Tick-borne encephalitis virus transmission between ticks cofeeding on specific immune natural rodent hosts.

To determine whether the portion of a vertebrate host population having specific immunity to tick-borne encephalitis (TBE) virus can participate in the TBE virus transmission cycle, natural hosts immunized against TBE virus were challenged with infected and uninfected ticks. Yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus) were either immunized with TBE virus by subcutaneous inoculation of the virus, or they were exposed to virus-infected Ixodes ricinus ticks. One month later, when serum neutralizing antibody was detectable, the animals were infested with infected (donor) adult female ticks and uninfected (recipient) nymphal ticks; recipients were allowed to feed either in close contact (chamber 1) or physically separated (chamber 2) from the infected donor ticks. Following challenge with infected (and uninfected) ticks, viremia developed in all the control, nonimmune animals, whereas viremia was undetectable in all those animals naturally immunized by previous exposure to infected ticks. Despite the presence of neutralizing antibodies in all the immunized animals, 89% (24/ 27) immune animals supported virus transmission between infected and uninfected cofeeding ticks. Most transmission was localized, occurring within chamber 1; disseminated transmission from chamber 1 to chamber 2 was reduced. Immunization by tick bite was more effective than immunization by syringe inoculation in blocking cofeeding virus transmission. Nevertheless 76% (9/12) animals with "natural" immunity still supported transmission. The results demonstrate that natural hosts having neutralizing antibodies to TBE virus (and no detectable viremia) can still support virus transmission between infected and uninfected ticks feeding closely together on the same animal. These observations have important epidemiological implications relating to the survival of TBE virus in Nature.

Importance of localized skin infection in tick-borne encephalitis virus transmission.

Arboviruses are transmitted to vertebrates by the "bite" of infected arthropods. Events at the site of virus deposition are largely unknown despite increasing evidence that blood-sucking arthropods immunomodulate their skin site of feeding. This question is particularly relevant for ixodid ticks that feed for several days. To examine events under conditions mimicking tick-borne encephalitis (TBE) virus transmission in nature (i.e., infected and uninfected Ixodes ricinus ticks feeding on the same animal), infected adult and uninfected nymphal ticks were placed in one retaining chamber (skin site A) and uninfected nymphs were placed within a second chamber posteriorly (skin site B) on two natural host species, yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus). Virus transmission from infected to uninfected cofeeding ticks was correlated with infection in the skin site of tick feeding. Furthermore, virus was recruited preferentially to the site in which ticks were feeding compared with uninfested skin sites. Viremia did not correspond with a generalized infection of the skin; virus was not detected in an uninfested skin site (C) of 12/13 natural hosts that had viremia levels > or = 2.0 log10 ic mouse LD50/0.02 ml blood. To characterize infected cells, laboratory mouse strains were infested with infected ticks and then explants were removed from selected skin sites and floated on culture medium. Numerous leukocytes were found to migrate from the skin explants of tick feeding sites. Two-color immunocytochemistry revealed viral antigen in both migratory Langerhans cells and neutrophils; in addition, the migratory monocyte/macrophages were shown to produce infectious virus. The results indicate that the local skin site of tick feeding is an important focus of viral replication early after TBE virus transmission by ticks. Cellular infiltration of tick feeding sites, and the migration of cells from such sites, may provide a vehicle for transmission between infected and uninfected cofeeding ticks that is independent of a patent viremia. The data support the hypothesis that viremia is a product, rather than a prerequisite, of tick-borne virus transmission.

Reverted virulence of attenuated tick-borne encephalitis virus mutant is not accompanied with the changes in deduced viral envelope protein amino acid sequence.

Serial passages of tick-borne encephalitis (TBE) virus strain 4387 isolated from the liver and lungs of the bank vole through the salivary glands of Ixodes ricinus ticks led to a reduction of its virulence for laboratory mice infected via peripheral route. When attenuated mutants were passaged through mouse brains, virulent phenotypes have appeared in the 3rd mouse passage. After 5 consecutive passages the virus was more pathogenic for mice after peripheral inoculation than the parental 4387 strain. The nucleotide sequence of the envelope proteins of the strain 4387 was studied after passaging through ticks salivary glands and subsequently through mice. The sequences coding for the envelope protein E of the virus from the first, third and fifth mouse passages were compared with those of parental virus and mutant attenuated in ticks. The attenuated mutant differing from the parental strain 4387 by the amino acid substitution from glutamic acid to lysine at position 84, and from isoleucine to threonine at amino acid position 319 revealed strongly reduced pathogenicity for adult laboratory mice after peripheral inoculation. The attenuated mutant regained its virulence after 3 - 5 mouse brain passages, but the two amino acid substitutions were still conserved.

Salivary gland extracts of partially fed Dermacentor reticulatus ticks decrease natural killer cell activity in vitro.

The salivary glands and saliva of ticks (Arachnida, Acari, Ixodida) play a vital role in blood feeding, including manipulation of the host's immune response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva. A factor synthesized in the salivary glands of feeding ticks potentiates the transmission of certain tick-borne viruses. We show that salivary gland extracts (SGE) derived from Dermacentor reticulatus female ticks fed for 6 days on laboratory mice (SGED6) induced a decrease in the natural killer (NK) activity of effector cells obtained from 16 healthy blood donors. The decreased activity ranged from 14 to 69% of NK activity observed with the respective untreated effector cells. Such a decrease was not observed after treatment of effector cells with SGE from unfed ticks. Ten-fold dilution of SGED6 significantly reduced the capacity to decrease NK activity and a further 10-fold dilution almost eliminated the effect. After addition of IFN-alpha 2, the SGED6-induced decrease in NK activity was restored to activity levels approaching those of untreated cells. The apparent reversibility of the inhibition indicates that the effect of SGED6 on NK activity was not due to cytotoxicity. The results demonstrate the presence of a factor(s) in the salivary gland products of feeding D. reticulatus female ticks that influences human NK activity in vitro. These data suggest a possible mechanism by which tick SGE potentiates the transmission of some tick-borne viruses through suppression of NK activity.

Change in phenotype of tick-borne encephalitis virus following passage in Ixodes ricinus ticks and associated amino acid substitution in the envelope protein.

Serial passage of an uncloned tick-borne encephalitis virus (strain 4387 isolated from the liver and lungs of a bank vole) in Ixodes ricinus ticks, was accompanied by gradual reduction in virulence of the virus, as indicated by transmission of virus by infected ticks feeding on laboratory mice. After the 7th serial passage in ticks (strain 4387/7), 95% of mice survived the bite of infected ticks. The surviving infected mice showed either no or only low viraemia although virus could be isolated from the brains of some mice 14 and 30 days after commencement of tick feeding, implying that the tick passaged virus might have established a persistent infection in the mice. Tests for haemagglutinating capacity were positive with TBE strain 4387 but strain 4387/7 exhibited no haemagglutinating activity over a wide pH range, suggesting that phenotypic changes, resulting from selection, had affected the site on the viral envelope protein that binds red blood cell receptors. Sequencing of the envelope protein gene of the virulent TBE strain 4387 showed 3 amino acid codon differences from western European TBE virus strain Neudorfl, which is also virulent for mice. The attenuated virus 4387/7, had an amino acid substitution that was different from 4387 and Neudorfl TBE virus (amino acid 84, E to K) and a second substitution different from 4387 but identical to Neudorfl virus (amino acid 319, I to T). Thus, the phenotypic change from virulence to attenuation was associated with a single amino acid codon change in the viral envelope gene of TBE virus. It is recognised, however, that amino acid substitutions in other parts of the viral genome have not been ruled out.

Non-viraemic transmission of tick-borne encephalitis virus: a mechanism for arbovirus survival in nature.

The vectors of arthropod-borne viruses (arboviruses) become infected by feeding on the viraemic blood of an infected animal. This theory is based on transmission studies involving artificial infection of vertebrate hosts by syringe inoculation. To reproduce natural conditions of virus transmission, infected and uninfected vectors (ticks) of tick-borne encephalitis virus, the most important arbovirus in Europe, were allowed to feed together on uninfected wild vertebrate hosts. The greatest numbers of infected ticks were obtained from susceptible host species that had undetectable or very low levels of viraemia. The results suggest that 'nonviremic transmission' is an important mechanism for the survival of certain arboviruses in nature.