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.

Peroral immunization of adult white mice with the Skalica strain from the tick-borne encephalitis virus complex.

Adult white mice immunized perorally with the infectious Skalica strain from the tick-borne encephalitis (TBE) virus complex did not show any clinical symptoms of illness. 56% of experimental animals immunized with two doses of the Skalica virus (the titer of virus was 6 x 10(10) LD50) were protected against the challenge with the Hypr strain of TBE virus. All mice immunized with the Skalica virus and having haemagglutination-inhibiting antibodies higher than 1:80 survived the challenge with the given dose of virulent TBE virus. No differences in the immunogenicity and protectivity were observed in experimental animals infected with infectious Skalica virus by oesophageal probe, or by drinking virus-containing medium. A higher protective activity against the virulent Hypr virus was observed in adult white mice immunized subcutaneously with the Skalica virus.

Comparison of skalica, Hypr, and Langat viruses by kinetic haemagglutination-inhibition test.

The Skalica virus has been compared with Hypr and Langat viruses by kinetic haemagglutination-inhibition (HI) test. Using Hypr antigen, differences were observed between Skalica, Hypr, and Langat viruses. By the use of Skalica antigen, a close relationship between Hypr and Skalica viruses was detected, however, it was possible to differentiate the Langat virus. When Langat antigen was tested, a close relationship among all the three viruses was found.

Infection of the central nervous system of mice by standard Sendai virus, defective interfering Sendai virus and the mixture of both: comparison of virus multiplication and pathogenicity.

The intracerebral (i.c.) infection of newborn mice with standard Sendai virus (SV), defective interfering Sendai virus (DV) and their mixture (SV + DV) has been used as a model for the possible role of defective interfering particles of paramyxoviruses in several chronic degenerative diseases of central nervous system (CNS). The dynamics of Sendai virus multiplication and virus distribution in CNS of mice, as well as the histological changes and the clinical symptoms were evaluated for up to 112 days post-infection (p.i.). The infectious virus was detected in the brains of animals inoculated i.c. either with SV, or DV, or SV + DV as soon as by 5 hr p.i., with maximum infectivity titre at 24 hr p.i. In brains of animals inoculated with SV, the virus was detected until 5th day p.i.; nevertheless in those, inoculated with SV + DV or DV, low infectious titres could be detected even at later intervals. In mice inoculated i.c. with DV, traces of Sendai virus were detected in subpassages, as late as 3 months p.i.

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.

Transmission of tick-borne bunyaviruses by cofeeding ixodid ticks.

Palma and Bhanja bunyaviruses replicated in Dermacentor marginatus, D. reticulatus, Rhipicephalus sanguineus, R. appendiculatus and Ixodes ricinus ticks after parenteral inoculation and appropriate incubation and feeding. Palma virus was transmitted to D. marginatus and D. reticulatus males, D. reticulatus and R. sanguineus females, and R. appendiculatus nymphs while cofeeding with infected ticks on laboratory mice. Bhanja virus was transmitted to D. marginatus males and R. appendiculatus nymphs. Laboratory mice developed low levels of viraemia detectable only by intracranial (i.c.) inoculation of newborne laboratory mice.

[Familial epidemics of tick-borne encephalitis in central Povazie]. / Rodinné epidémie kliest'ovej encefalitídy na strednom Povazí.

The authors describe two cases of tick-born encephalitis family epidemies in the natural focus of tick-born encephalitis (TbE) in the central part of Povazie in the district of Povazká Bystrica in the years 1989 and 1993. The infection took place due to consumption of raw goat milk. The best prevention against tick-born encephalitis is represented by vaccination. (Tab. 4, Ref. 6.)

Family outbreak of alimentary tick-borne encephalitis in Slovakia associated with a natural focus of infection.

A family outbreak of tick-borne encephalitis involving 7 people, all of them hospitalized, was observed in the district of Povazská Bystrica (central Slovakia). The disease was associated with the drinking of unboiled goat milk and tick-borne encephalitis virus was recovered from Ixodes ricinus ticks collected from places where goats were grazing.

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.

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.