear. We infected colonized bank voles subcutaneously to study and compare the infection kinetics, acute infection, and potential viral persistence of the three known TBEV subtypes: European (TBEV-Eur), Siberian (TBEV-Sib) and Far Eastern (TBEV-FE). All strains representing the three subtypes were infective and highly neurotropic. They induced (meningo)encephalitis in some of the animals, however most of the cases did not present with apparent clinical symptoms. TBEV-RNA was cleared significantly slower from the brain as compared to other organs studied. Supporting our earlier findings in natural rodent populations, TBEV-RNA could be detected in the brain for up to 168 days post infection, but we could not demonstrate infectivity by cell culture isolation. Throughout all time points post infection, RNA of the TBEV-FE was detected significantly more often than RNA of the other two strains in all organs studied. TBEV-FE also induced prolonged viremia, indicating distinctive kinetics in rodents in comparison to the other two subtypes. This study shows that bank voles can develop a neuroinvasive TBEV infection with persistence of viral RNA in brain, and mount an anti-TBEV IgG response. The findings also provide further evidence that bank voles can serve as sentinels for TBEV endemicity.     

The Relationship between the Structure of the Tick-Borne Encephalitis Virus Strains and Their Pathogenic Properties

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.