Detection of Leptospira species in bat cadavers, Czech and Slovak Republics.

Kidney samples from 300 bat cadavers from the Czech and Slovak Republics were tested for Leptospira DNA using PCR and sequencing of three genes (lipL32, flab, and 16S ribosomal RNA). Overall detection rate was 4.7% and two bat species (Myotis myotis and Nyctalus noctula) were PCR-positive for at least one gene. Detected Leptospira sequences were similar to L. interrogans and L. borgpetersenii, and included a potentially novel species related to L. weilii.

Diphyllin Shows a Broad-Spectrum Antiviral Activity against Multiple Medically Important Enveloped RNA and DNA Viruses.

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

Clinical Characteristics of Patients with Tick-Borne Encephalitis (TBE): A European Multicentre Study from 2010 to 2017.

Tick-borne encephalitis (TBE) virus is a major cause of central nervous system infections in endemic countries. Here, we present clinical and laboratory characteristics of a large international cohort of patients with confirmed TBE using a uniform clinical protocol. Patients were recruited in eight centers from six European countries between 2010 and 2017. A detailed description of clinical signs and symptoms was recorded. The obtained information enabled a reliable classification in 553 of 555 patients: 207 (37.3%) had meningitis, 273 (49.2%) meningoencephalitis, 15 (2.7%) meningomyelitis, and 58 (10.5%) meningoencephalomyelitis; 41 (7.4%) patients had a peripheral paresis of extremities, 13 (2.3%) a central paresis of extremities, and 25 (4.5%) had single or multiple cranial nerve palsies. Five (0.9%) patients died during acute illness. Outcome at discharge was recorded in 298 patients. Of 176 (59.1%) patients with incomplete recovery, 80 (27%) displayed persisting symptoms or signs without recovery expectation. This study provides further evidence that TBE is a severe disease with a large proportion of patients with incomplete recovery. We suggest monitoring TBE in endemic European countries using a uniform protocol to record the full clinical spectrum of the disease.

Mannitol treatment is not effective in therapy of rabies virus infection in mice.

Rabies is a deadly viral disease with an extremely high fatality rate in humans. Previously, it was suggested that an enhancement of the blood-brain barrier (BBB) permeability, which allows immune cells and/or antibodies to enter the central nervous system (CNS) tissue, is critical to clear the infection. In this study, we utilised mannitol to increase BBB permeability in mice infected with highly pathogenic silver-haired bat rabies virus (SHBRV). We found that intraperitoneal injection of mannitol causes a slight, transient increase of BBB permeability in the treated mice. SHBRV-infected mice were treated with intraperitoneally administered mannitol daily from day 3 or day 4 post-infection, but no effect of this treatment on the time of disease onset, clinical signs or survival was observed. This data indicates that the increase of BBB permeability by mannitol is not efficient in promoting CNS virus clearance in SHBRV-infected mice.

Combination therapy of rabies-infected mice with inhibitors of pro-inflammatory host response, antiviral compounds and human rabies immunoglobulin.

Recent studies demonstrated that inhibitors of pro-inflammatory molecular cascades triggered by rabies infection in the central nervous system (CNS) can enhance survival in mouse model and that certain antiviral compounds interfere with rabies virus replication in vitro. In this study different combinations of therapeutics were tested to evaluate their effect on survival in rabies-infected mice, as well as on viral load in the CNS. C57Bl/6 mice were infected with Silver-haired bat rabies virus (SHBRV)-18 at virus dose approaching LD50 and LD100. In one experimental group daily treatments were initiated 4 h before-, in other groups 48 or 96 h after challenge. In the first experiment therapeutic combination contained inhibitors of tumour necrosis factor-α (infliximab), caspase-1 (Ac-YVAD-cmk), and a multikinase inhibitor (sorafenib). In the treated groups there was a notable but not significant increase of survival compared to the virus infected, non-treated mice. The addition of human rabies immunoglobulins (HRIG) to the combination in the second experiment almost completely prevented mortality in the pre-exposure treatment group along with a significant reduction of viral titres in the CNS. Post-exposure treatments also greatly improved survival rates. As part of the combination with immunomodulatory compounds, HRIG had a higher impact on survival than alone. In the third experiment the combination was further supplemented with type-I interferons, ribavirin and favipiravir (T-705). As a blood-brain barrier opener, mannitol was also administered. This treatment was unable to prevent lethal consequences of SHBRV-18 infection; furthermore, it caused toxicity in treated mice, presumably due to interaction among the components. In all experiments, viral loads in the CNS were similar in mice that succumbed to rabies regardless of treatment. According to the findings, inhibitors of detrimental host response to rabies combined with antibodies can be considered among the possible therapeutic and post-exposure options in human rabies cases.

Novel hantavirus identified in European bat species Nyctalus noctula.

Hantaviruses are emerging RNA viruses that cause human diseases predominantly in Asia, Europe, and the Americas. Besides rodents, insectivores and bats serve as hantavirus reservoirs. We report the detection and genome characterization of a novel bat-borne hantavirus isolated from insectivorous common noctule bat. The newfound virus was tentatively named as Brno virus.

Epidemiological survey of enteric viruses in wild boars in the Czech Republic: First evidence of close relationship between wild boar and human rotavirus A strains.

Population of wild boar is increasing in the whole Europe, the animals migrate close to human habitats which greatly increases the possibility of natural transmission between domestic animals or humans and wild boars. The aim of the study was to estimate in population of free-living wild boar in the Czech Republic the prevalence of enteric viral pathogens, namely rotavirus groups A and C (RVA and RVC), porcine reproductive and respiratory syndrome virus (PRRSV), and members of family Coronaviridae (transmissible gastroenteritis virus - TGEV, porcine epidemic diarrhea virus - PEDV, porcine respiratory coronavirus - PRCV, and porcine hemagglutination encephalomyelitis virus - PHEV) and Picornaviridae,(teschovirus A - PTV, sapelovirus A - PSV, and enterovirus G - EV-G). In our study, stool samples from 203 wild boars culled during hunting season 2014-2015 (from October to January) were examined by RT-PCR. RVA was detected in 2.5% of tested samples. Nucleotide analysis of VP7, VP4, and VP6 genes revealed that four RVA strains belong to G4P[25]I1, G4P[6]I5, G11P[13]I5, and G5P[13]I5 genotypes and phylogenetic analysis suggested close relation to porcine and human RVAs. The prevalence of RVC in wild boar population reached 12.8%, PTV was detected in 20.2%, PSV in 8.9%, and EV-G in 2.5% of samples. During our study no PRRSV or coronaviruses were detected. Our study provides the first evidence of RVC prevalence in wild boars and indicates that wild boars might contribute to the genetic variability of RVA and also serve as an important reservoir of other enteric viruses.

Detection of Diverse Novel Bat Astrovirus Sequences in the Czech Republic.

Astroviruses are a major cause of gastroenteritis in humans and animals. Recently, novel groups of astroviruses were identified in apparently healthy insectivorous bats. We report the detection of diverse novel astrovirus sequences in nine different European bat species: Eptesicus serotinus, Hypsugo savii, Myotis emarginatus, M. mystacinus, Nyctalus noctula, Pipistrellus nathusii or P. pygmaeus, P. pipistrellus, Vespertilio murinus, and Rhinolophus hipposideros. In six bat species, astrovirus sequences were detected for the first time. One astrovirus strain detected in R. hipposideros clustered phylogenetically with Chinese astrovirus strains originating from bats of the families Rhinolophidae and Hipposideridae. All other Czech astrovirus sequences from vesper bats formed, together with one Hungarian sequence, a separate monophyletic lineage within the bat astrovirus group. These findings provide new insights into the molecular epidemiology, ecology, and prevalence of astroviruses in European bat populations.

Diversity of VP7, VP4, VP6, NSP2, NSP4, and NSP5 genes of porcine rotavirus C: phylogenetic analysis and description of potential new VP7, VP4, VP6, and NSP4 genotypes.

Rotavirus C (RVC) is a cause of gastroenteritis in swine and has a worldwide distribution. A total of 448 intestinal or faecal samples from pigs of all ages were tested for viruses causing gastroenteritis. RVC was detected in 118 samples (26.3%). To gain information on virus diversity, the complete coding nucleotide sequences of the VP7, VP4, VP6, NSP2, NSP4, and NSP5 genes of seven RVC strains were determined. Phylogenetic analysis of VP7 nucleotide sequence divided studied Czech strains into six G genotypes (G1, G3, G5-G7, and a newly described G10 genotype) based on an 85% identity cutoff value at the nucleotide level. Analysis of the VP4 gene revealed low nucleotide sequence identities between two Czech strains and other porcine (72.2-75.3%), bovine (74.1-74.6%), and human (69.1-69.3%) RVC strains. Thus, we propose that those two Czech porcine strains comprise a new RVC VP4 genotype, P8. Analysis of the VP6 gene showed 79.9-86.8% similarity at the nucleotide level between the Czech strains and other porcine RVC strains. According to the 87% identity cutoff value, we propose the existence of three new RVC VP6 genotypes, I8-I10. Analysis of the NSP4 gene divided porcine RVC strains into two clusters (the E1 genotype and the new E4 genotype, based on an 85% nucleotide sequence identity cutoff value). Our results indicate a degree of high genetic heterogeneity, not only in the variable VP7 and VP4 genes encoding the outer capsid proteins, but also in more-conserved genes encoding the inner capsid protein VP6 and the non-structural proteins NSP2, NSP4, and NSP5. This emphasizes the need for a whole-genome-sequence-based classification system.

Full-length genome analysis of Calovo strains of Batai orthobunyavirus (Bunyamwera serogroup): implications to taxonomy.

Batai virus (BATV) is a poorly studied arthropod-borne virus belonging to the genus Orthobunyavirus (Bunyamwera serogroup) within the family Bunyaviridae. It has been associated with human influenza-like febrile illness in several Asian, African, and European countries. Calovo virus (CVOV), isolated in 1960 in Slovakia, has been classified as BATV based on high antigenic similarity, and since then both CVOV and BATV were used as synonyms. In order to fully clarify the phylogenetic relationships between CVOV, BATV, and other members of the Bunyamwera serogroup, we performed whole genome sequencing of four CVOV strains isolated in Europe and phylogenetic analyses of all related viruses. The nucleocapsid protein, encoded by the S genomic segment, contains 233 amino acids, 60 of which, putatively critical for protein function, are conserved. Within the CVOV polyprotein encoded by the M genomic segment, putative cleavage sites, N-glycosylation sites, and seven transmembrane regions were identified. The RNA-dependent RNA polymerase, encoded by the L genome segment, exhibits conservation of the three regions known to be conserved among bunyavirus and arenavirus L proteins. Phylogenetic analyses of all three genomic segments of selected orthobunyaviruses clearly revealed that European and Asian/African strains of BATV are phylogenetically different and form two distinct lineages, indicating the existence of two different genotypes of BATV, tentatively named European genotype (with CVOV as a type strain) and Afro-Asian genotype (with BATV as a type strain) of BATV.

Prevalence study and phylogenetic analysis of group C porcine rotavirus in the Czech Republic revealed a high level of VP6 gene heterogeneity within porcine cluster I1.

Group C rotavirus (RVC) has been described to be a causative agent of gastroenteritis in humans and animals. In the current study, the presence of porcine RVC was confirmed in 25.6 % of 293 porcine faecal samples collected from seven Czech farms. A significantly larger (p < 0.05) number of RVC-positive samples was detected in groups of finisher pigs and post-weaning piglets (4-12 weeks of age). Phylogenetic analysis of nine RVC-positive Czech strains and their comparison with available sequence data for the gene encoding RVC group antigen VP6 revealed two separate lineages within porcine cluster I1.

Genetic diversity of porcine sapoviruses, kobuviruses, and astroviruses in asymptomatic pigs: an emerging new sapovirus GIII genotype.

Small, non-enveloped RNA viruses belonging to the genera Sapovirus, Kobuvirus, and Mamastrovirus are usually associated with gastroenteritis in humans and animals. These enteric pathogens are considered potential zoonotic agents. In this study, the prevalence and genetic diversity of sapoviruses (SaVs), kobuviruses (KoVs), and astroviruses (AstVs) in asymptomatic pigs were investigated using a PCR approach. KoV was found to be the most prevalent virus (87.3 %), followed by AstV (34.2 %) and SaV (10.2 %). Interestingly, the intra- and inter-cluster distances between porcine SaV capsid sequences revealed one strain (P38/11/CZ) that formed a new genotype within genogroup III of porcine SaVs, and it is tentatively called "P38/11-like" genotype. Moreover, this is the first report of porcine kobuvirus detection on Czech pig farms. The high prevalence rate of gastroenteritis-producing viruses in clinically healthy pigs represents a continuous source of infection of pigs, and possibly to humans.