High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses.

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

Human Diploid Fibroblast Cell Lines - a Model System for Studying Immunodulatory Properties of Modern Immunobiological Drugs and Viruses.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 µg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1ß. Phosprenyl, on the contrary, increased production of IL-1ß and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1ß, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

Inhibitory Activity of Scutellaria baicalensis Flavonoids against Tick-Borne Encephalitis Virus.

We studied virus-inhibiting activity of Baikal skullcap (Scutellaria baicalensis) flavonoids against tick-borne encephalitis virus using various model schemes. The half-maximum cytotoxic concentration (CC50) for the plant extract was found (363.9±58.6 µg/ml). Based on the CC50 and IC50, selective index (SI) was calculated for viricidal (53.4), preventive (50.5), and direct antiviral actions (39.1) and for-intracellular replication of the virus (40.4). Suppression of virus reproduction ≥2.0 lg TCID50 was observed at extract concentration ≥5 µg/ml (viricidal effect), ≥11.2 µg/ml (preventive and direct antiviral effects), and ≥9 µg/ml (intracellular replication). Flavonoids of Baikal skullcap extract produced an in vitro inhibitory effect on tick-borne encephalitis virus due to their direct viricidal activity and direct inhibition of adsorption and intracellular replication of tick-borne encephalitis virus, which determines their value as highly effective antiviral drugs.

Revealing new tick-borne encephalitis virus foci by screening antibodies in sheep milk.

BACKGROUND: Tick distribution in Sweden has increased in recent years, with the prevalence of ticks predicted to spread towards the northern parts of the country, thus increasing the risk of tick-borne zoonoses in new regions. Tick-borne encephalitis (TBE) is the most significant viral tick-borne zoonotic disease in Europe. The disease is caused by TBE virus (TBEV) infection which often leads to severe encephalitis and myelitis in humans. TBEV is usually transmitted to humans via tick bites; however, the virus can also be excreted in the milk of goats, sheep and cattle and infection may then occur via consumption of unpasteurised dairy products. Virus prevalence in questing ticks is an unreliable indicator of TBE infection risk as viral RNA is rarely detected even in large sample sizes collected at TBE-endemic areas. Hence, there is a need for robust surveillance techniques to identify emerging TBEV risk areas at early stages. METHODS: Milk and colostrum samples were collected from sheep and goats in Örebro County, Sweden. The milk samples were analysed for the presence of TBEV antibodies by ELISA and validated by western blot in which milk samples were used to detect over-expressed TBEV E-protein in crude cell extracts. Neutralising titers were determined by focus reduction neutralisation test (FRNT). The stability of TBEV in milk and colostrum was studied at different temperatures. RESULTS: In this study we have developed a novel strategy to identify new TBEV foci. By monitoring TBEV antibodies in milk, we have identified three previously unknown foci in Örebro County which also overlap with areas of TBE infection reported during 2009-2018. In addition, our data indicates that keeping unpasteurised milk at 4 °C will preserve the infectivity of TBEV for several days. CONCLUSIONS: Altogether, we report a non-invasive surveillance technique for revealing risk areas for TBE in Sweden, by detecting TBEV antibodies in sheep milk. This approach is robust and reliable and can accordingly be used to map TBEV "hotspots". TBEV infectivity in refrigerated milk was preserved, emphasising the importance of pasteurisation (i.e. 72 °C for 15 s) prior to consumption.

Tick-borne Encephalitis: Stroke-like Presentation.

Tick-borne encephalitis, caused by the tick-borne virus (TBEV), is endemic in central, eastern, and northern Europe eastwards through Russian Siberia and China. For the year 2009, the highest incidence in Scandinavian countries was in Sweden. The clinical symptoms have a wide spectrum. We report a unique case of clinical symptoms and radiological findings compatible with a stroke-like inflammatory lesion in the thalamus, suggesting microangiopathy from TBEV. Our case shows that TBEV could be a possible cause of stroke-like lesions.

Structural modeling of the NS 3 helicase of Tick-borne encephalitis virus and their virtual screening of potent drugs using molecular docking.

Homology modeling of NS3 helicase in Tick-borne encephalitis virus using known protein crystal structure was done. Its 3-D structure was evaluated and validated using PROCHECK comprising amino acid residues in favored region of Ramachandran plot. Helicase forms a large family of proteins which ubiquitously distributes in wide variety of organisms. It plays crucial role in transcription and replication of single-stranded viral RNA genomes. Consequently, NS3 represents an interesting target for the development of specific antiviral inhibitors. Several helicase inhibiting effective drugs and analogs were selected and the active amino acid residues were targeted. Levovirin, Ribamidine and Ribavirin were found more potent to inhibit TBEV on the basis of robust binding affinity between protein-drug interactions. This finding may help to understand the nature of helicase and development of specific anti-TBEV therapies.

Severe tick borne encephalitis with simultaneous brain stem, bithalamic, and spinal cord involvement documented by MRI.

A case of tick borne encephalitis (TBE) is reported, with simultaneous brain stem, spinal cord, and bilateral thalamic involvement confirmed by magnetic resonance imaging (MRI). After exposure to a TBE endemic region, the patient developed a biphasic clinical course with initial flu-like symptoms followed by a severe brain stem syndrome. The diagnosis of TBE was confirmed serologically. Repeated MRI scans showed brain stem, bithalamic, and spinal cord involvement. The outcome was favourable. TBE cases with concomitant myelitis tend to have a more severe clinical course and more likelihood of needing intensive care support. They should therefore be identified early in order to be prepared for life threatening respiratory complications.

[A comparison of the immune response induced by DNA or by an inactivated vaccine against tick-borne encephalitis]. / Sravnenie immunnogo otveta, indutsirovannogo DNK ili inaktivirovannoi vaktsinoi protiv kleshchevogo éntsefalita.

BALB/c mice were immunized with recombinant plasmid DNA pSVK3-ENS1 and pcDNAI-NS3 containing, respectively, genes E-NS1 and NS3 of tick-borne encephalitis (TBE) virus. Antibodies to TBE virus proteins were detected in the blood sera of the immunized animals by the method of the enzyme immunoassay. Though the titers of virus-specific antibodies in the sera of mice immunized with protein vaccines exceeded those registered after immunization with DNA vaccines, essential protective immunity was observed after the use of both vaccines.

The role of lambs in louping-ill virus amplification.

In some areas of Scotland, the prevalence of louping-ill virus has not decreased despite the vaccination of replacement ewes for over 30 years. The role of unvaccinated lambs in viral persistence was examined through a combination of an empirical study of infection rates of lambs and mathematical modelling. Serological sampling revealed that most lambs were protected by colostral immunity at turnout in May/June but were fully susceptible by the end of September. Between 8 and 83% of lambs were infected over the first season, with seroconversion rates greater in late rather than early summer. The proportion of lambs that could have amplified the louping-ill virus was low, however, because high initial titres of colostral antibody on farms with a high force of infection gave protection for several months. A simple mathematical model suggested that the relationship between the force of infection and the percentage of lambs that became viraemic was not linear and that the maximum percentage of viraemic lambs occurred at moderately high infection rates. Examination of the conditions required for louping-ill persistence suggested that the virus could theoretically persist in a sheep flock with over 370 lambs, if the grazing season was longer than 130 days. In practice, however, lamb viraemia is not a general explanation for louping-ill virus persistence as these conditions are not met in most management systems and because the widespread use of acaracides in most tick-affected hill farming systems reduces the number of ticks feeding successfully.

Evaluation of tick-borne encephalitis DNA vaccines in monkeys.

Tick-borne encephalitis is usually caused by infection with one of two flaviviruses: Russian spring summer encephalitis virus (RSSEV) or Central European encephalitis virus (CEEV). We previously demonstrated that gene gun inoculation of mice with naked DNA vaccines expressing the prM and E genes of these viruses resulted in long-lived homologous and heterologous protective immunity (Schmaljohn et al., 1997). To further evaluate these vaccines, we inoculated rhesus macaques by gene gun with the RSSEV or CEEV vaccines or with both DNA vaccines and compared resulting antibody titers with those obtained by vaccination with a commercial, formalin-inactivated vaccine administered at the human dose. Vaccinations were given at days 0, 30, and 70. All of the vaccines elicited antibodies detected by ELISA and by plaque-reduction neutralization tests. The neutralizing antibody responses persisted for at least 15 weeks after the final vaccination. Because monkeys are not uniformly susceptible to tick-borne encephalitis, the protective properties of the vaccines were assessed by passive transfer of monkey sera to mice and subsequent challenge of the mice with RSSEV or CEEV. One hour after transfer, mice that received 50 microl of sera from monkeys vaccinated with both DNA vaccines had circulating neutralizing antibody levels <20-80. All of these mice were protected from challenge with RSSEV or CEEV. Mice that received 10 microl of sera from monkeys vaccinated with the individual DNA vaccines, both DNA vaccines, or a commercial vaccine were partially to completely protected from RSSEV or CEEV challenge. These data suggest that DNA vaccines may offer protective immunity to primates similar to that obtained with a commercial inactivated-virus vaccine.

Humoral and cellular immunity induced by antigens adjuvanted with colloidal iron hydroxide.

The immunopotentiating activities of colloidal iron hydroxide, a novel, experimental mineral adjuvant, and of aluminium hydroxide. the licensed adjuvant for human vaccines, were compared. Our studies revealed that colloidal iron hydroxide and aluminium hydroxide behaved comparably with respect to supporting induction of an antibody response to tetanus toxoid. Furthermore, mice immunized with both, the experimental vaccine (tick-borne encephalitis virus (TBEV) antigen adsorbed to colloidal iron hydroxide) or with a commercially available TBEV vaccine (adjuvanted with aluminium hydroxide), developed long-lasting antibody responses which protected the animals from TBEV infection even one year after vaccination. The use of colloidal iron hydroxide as adjuvant had the additional advantage to reproducibly support induction of HIV-1 envelope-specific cytotoxic T lymphocytes (CTL), when used as adjuvant for a HIV-1 env-carrying recombinant fowlpox virus and being applied via the subcutaneous route. Aluminium hydroxide was much less active in this respect. Non-adjuvanted recombinant fowlpox elicited CTLs only when given intravenously or intraperitoneally, vaccination routes considered not to be suitable for routine use in humans. Further studies to evaluate the use of colloidal iron as possible alternative and/or supplement for routinely used mineral adjuvants may therefore be warranted.

[An immunomodifier--staphylococcal anatoxin--prevents the development of immunosuppression caused by informational stress in mice]. / Immunomodifikator--stafilokokkovyi anatoksin--preduprezhdaet razvitie immunosupressii, vyzvannoi informatsionnym stressom u myshei.

The action of information stress for 14 days leads to the development of immunosuppression, which is manifested by the suppression of humoral response to sheep red blood cells (SRBC) and the decrease of resistance to Langat virus having low pathogenicity. As shown in this investigation, an immunomodifier, purified staphylococcal toxoid (PST), protects experimental animals from the immunosuppressive effect of information stress. After the injection of PST to stress-affected mice in doses of 15 or 1.5 binding units per animal on days 9, 11 and 13 of the experiment their humoral response to SRBC and resistance to Langat virus are partially restored (by 45-60%).

[Strategy for choosing antibiotics for treating bacterial infections associated with chronic tick-borne encephalitis]. / Strategiia vybora antibiotikov dlia terapii bakterial'nykh infektsii, assotsiiruiushchikhsia s khronicheskim kleshchevym éntsefalitom.

The capacity of wide-spectrum antibiotics kefzol and ristomycin to activate the persisting tick-borne encephalitis (TBE) virus and cause an exacerbation of chronic process was investigated in Syrian hamsters in whom a prolonged (77 to 270 days) persistent TBE infection was induced by three TBE strains: Vasilchenko, V-383, and 205. The degree of antibiotic-induced activation was assessed using the criteria characterizing the reproduction and peculiarities of persisting TBE virus, immunodepression, and morphologic changes in the central nervous system. Effects of kefzol and ristomycin were compared with those of 8 antibiotics studied previously. Ristomycin, levomycetin (chloramphycin), penicillin, ampicillin (ampital), and levoridan were referred to drugs devoid of evident provoking effect. Kefzol (cefamezin), florimycin (viomycin), and kanamycin (kanamytrex) were characterized as weak activators and streptomycin and tetracycline as potent activators of the persisting TBE virus. These data may be used when selecting alternative agents for therapy of secondary bacterial infections concomitant with TBE.

[Thymus hormones in the treatment and prevention of flavivirus infection under experimental conditions]. / Gormony timusa v lechenii i profilaktike flavivirusnoi infektsii v usloviiakh éksperimenta.

The evaluation and selection of the preparations of thymic hormones for the treatment and prophylaxis of acute tick-borne encephalitis (TBE) caused by Far Eastern TBE virus strains have been carried out on mice under different experimental conditions. These virus strains, highly and faintly virulent with respect to noninbred mice, produce a different modulating effect of the immune responsiveness of the host, respectively suppressing or simulating immune response to sheep red blood cells. A high prophylactic effect produced by thymic hormones (having protective index equal to 50-67%) with respect to highly virulent TBE virus stains has been established, which is seemingly indicative of the fact that such course of TBE leads to the formation of the state of severe immunodeficiency due to the lesion of the thymus. A high therapeutic effect resulting from the clinical use of thymic hormones is suggested.

[Highly-sensitive nonradioactive detection of the tick-borne encephalitis virus]. / Vysokochuvstvitel'naia neradioaktivnaia detektsiia virusa kleshchevogo éntsefalita.

The non-radioactive reverse dot-blot method was used for the detection of tick-borne encephalitis virus (TBEV) in clinical specimens. The method involves reverse transcription (RT) and polymerase chain reaction (PCR) using a pair of biotin-labelled oligonucleotide primers. These primers flank a region in the gene of the envelope protein E, which is more conserved than other regions, and initiate the polymerisation with RNAs of all the investigated strains. The amplified cDNA was captured from solution on a solid support using complementary oligonucleotides covalently bound to a polyamide membrane. The biotin labels of the resulting hybrids were visualized by means of the streptavidin-horseradish peroxidase conjugate. The detection limit of the test was about 10(3)-10(4) molecules of target RNA. The sensitivity was comparable to that obtained by dot-hybridization of PCR-product with 32P-labelled DNA probe. The method was used for the detection of RNA in specimens of tick and blood.

[A live vaccine against tick-borne encephalitis: the principles of selecting the vaccinal strains]. / Zivaia vaktsina protiv kleshchevogo éntsefalita: printsipy otbora vaktsinnykh shtammov.

The comparative, semiquantitative, pathomorphological study of the neurovirulence of clones of Elantsev virus and Langat virus TP-21 for intracerebrally infected monkeys has been carried out. The study has revealed that the viruses may be differentiated by their neurovirulence for primates according to the average statistical data on the degree of pathomorphological changes in the central nervous system, but not to maximum lesions in cerebral structures. The level of neurovirulence of yellow fever virus 17D was formerly considered to be the highest admissible limit of residual neurovirulence of encephalitogenic viruses (flaviviruses). According to our data, Elantsev virus, used for the immunization of humans and known to have caused some cases of encephalitis, is similar to yellow fever virus with respect to its neurovirulence for primates: therefore, a candidate strain intended for the preparation of tick-borne encephalitis (TBE) vaccine must be significantly less neurovirulent. The neurovirulence of clones isolated from Langat virus TP-21 has proved to be essentially lower than that of Elantsev virus clones. Langat virus TP-21 is a promising source of clones suitable for use as candidates for live TBE vaccine. Search for vaccine strains by testing their neurovirulence in experiments on several strains of mice and their hybrids, on hamsters and on immunosuppressed animals is methodologically groundless. The adequate evaluation of the level of residual neurovirulence of viruses to be used as candidates for live TBE vaccine can be made only on monkeys.

[The relationship of the antigenic characteristics of the tick-borne encephalitis virus to the level of the protective activity of inactivated cultured vaccines]. / Izuchenie vzaimosviazi antigennoi kharakteristiki virusa kleshchevogo éntsefalita s urovnem protektivnoi aktivnosti inaktivirovannykh kul'tural'nykh vaktsin.

As shown in this study, the immunization of animals with killed vaccines against tick-borne encephalitis (TBE) leads to the formation of specific immunity, depending on the antigenic structure of the vaccine strain and the test strains used for challenge. Vaccines obtained on the basis of the TBE virus strain of the Eastern antigenic variant induced the development of a wider spectrum of specific protective activity than vaccines obtained on the basis of the TBE virus strain of the Western antigenic variant.