[New findings on the ecology and epidemiology of murine herpes virus isolated in Slovakia]. / Nové poznatky o ekológii a epidemiológii mysieho herpetického vírusu izolovaného na Slovensku.

Rodents are important reservoir animals of many human microbial pathogens. Of small rodents, trapped on Slovakia territory, were several strains of murine herpes virus (MHV) isolated. Our purpose was to complete the existing knowledge about circulation of MHV in rodents and to find out, whether also other animal species including man are MHV sensitive or not. The presence of antibodies against MHV in serum of the tested animals and men was followed by virus neutralization test (VNT) and ELISA. Pathological changes in differential white blood cell count of the trapped rodents, were also observed because it is known, that MHV induces them in laboratory mice. A total of 627 small terrestrial mammals of nine species were collected in four localities of western and eastern Slovakia during 1984-1988. Neutralizing antibodies to MHV were detected in five species of rodents in 130 cases (20.7%). Antibodies were most frequently detected in Apodemus flavicollis (34.9%). Pathological changes in differential white blood cell count of trapped rodents were detected in 37% (34/92). Neutralizing antibodies were found also in serum of fallow deers (Dama dama), wild boars (Sus scrofa), deers (Cervus elaphus) and sheep but not in serum of pheasants (Phasianus colchicus) and muflons (Ovis musimon). ELISA and VNT tests were used to investigate 20 serums of employees of the Institute of Virology, Slovak Academy of Sciences and Faculty of Natural Sciences of Comenius University. There were eight samples positive (40%). The titers of antibodies were 4-32 in VNT and 1000 in ELISA.

Short report: simultaneous occurrence of Dobrava, Puumala, and Tula Hantaviruses in Slovakia.

The prevalence of antibody to hantaviruses in Slovakia (serum panel n = 2,133) was lower in the western part (0.54%) and higher in the eastern part (1.91%) of the country and was found to be significantly enhanced in a group of forest workers from eastern Slovakia (5.88%). One-third of the IgM-negative convalescent phase sera from patients with hemorrhagic fever with renal syndrome exhibited antibodies reacting predominantly with Puumala virus antigen, while two-thirds had antibodies directed mainly against Hantaan virus antigen. Fine analysis of two Hantaan virus-reactive sera by a focus reduction neutralization test showed that Dobrava hantavirus was the source of these human infections. Initial results of rodent screening indicated the circulation of Dobrava virus in populations of striped field mice (Apodemus agrarius) in eastern Slovakia.

Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia.

To examine the evolution of Tula hantavirus (TUL), carried by the European common vole (Microtus arvalis and M. rossiaemeridionalis), we have analyzed genetic variants from Slovakia, the country where the virus is endemic. Phylogenetic analysis (PHYLIP) based on either partial (nucleotides [nt] 441 to 898) or complete N-protein-encoding sequences divided Slovakian TUL variants into two main lineages: (i) strains from eastern Slovakia, which clustered with Russian strains, and (ii) strains from western Slovakia situated closer to those from the Czech Republic. We found genetic diversity of 19% between the two groups and 4% within the western Slovakian TUL strains. Phylogenetic analysis of the 3' noncoding region (3'-NCR), however, placed the eastern Slovakian strains closer to those from western Slovakia and the Czech Republic, with a greater distance to the Russian strains, suggesting a recombinant nature of the S segment in the eastern Slovakian TUL lineage. A bootscan search of the S-segment sequences of TUL strains revealed at least two recombination points in the S sequences of eastern Slovakian TUL strains (nt 400 to 415 and around 1200) which agreed well with the pattern of amino acid substitutions in the N protein and deletions/insertions in the 3'-NCR of the S segment. These data suggest that homologous recombination events occurred in the evolution of hantaviruses.

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.

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.

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.

Genetic relatedness of Sindbis virus strains from Europe, Middle East, and Africa.

The relatedness of 40 strains of Sindbis virus (SIN) from Europe, the Middle East, and Africa was investigated by limited sequencing within the gene encoding the E2 glycoprotein corresponding to amino acid residues 117 to 229 and encompassing one of the major neutralization epitopes. Phylogenetic analyses using distance matrix and parsimonious methods identified two major genetic clusters of western SIN strains, although the variability was less than that of the corresponding region for Venezuelan equine encephalomyelitis virus with a maximum divergence of 12.4% versus 28.5%, respectively. One cluster comprising 19 strains included the HR derivate of the Egypt SIN prototype, AR339, and strains from Israel, Saudi-Arabia, Italy, Slovak Republic, Azerbaijan, as well as three Swedish strains. Another cluster of 17 strains included the Ockelbo virus (OCK) prototype, Edsbyn 5/82, and the majority of SIN strains from northern Europe including strains from Sweden, Norway, and Karelia, as well as two strains from South Africa. A third cluster, supported by the Neighbor joining method, was made up of four strains from South Africa, Uganda, and Cameroon. Residue 212, either Ser or Thr, previously appointed important for the differences in neutralization assays between SIN and Edsbyn 5/82, respectively, correlated with the two major genetic clusters, but was a Thr for two of the three Swedish strains in the SIN prototype cluster, and a Ser in one Swedish and one Karelian strain in the OCK cluster. The finding of strains similar to prototype SIN in Middle Sweden and of strains in South Africa relating to the northern cluster of SIN strains supports the notion of the dispersal of SIN by migrating birds as previously suggested for New World alphaviruses.

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.

Nucleotide sequence of the protein E gene of the tick-borne encephalitis virus strain 595 isolated in Slovakia.

Tick-borne encephalitis (TBE) virus, strain 595 was isolated from Ixodes ricinus ticks in southern Slovakia. A part of the protein E gene was sequenced and compared with the prototype strain Neudorfl. Seventeen silent mutations and two amino acid changes (Ile-->Val, residue 167; Asn-->Thr, residue 366) were found. The nucleotide homology in the sequenced part of protein E gene of the strain 595 and the prototype strain Neudorfl is 98.6%. These findings indicate that the strain 595 is closely related to the strain Neudorfl.

Mixed natural focus of tick-borne encephalitis, tularemia and haemorrhagic fever with renal syndrome in west Slovakia.

Total of 923 small mammals of 7 species were collected in locality Záhorská Ves, West Slovakia, in 1990-1992. Among examined small mammal species it was Clethrionomys glareolus (48.7% of total, 17.5% positive for tick-borne encephalitis (TBE) virus antibodies), Apodemus flavicollis (29.7% of total, 17.5% positive), A. sylvaticus (11.3% of total, 16.3% positive), and Microtus arvalis (6.2% of total, 10.5% positive). The most abundant tick species (larval and nymphal stages) on small mammals was Ixodes ricinus. The extensity of infestation was 35.1-50.7%, and the intensity of infestation ranged in average from 4.1 to 7.8 ticks per animal. Out of 884 small rodent serum samples 16.9% had neutralizing antibody to TBE virus. Eight TBE virus isolates were recovered, six from C. glareolus and one each from A. flavicollis and A. sylvaticus; seven isolates were from brain tissue and one was from a pool of lung and liver tissues. One strain of Francisella tularensis was isolated from a pool of spleens of four C. glareolus collected in August 1991. Hantavirus antigens were detected in lung tissues of four M. arvalis collected in July and November 1990-1992. Antibody to Hantaan virus was detected by ELISA in one serum sample of A. flavicollis (titer 1:256) and antibody to Puumala virus in one serum sample of C. glareolus (titer 1:16).

Genetic characterization of a new hantavirus detected in Microtus arvalis from Slovakia.

A new hantavirus, called Malacky, has been identified in lung tissue specimens of a vole, Microtus arvalis, by the reverse transcriptase polymerase chain reaction (RT-PCR). The voles were trapped in a geographical area in Slovakia where hemorrhagic fever with renal syndrome (HFRS) is endemic in the human population. Sequence analysis of a major part of the S segment showed this virus to represent a new subtype within Tula, a new hantavirus genetic group defined very recently.

[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.)

Serological evidence of Hantaan virus in Slovakia during 1989-1991.

Nephropathia epidemica was supposed to be an European form of haemorrhagic fever with renal syndrome (HFRS). However, in 1989-1991 we found serological evidence of infection with Hantaan virus in patients' sera from Slovakia. Higher titers of antibodies against Hantaan virus were detected in sera of 6 out of 75 patients with febrile disease, renal failure and clinical diagnosis of HFRS. The higher titers of antibodies against Puumala virus were detected in sera of 6 out of 75 patients. Serologic differentiation between Hantaan and Puumala infection in patients was based on the detection of higher antibody titers (4-fold or greater differences).

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.

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.

[Long-term study of natural foci of tick-borne encephalitis in the District of Olomouc]. / Dlouhodobé sledování aktivity prírodních ohnisek klíst'ové encefalitidy v okrese Olomouc.

The incidence of natural foci of tick-borne encephalitis in the Olomouc district has been characterized as general. This finding is based on analyses of results of earlier reports and results of serological blood tests of 397 small mammals from localities where probably transmission of the infection to humans occurred. The incidence almost corresponds to the incidence of the tick Ixodes ricinus. It has a long-lasting and also cumulative character; the number of reported cases from the main focal areas is proportional to the frequency of localities infestated with Ixodes ricinus ticks, assuming an approximately equal number of visitors in the woods of the district.

The prevalence of small terrestrial mammals infected with tick-borne encephalitis virus and leptospirae in the foothills of the southern Bavarian forest, Germany.

In the district of Grafenau/Freyung (Bavaria, Germany), 266 specimens of small terrestrial mammals of 8 species were captured using live traps. From these mammals, Apodemus flavicollis (42.1%) and Clethrionomys glareolus (39.5%) were prevalent. All animals were tested for neutralizing antibodies to tick-borne encephalitis (TBE) virus and agglutinating antibodies to leptospirae. Seropositivity against TBE virus was 14.0% and against leptospirae 7.9%, respectively. Seropositivity to leptospirae appeared to be primarily to Leptospira grippotyphosa, less to Australis and occasionally to Javanica serovars. Only one A. flavicollis specimen was positive to both pathogens tested. The parasitocoenosis of trapped micromammals with ectoparasites consisted of 69.5% from ticks (mainly Ixodes ricinus, less from I. trianguliceps), 16.1% of mites (primarily Laelaps agilis) and 14.3% of fleas (mostly Ctenophthalmus agyrtes).

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.

Tick-borne encephalitis virus activity in Styria, Austria.

From 3,404 Ixodes ricinus ticks collected in 12 localities of Styria, Austria in 1990, 15 tick-borne encephalitis (TBE) virus isolates were recovered. Minimal field infection rate reached 4.4 virus containing ticks out of 1,000 collected ticks. Five isolates of TBE virus were obtained from target organs of Apodemus flavicollis trapped in locality Wagnitz. In a serosurvey based on virus neutralizing antibodies high prevalence of TBE virus was demonstrated in A. flavicollis (47.9%) and Clethrionomys glareolus (29.4%). These rodents formed 57.8% and 41.0% of 83 trapped small mammals.

Further isolation of murine herpesviruses from small mammals in southwestern Slovakia.

A total of 69 small mammals of 6 species were collected in localities Marcelová and Kopác (southwestern Slovakia) and investigated. Two strains of murine herpesvirus (MHV) have been isolated and reisolated from Apodemus flavicollis. Both virus strains killed suckling mice after i.c. and i.p. inoculation. Adult mice were killed 4-7 days after i.c. inoculation. Cross antigenic reactions among 14C aminoacid hydrolysate labelled MHV-72 infected Vero cells and mouse immune sera against the two new isolates and rabbit immune sera raised against purified MHV strains No. 72 and No. 76 were done. Profiles of immunoprecipitated proteins are almost identical. All used immune sera crossreacted with major proteins of MHV-72 strain. We conclude that the two virus isolates from Apodemus flavicollis are new strains of MHV.