RESUMO
The synthesis of new, artificial ribonucleases containing two amino acid residues connected by an aliphatic linker has been developed. Target molecules were synthesized via a catalytic three-component Ugi reaction from aliphatic diisocyanides. Preliminary investigations proved unspecific nuclease activity of the new compounds towards single-stranded RNA and double-stranded circular DNA.
RESUMO
Two dominant species of wild small rodents trapped in Novosibirsk region, South-Western Siberia, Russia differed in their susceptibility to the tick-borne encephalitis virus (TBEV) infection. TBEV RNA average detection rate for Northern red-backed vole Myodes rutilus (Pallas, 1779) (82.2 ± 5.8% blood samples and 63.1 ± 2.7% organ samples) significantly exceeded the corresponding values for the striped field mouse Apodemus agrarius (Pallas, 1771) (47.0 ± 8.7% blood and 24.5 ± 2.8% organ samples) (p <0.001). Innate immunity may be one of possible reasons of the differences. Th1 cytokine gene expression distinguished between M. rutilus (12.5 ± 8.5%) and A. agrarius (66.6 ± 11.4%), whereas Th2 cytokine frequencies were statistically similar (81.8 ± 12.2% and 100.0%, respectively). Polarization indexes (PI) of the innate immunity calculated as ratio of Th2 to Th1 cytokine RNA detection rates for both M. rutilus (6.5) and A. agrarius (1.5) suggested Th2 mainly humoral immune response against persistent TBEV in natural mammalian hosts. Therefore, the TBEV-induced antibodies were analyzed by ELISA and hemagglutination inhibition (HI) tests. The TBEV-specific antibodies were detected in 74.8 ± 4.3% sera of M. rutilus and 67.3 ± 6.8% of A. agrarius. Among them HI antibodies were found in 4.8 ± 2.1% of the same analyzed sera of M. rutilus and in 6.0 ± 3.4% blood samples of A. agrarius only. To model the TBEV persistence both M. rutilus and A. agrarius were infected with the suspensions of the TBEV-infected ticks with further observations during 4 subsequent months. Detection rate of the TBEV RNA and antigen E remained high during the whole period, however, pathogenic for laboratory suckling mice virus was isolated up to 8 days postinfection. At late stages of the persistent infection (1-4 months) the TBEV RNA detection rate in northern red-backed voles remained high 70.6 ± 7.9% whereas in striped field mice significantly declined to 26.7 ± 9.2% (p < .001). Comparative analysis of the innate immunity of the wild rodents in 2 months postinfection showed similar frequencies of Th2 cytokine gene expression for M. rutilus (77.8 ± 10.1%) and A. agrarius (71.4 ± 12.5%) (p > .05) but Th1 cytokine mRNA detection rates were different (44.4 ± 12.5% and 85.7 ± 9.7%, respectively) (p < .05). In 2 months PI decreased from 6.5 until 1.75 for M. rutilus and from 1.5 until 0.83 for A. agrarius. Nevertheless, Th2 mainly humoral immune response was confirmed by direct detection of the TBEV-specific antibodies. HI and neutralizing antibodies were revealed in blood sera of the small rodents of both studied species in 30 days postinfection and remained at detectable levels during 4 months of observations. Accordingly, Th2 polarized innate immunity of small rodents might facilitate the TBEV intracellular persistence in the presence of HI and neutralization antibodies.
Assuntos
Imunidade Adaptativa , Animais Selvagens , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/veterinária , Imunidade Inata , Doenças dos Roedores/virologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Camundongos , RNA Viral , Doenças dos Roedores/imunologia , Doenças dos Roedores/metabolismoRESUMO
Infestation of small mammals, including common shrews Sorex araneus L., field mice Apodemus agrarius Pallas, and red voles Clethrionomus rutilus Schreber, with immature Ixodes persulcatus ticks and their infection with tickborne encephalitis virus (TBEV) were studied in the forest-steppe habitat in the vicinity of Novosibirsk, Russia. Larval ticks parasitize all three host species, but virtually all nymphs were found only on field mice and red voles. Detection of the viral RNA using reverse transcription (RT) with subsequent nested polymerase chain reaction (nPCR) and of viral antigen using enzyme-linked immunosorbent assay (ELISA) revealed a high prevalence of TBEV-positive animals in both the summer and winter. The proportion of small mammals with hemagglutination inhibition (HI) antibodies was significantly lower than with ELISA-detected antibodies. Taken together, the data suggest that small mammals may maintain TBEV as a persistent infection throughout the year.
Assuntos
Arvicolinae/virologia , Encefalite Transmitida por Carrapatos/veterinária , Murinae/virologia , Doenças dos Roedores/epidemiologia , Musaranhos/virologia , Animais , Anticorpos Antivirais/sangue , Arvicolinae/parasitologia , Sequência de Bases , Primers do DNA/química , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Testes de Inibição da Hemaglutinação/veterinária , Larva , Dados de Sequência Molecular , Murinae/parasitologia , Ninfa , RNA Viral/química , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Estações do Ano , Alinhamento de Sequência/veterinária , Musaranhos/parasitologia , Sibéria/epidemiologia , Carrapatos/crescimento & desenvolvimento , Carrapatos/fisiologiaRESUMO
UNLABELLED: The persistence of tick-borne encephalitis virus (TBEV) in nature is maintained by numerous species of reservoir hosts, multiple transmissions between vertebrates and invertebrates, and the virus adaptation to its hosts. Our Aim: was to compare TBEV isolates from ticks and small wild mammals to estimate their roles in the circulation of the viral subtypes. METHODS: TBEV isolates from two species of ixodid ticks, four species of rodents, and one species of shrews in the Novosibirsk region, South-Western Siberia, Russia, were analyzed using bioassay, hemagglutination, hemagglutination inhibition, neutralization tests, ELISA, reverse transcription with real-time PCR, and phylogenetic analysis. RESULTS: TBEV RNA and/or protein E were found in 70.9% ± 3.0% of mammals and in 3.8% ± 0.4% of ticks. The TBEV infection rate, main subtypes, and neurovirulence were similar between ixodid tick species. However, the proportions of the virus that were pathogenic for laboratory mice and of the Far-Eastern (FE) subtype, as well as the viral loads with the Siberian and the European subtypes for the TBEV in Ixodes pavlovskyi Pomerantsev, 1946 were higher than in Ixodes persulcatus (P. Schulze, 1930). Percentages of infected Myodes rutilus, Sicista betulina, and Sorex araneus exceeded those of Apodemus agrarius and Myodes rufocanus. Larvae and nymphs of ticks were found mainly on rodents, especially on Myodes rufocanus and S. betulina. The proportion of TBEV-mixed infections with different subtypes in the infected ticks (55.9% ± 6.5%) was higher than in small mammals (36.1% ± 4.0%) (p < 0.01). CONCLUSIONS: Molecular typing revealed mono- or mixed infection with three main subtypes of TBEV in ticks and small mammals. The Siberian subtype was more common in ixodid ticks, and the FE subtype was more common in small mammals (p < 0.001). TBEV isolates of the European subtype were rare. TBEV infection among different species of small mammals did not correlate with their infestation rate with ticks in the Novosibirsk region, Russia.
Assuntos
Reservatórios de Doenças , Vírus da Encefalite Transmitidos por Carrapatos/genética , Ixodidae/microbiologia , Mamíferos/microbiologia , Animais , Animais Recém-Nascidos , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Camundongos , Camundongos Endogâmicos ICR , Filogenia , Sibéria , VirulênciaRESUMO
The tick-borne encephalitis virus (TBEV) strains have been isolated from unfed adult ticks Ixodes persulcatus Schulze in Novosibirsk region (South-Western Siberia, Russia) beginning from 1980 till 2006. The TBEV 3'-untraslated region (3'UTR) variable fragment was amplified with primers corresponding to conserved flanking areas. The RT-PCR product lengths varied in range from 100 to 400 bp. Comparative analysis of 3'UTR nucleotide sequences revealed a few groups of the TBEV strains within Siberian genetic subtype with significant intra-group homology and essential differences between groups. Correlation between lengths of the 3'UTR fragments and hemagglutination (HA) titers for subsequent passages of the TBEV strains was not found. However, for the viral strains with shorter 3'UTR (less than 200 nucleotides) incubation period for suckling mice was longer than 5 days. It might be resulted from decreased RNA synthesis or reduced neuroinvasiveness.
RESUMO
PCR assays were used to test adult Ixodes persulcatus ticks from Western Siberia, Russia, for Borrelia burgdorferi sensu lato, tick-borne encephalitis virus (TBEV), and the human granulocytic ehrlichiosis (HGE) agent. Of the 150 ticks that were studied, 38% were infected with B. burgdorferi, 46% were infected with TBEV, and 8% were infected with the HGE agent. These three pathogens were distributed in the ticks independently of one another.