Kyasanur Forest disease virus infection in mice is associated with higher morbidity and mortality than infection with the closely related Alkhurma hemorrhagic fever virus.

BACKGROUND: Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic fever virus (AHFV) are closely related members of the Flavivirus genus and are important causes of human disease in India and the Arabian Peninsula, respectively. Despite high genetic similarity, the viruses have distinctly different host ranges and ecologies. Human cases of KFDV or AHFV develop a spectrum of disease syndromes ranging from liver pathology to neurologic disease. Case reports suggest KFDV is more commonly associated with hepatic and gastrointestinal manifestations whereas AHFV is more commonly associated with neurologic disease. METHODOLOGY/PRINCIPAL FINDINGS: Inoculation of three immunocompetent laboratory mouse strains revealed that KFDV was consistently more lethal than AHFV. In subsequent studies utilizing C57BL/6J mice, we demonstrated that KFDV infection was associated with higher viral loads and significantly higher mortality. KFDV-infected mice rapidly developed more severe disease than AHFV-infected mice, as evidenced by significant abnormalities on clinical chemistry panels and more severe pathology in the brain and gastrointestinal tract. CONCLUSIONS/SIGNIFICANCE: Infections of C57BL/6J mice with KFDV or AHFV resulted in clinical disease syndromes that closely approximate the diseases seen in human cases. Despite high genetic similarity, there were clear differences in survival, viral kinetics, clinical chemistry data and histology. These results suggest that distinct mouse models for AHFV and KFDV are necessary in order to gain a better understanding of the unique pathogenesis of each virus, as well as to provide platforms for testing promising vaccines and therapeutics.

Serum matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels in patients with tick-borne encephalitis.

OBJECTIVES: Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) play important roles in the function of the blood-brain barrier (BBB). To investigate the function of the BBB during tick-borne encephalitis (TBE), the levels of MMP-9 and its common tissue inhibitor, TIMP-1, were measured in serum from patients with acute phase of TBE. METHODS: Serum MMP-9 and TIMP-1 levels were measured in 147 patients with TBE and 153 controls by ELISA. RESULTS: Serum MMP-9 levels and MMP-9/TIMP-1 ratios of TBE patients were significantly higher than controls (p < 0.0001 and p < 0.005, respectively). There were no significant differences in serum TIMP-1 levels between TBE patients and controls. Serum MMP-9 and TIMP-1 levels and MMP-9/TIMP-1 ratios were not associated with age of the patients. However, TBE-positive males with TBE had higher levels of MMP-9 than TBE-positive females (p < 0.05). CONCLUSIONS: Our results suggest that the increased serum level of MMP-9 and MMP-9/TIMP-1 ratio is associated with the pathogenesis of TBE. Serum MMP-9 can serve as an indicator of breakdown of the BBB and inflammatory brain damage during TBE.

Changes in the metabolic activity of macrophages under the influence of tick-borne encephalitis virus.

The metabolic activity of macrophages infected with tick-borne encephalitis virus (TBEV) affecting the human nervous system has been studied for the first time. The penetration and reproduction of TBEV in the macrophages stimulated their oxygen metabolism, increasing the activity of NADPH-oxidase complex, as well as the mitochondrial enzymes lactate dehydrogenase, succinate dehydrogenase, and cytochrome oxidase. A wave-like change in the activity of these enzymes in the macrophages reflected the reaction of the cells to the penetration of the virus in the first period (within 3 h) and to the synthesis of the virus particles and their exit into the extracellular space in the second period (from 5 to 48 h). In the macrophages infected with TBEV, accumulation of NO metabolites was observed. In the late period of the examination (1-4 days), the activities of superoxide dismutase and lysosomal enzymes (nonspecific esterase and acid phosphatase) were detected. Thus, the early increase in the activity of the cell enzymes indicates the activation of the macrophages, and the subsequent increase in their activity corresponds to the enhanced synthetic activity of the macrophages.

[Decreased antioxidant-defence mechanisms in cerebrospinal fluid (CSF) in patients with tick-borne encephalitis (TBE)]. / Obnizenie mechanizmów obrony antyoksydacyjnej w plynie mózgowo-rdzeniowym u chorych z kleszczowym zapaleniem mózgu.

14 patients with Tick-borne Encephalitis (TBE) aged 21-64 (mean = 42.3) were analysed. The activity of superoxide dismutase (SOD), glutathione reductase (GSSG-R), glutathione peroxidase (GSH-Px), concentrations of malondialdehyde (MDA) and total sulphydryl groups (-SH) were measured in cerebrospinal fluid (CSF). Control group consisted of 10 patients whose CSF parameters remained in normal range. The CSF examination was performed twice: before and 3 weeks after treatment. The analysed activity of SOD, GSH-Px, GSSG-R, MDA and total sulphydryl groups (-SH) during the acute stage of the disease was significantly lower comparing to the control group. Despite the treatment, GSSG-R activity, MDA concentration and total sulphydryl groups--SH further lowered significantly. Although the SOD activity in CSF was higher in the second examination, it remained significantly lower comparing to the control group. We showed that the GSH-Px and GSSG-R activity in CSF after the acute stage of the TBE remained significantly lower than in the control group. Our examinations prove that during the TBE an increased generation of oxygen-derived free radicals occurs what shows decreased activity of the antioxidant parameters (SOD, GSH-Px, GSSG-R) and decreased concentration of total sulphydryl groups--SH in CSF. Our results suggest that during TBE, molecular structures injury of enzymes and antioxidative reactive cofactors may occur.

NS3 protease of Langat tick-borne flavivirus cleaves serine protease substrates.

Langat (LGT) virus, initially isolated in 1956 from ticks in Malaysia, is a naturally occurring nonpathogenic virus with a very close antigenicity to the highly pathogenic tick-borne encephalitis (TBE) Western subtype virus and TBE Far Eastern subtype virus. NS3, the second largest viral protein of LGT virus, is highly conserved among flaviviruses and contains a characteristic protease moiety (NS3 pro). NS3 pro represents an attractive target for anti-protease molecules against TBE virus. We report herein a purification method specially designed for NS3 pro of LGT using a strategy for proper refolding coupled with the enzymatic characterisation of the protein. Different p-nitroanilide substrates, defined on canonic sequences for their susceptibility to Ser-protease, were applied to the proteolytic assays of the protein. The highest values were obtained from substrates containing an Arg or Lys (amino acid) residue at the P1 position. This purification method will facilitate the future development of reliable testing procedures for anti-proteases directed to NS3 proteins.

Serum time course of two brain-specific proteins, alpha(1) brain globulin and neuron-specific enolase, in tick-born encephalitis and Lyme disease.

METHODS: Time courses of the serum concentrations of two brain-specific proteins (BSP), alpha(1) brain globulin (alpha(1)BG, an astroglial marker) and neuron-specific enolase (NSE), were studied in patients with severe tick-born encephalitis (TBE) and Lyme disease (LD; neuroborreliosis). The concentrations were determined on the second day of the acute phase and then on the 7th, 12th, 18th, and 23rd days. Apparent rate constants for the elimination of the BSP from blood (k(e)) were calculated with the non-linear regression. RESULTS: In patients with TBE, the highest serum concentrations of alpha(1)BG and NSE, observed on the second day, were followed by their monotonic decrease to the normal levels reached by the 23rd day. The mean k(e) values for alpha(1)BG and NSE were found to be significantly different (0.086+/-0.003 vs. 0.057+/-0.006 day(-1), respectively; p<0.05). Higher serum levels of both BSP were observed in the more severe clinical cases and in the cases with unfavorable outcomes. Similar profiles were also observed for the serum alpha(1)BG and NSE in LD. CONCLUSIONS: These results suggest that, in the patients examined, the blood-brain barrier was partially impaired; the quantitative parameters of the serum BSP time courses can be indicative of the extents of the neuronal and/or glial lesions.

[Histochemical study of the activity of several enzymes in the process of immunogenesis in monkeys orally infected with viruses of the tick-borne encephalitis complex]. / Gistokhimicheskoe izuchenie aktivnostinekotorych fermentov v protsesse immunogeneza u obez'ian, peroral'no zarazhennykh virusami kompleksa kleshchevogo éntsefalita

The activity of succinate dehydrogenase, acid and alkaline phosphatase was studied histochemically in immunocompetent organs of Macacus rhesus monkeys orally infected with viruses of the tick-borne encephalitis complex with different biological properties. A gradual increase in the activity of acid phosphatase and succinate dehydrogenase was observed by the 10th--15th day of the experiment followed by its decline to normal by 90 days. The content of alkaline phosphatase in lympoid organs diminished markedly in the first day of the study and then increased by 15--30 days after antigenic stimulation.

[Clinico-immunologic features of tick-borne encephalitis in the Tomsk region]. / Kliniko-immunologicheskie osobennosti kleshchevogo éntsefalita v Tomskoi oblasti

In 243 patients with tick-borne encephalitis 60.9% had an oblivated form, 21.3%--the meningeal, 2.5% encephalitic, 2.9%--polyencephalomyelitic and 12.4%--doublewave development of the disease. In all cases the clinical picture, blood and CSF content of asparite-aminotransferase and alanine aminotransferase was studied. The study included the level of urine diastase, blood histamine, autoantibodies and indices of destructed blood neutrophils. It was established that tick-borne encephalitis remains one of the most serious diseases of the nervous system which is accompanied by lesions of the internal organs. Changes in the studied indices are in direct correlation with the form and severity of the disease. They do not come to norm even at the moment of hospital discharge which indicates the continuation of the process.