β-Sitosterol: Supercritical Carbon Dioxide Extraction from Sea Buckthorn (Hippophae rhamnoides L.) Seeds

Supercritical fluid extraction represents an efficient and environmentally friendly technique for isolation of phytosterols from different plant sources. Sea buckthorn (Hippophae rhamnoides L.) seeds were extracted with supercritical carbon dioxide at pressures ranging from 15–60 MPa and temperatures of 40–80 °C. Oil and β-sitosterol yields were measured in the extraction course and compared with Soxhlet extraction with hexane. The average yield of β-sitosterol was 0.31 mg/g of seeds. The maximum concentration of β-sitosterol in the extract, 0.5% w/w, was achieved at 15 MPa, 40 °C, and a carbon dioxide consumption of 50 g/g of seeds. The extraction rate was maximal at 60 MPa and 40 °C. Both β-sitosterol yield and its concentration in the extract obtained with hexane were lower than with carbon dioxide

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Alimentary Infections by Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) causes serious the neurological disease, tick-borne encephalitis (TBE). TBEV can be transmitted to humans by ticks as well as by the alimentary route, which is mediated through the consumption of raw milk products from infected ruminants such as sheep, goats, and cows. The alimentary route of TBEV was recognized in the early 1950s and many important experimental studies were performed shortly thereafter. Nowadays, alimentary TBEV infections are recognized as a relevant factor contributing to the overall increase in TBE incidences in Europe. This review aims to summarize the history and current extent of alimentary TBEV infections across Europe, to analyze experimental data on virus secretion in milk, and to review possible alimentary infection preventive measures

Co-Circulation of West Nile, Usutu, and Tick-Borne Encephalitis Viruses in the Same Area: A Great Challenge for Diagnostic and Blood and Organ Safety

Viral infections caused by viruses from the family Flaviviridae such as Zika (ZIKV), Dengue (DENV), yellow fever (YFV), tick-borne encephalitis (TBEV), West Nile (WNV), and Usutu (USUV) are some of the most challenging diseases for recognition in clinical diagnostics and epidemiological tracking thanks to their short viremia, non-specific symptoms, and high cross-reactivity observed in laboratory techniques. In Central Europe, the most relevant endemic flaviviruses are mosquito-borne WNV and USUV, and tick-borne TBEV. All three viruses have been recognised to be responsible for human neuroinvasive diseases. Moreover, they are interrupting the blood and transplantation safety processes, when the great efforts made to save a patient’s life could be defeated by acquired infection from donors. Due to the trend of changing distribution and abundance of flaviviruses and their vectors influenced by global change, the co-circulation of WNV, USUV, and TBEV can be observed in the same area. In this perspective, we discuss the problems of flavivirus diagnostics and epidemiology monitoring in Slovakia as a model area of Central Europe, where co-circulation of WNV, USUV, and TBEV in the same zone has been recently detected. This new situation presents multiple challenges not only for diagnostics or surveillance but particularly also for blood and organ safety. We conclude that the current routinely used laboratory diagnostics and donor screening applied by the European Union (EU) regulations are out of date and the novel methods which have become available in recent years, e.g., next-gene sequencing or urine screening should be implemented immediately

Co-Circulation of West Nile, Usutu, and Tick-Borne Encephalitis Viruses in the Same Area: A Great Challenge for Diagnostic and Blood and Organ Safety

Viral infections caused by viruses from the family Flaviviridae such as Zika (ZIKV), Dengue (DENV), yellow fever (YFV), tick-borne encephalitis (TBEV), West Nile (WNV), and Usutu (USUV) are some of the most challenging diseases for recognition in clinical diagnostics and epidemiological tracking thanks to their short viremia, non-specific symptoms, and high cross-reactivity observed in laboratory techniques. In Central Europe, the most relevant endemic flaviviruses are mosquito-borne WNV and USUV, and tick-borne TBEV. All three viruses have been recognised to be responsible for human neuroinvasive diseases. Moreover, they are interrupting the blood and transplantation safety processes, when the great efforts made to save a patient’s life could be defeated by acquired infection from donors. Due to the trend of changing distribution and abundance of flaviviruses and their vectors influenced by global change, the co-circulation of WNV, USUV, and TBEV can be observed in the same area. In this perspective, we discuss the problems of flavivirus diagnostics and epidemiology monitoring in Slovakia as a model area of Central Europe, where co-circulation of WNV, USUV, and TBEV in the same zone has been recently detected. This new situation presents multiple challenges not only for diagnostics or surveillance but particularly also for blood and organ safety. We conclude that the current routinely used laboratory diagnostics and donor screening applied by the European Union (EU) regulations are out of date and the novel methods which have become available in recent years, e.g., next-gene sequencing or urine screening should be implemented immediately

An antivector vaccine protects against a lethal vector-borne pathogen

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a “dual action” in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: “transmission,” number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; “support,” number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and “survival,” number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding sit

Phylogenetic analysis of Puumala virus strains from Central Europe highlights the need for a full-genome perspective on hantavirus evolution

Puumala virus (PUUV), carried by bank voles (Myodes glareolus), is the medically most important hantavirus in Central and Western Europe. In this study, a total of 523 bank voles (408 from Germany, 72 from Slovakia, and 43 from Czech Republic) collected between the years 2007-2012 were analyzed for the presence of hantavirus RNA. Partial PUUV genome segment sequences were obtained from 51 voles. Phylogenetic analyses of all three genome segments showed that the newfound strains cluster with other Central and Western European PUUV strains. The new sequences from Šumava (Bohemian Forest), Czech Republic, are most closely related to the strains from the neighboring Bavarian Forest, a known hantavirus disease outbreak region. Interestingly, the Slovak strains clustered with the sequences from Bohemian and Bavarian Forests only in the M but not S segment analyses. This well-supported topological incongruence suggests a segment reassortment event or, as we analyzed only partial sequences, homologous recombination. Our data highlight the necessity of sequencing all three hantavirus genome segments and of a broader bank vole screening not only in recognized endemic foci but also in regions with no reported human hantavirus disease cases.status: publishe

Skin Immunocytochemical Profile of Immunized Mice Infested with Virus-Infected and -Uninfected Ticks

<p>Immunocytochemical profiles of skin sections taken at d 4 of TBEV-infected I. ricinus tick challenge on Balb/c mice immunized with either GST (A, B) or TRP5 (C, D), or unimmunized (E, F), using rat anti-mouse CD4⁺ antiserum (B, D, and F) and rat anti-mouse CD8⁺ (A, C, and E) antiserum, with a negative control sample (G, PBS plus normal rabbit serum). (C) TRP5-immunized mice: red arrowheads = numerous CD8⁺ T cells; red circles = CD8⁺ T cells occluding the dermal blood vessels; and (D) yellow arrows = CD4⁺ T cells. (E) and (F) unimmunized mice, few CD8⁺ T cells = red arrows and CD4⁺ T cells = yellow arrows/yellow circle, respectively. (B) Control GST-immunized mice yellow arrows = few CD4⁺ T cells. (G) PBS-negative control skin sample = no T cells. Magnification 20×.</p

Skin Histological Response in Immunized Mice Infested with Virus-Infected and -Uninfected Ticks

<p>Histological profiles of skin sections taken at d 4 of TBEV-infected I. ricinus tick challenge on Balb/c mice immunized with either TRP2 (A, D), TRP5 (E), or GST (C, F), or unimmunized (B). Stained with hematoxylin and eosin (A–D) or “Hema Gurr” Rapid stain BDH (E, F) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020027#ppat-0020027-b019" target="_blank">19</a>]. (A) (magnification 20×) TRP2-immunized animals, (B) (magnification 20×) control unimmunized, and (C) (magnification 20×) GST-immunized animals. Ep, epidermis; De, dermis; CC, cement cone of I. ricinus. (D–E) (magnification 63×) denote skin sections from TRP2 and TRP5 immunized mice, respectively, showing: perivascular cuffing = yellow circle; degranulating mast cells = green arrow; numerous lymphocytes = blue arrow; frequent macrophages = yellow arrow; and some eosinophils = white arrow; dermal dendrocytes = black arrow; neutrophils = gray arrow; and basophils = light blue arrow. (F) (magnification 63×) skin sections from GST control immunized mice showing lymphocytes, macrophages, and dermal dendrocytes.</p

Effect of Immunization on Mice Infested with Virus-Infected and -Uninfected Ticks

<p>Comparison of immunization with either 64TRP antigens, a commercial TBEV vaccine, or the commercial anti-tick vaccine (TickGARD) on (A) transmission = % uninfected nymphal ticks that became infected; (B) support = % mice supporting cofeeding virus transmission between an infected adult female tick and uninfected nymphs; and (C) survival = % mice that survived an infected tick bite (only animals surviving subsequent i.p. inoculation with 1,000 PFU TBEV were included in the analyses).</p