Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease of swine caused by the eponymous virus (PEDV) which belongs to the genus
within the
virus family. Following the ...disastrous outbreaks in Asia and the United States, PEDV has been detected also in Europe. In order to better understand the overall situation, the molecular epidemiology, and factors that might influence the most variable disease impact; 40 samples from swine feces were collected from different PED outbreaks in Germany and other European countries and sequenced by shot-gun next-generation sequencing. A total of 38 new PEDV complete coding sequences were generated. When compared on a global scale, all investigated sequences from Central and South-Eastern Europe formed a rather homogeneous PEDV S INDEL cluster, suggesting a recent re-introduction. However, in-detail analyses revealed two new clusters and putative ancestor strains. Based on the available background data, correlations between clusters and location, farm type or clinical presentation could not be established. Additionally, the impact of secondary infections was explored using the metagenomic data sets. While several coinfections were observed, no correlation was found with disease courses. However, in addition to the PEDV genomes, ten complete viral coding sequences from nine different data sets were reconstructed each representing new virus strains. In detail, three pasivirus A strains, two astroviruses, a porcine sapelovirus, a kobuvirus, a porcine torovirus, a posavirus, and an enterobacteria phage were almost fully sequenced.
Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic ...losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.
•Rotavirus typing in German pig population provides epidemiologic data.•Porcine rotavirus types show regional but also supraregional patterns.•Possible starting points for porcine rotavirus vaccine ...development.•Pig populations are promoting emergence of genotypes and their combinations.
Group A porcine rotaviruses are a global threat to animal health in stock breeding. While certain genotypes have shown predominance in other countries, data from Europe's second largest swine population is still scarce. Therefore, porcine rotaviruses taken from different areas of Germany were genotyped to create a basis for comparison with data from neighboring countries. In addition, the potential predominance and regionality based on regions (federal states) have been investigated by examining 101 samples.
The study revealed the dominance of the VP7 genotypes G9, G4, G5 as well as VP4 genotypes P23, P6, P32. The most common genotype combinations were G9P23, G4P6, and G9P32. Analysis focusing on the regionality aspect revealed that areas with high pig populations promote the emergence of dominant genotype combinations. However, pig populations in Germany cannot be considered individually and therefore results were put into international context, taken from already published genotyping data. In consequence, our data contributes to the fundamental understanding of regional and supraregional rotavirus epidemiology. The detected genotypes provide a basis for prospective porcine rotavirus surveillance, that first of all helps to identify interspecies transmission. Furthermore it may provide supporting data for the selection of particular genotypes, suitable for the production of porcine rotavirus A vaccine candidates.
Respiratory disease is a significant economic issue in pig farming, with a complex aetiology that includes swine influenza A viruses (swIAV), which are common in European domestic pig populations. ...The most recent human influenza pandemic in 2009 showed swIAV's zoonotic potential. Monitoring pathogens and disease control are critical from a preventive standpoint, and are based on quick, sensitive, and specific diagnostic assays capable of detecting and distinguishing currently circulating swIAV in clinical samples. For passive surveillance, a set of multiplex quantitative reverse transcription real-time PCRs (mRT-qPCR) and MinION-directed sequencing was updated and deployed. Several lineages and genotypes of swIAV were shown to be dynamically developing, including novel reassortants between human pandemic H1N1 and the avian-derived H1 lineage of swIAV. Despite this, nearly 70% (842/1216) of individual samples from pigs with respiratory symptoms were swIAV-negative, hinting to different aetiologies. The complex and synergistic interactions of swIAV infections with other viral and bacterial infectious agents contribute to the aggravation of pig respiratory diseases. Using a newly developed mRT-qPCR for the combined detection of swIAV and the recently described porcine respirovirus 1 (PRV1) and swine orthopneumovirus (SOV) widespread co-circulation of PRV1 (19.6%, 238/1216 samples) and SOV (14.2%, 173/1216 samples) was evident. Because of the high incidence of PRV1 and SOV infections in pigs with respiratory disease, these viruses may emerge as new allies in the porcine respiratory disease syndrome.
Sequential application of solvent extraction, gel permeation chromatography, and RP-HPLC in combination with taste dilution analyses, followed by LC-MS and 1D/2D NMR experiments, led to the discovery ...and structure determination of 25 key astringent compounds of red currant juice. Besides several flavonol glycosides, in particular, 3-carboxymethyl-indole-1-N-β-d-glucopyranoside, 3-methylcarboxymethyl-indole-1-N-β-d-glucopyranoside, and a family of previously not identified compounds, namely, 2-(4-hydroxybenzoyloxymethyl)-4-β-d-glucopyranosyloxy-2(E)-butenenitrile, 2-(4-hydroxy-3-methoxybenzoyloxymethyl)-4-β-d-glucopyranosyloxy-2(E)-butenenitrile, (E)-6-3-hydroxy-4-(O-β-d-glucopyranosyl)phenyl-5-hexen-2-one named dehydrorubrumin, and (3E,5E)-6-3-hydroxy-4-(O-β-d-glucopyranosyl)phenyl-3,5-hexadien-2-one named rubrumin, have been identified. Determination of the oral astringency thresholds by means of the half-tongue test revealed that the lowest thresholds of 0.3 and 1.0 nmol/L were found for the nitrogen-containing 3-carboxymethyl-indole-1-N-β-d-glucopyranoside and 3-methylcarboxymethyl-indole-1-N-β-d-glucopyranoside, which do not belong to the group of plant polyphenols. Keywords: Red currant; astringency; taste; taste dilution analysis
Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown ...hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.
Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.
F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.
Caliciviruses (CV) were identified in the intestinal contents of five chickens and one turkey from various regions in Germany between 2009 and 2011 by degenerate reverse transcription PCR. The full ...7,656-nt-long genomic sequence of the turkey CV L11043 was determined. Partial nucleotide sequences were determined for nine chicken strains. Phylogenetic analysis based on partial deduced amino acid sequences of the protease and RNA polymerase and the complete VP1 capsid sequence identified two distinct clusters of avian CVs, the first of which contained chicken CVs that were closely related to strains found in German chickens in Bavaria and that had been proposed to form a novel CV genus (proposed name: Bavovirus). In contrast, the turkey CV strain L11043 and three chicken CV strains formed a genetically distinct second cluster. Distance analysis suggested that the strains of the second cluster may represent members of two distinct genogroups of another novel CV genus (proposed name: Nacovirus). Based on the newly obtained sequence information, two real-time RT-PCR assays were developed and used to identify bavovirus and nacovirus in pooled intestinal contents from 24 chicken farms in Germany and the Netherlands. Of these, 20 (83 %) were positive for bavovirus, 11 (46 %) were positive for nacovirus, and nine (38 %) were positive for both bavovirus and nacovirus. Attempts were made to propagate chicken and turkey CVs from both the bavovirus and nacovirus clusters in primary chicken cecal cells, embryonal liver cells and fibroblast cells, but these attempts were not successful.
For decades, it is believed that astringency is due to the polyphenol-induced complexation of proline-rich salivary proteins in the oral cavity. In order to compare for the first time the human ...sensory threshold concentrations and the salivary protein binding activity of a series of astringent stimuli, human saliva protein was incubated for 5 min at 37 °C in the presence of astringent food-derived compounds and, after micro-centrifugation, the amount of the target molecules in the supernatant was quantitatively determined by HPLC-UV/Vis. Significant protein binding was observed for (-)-epigallocatechin-3-gallate, (-)-gallocatechin-3-gallate, (+)-gallocatechin, and (-)-catechin-3-gallate, all of which containing at least one galloyl moiety in the molecule and exhibiting rather high sensory thresholds of more than 200 μmol/L. In comparison, (+)-catechin and procyanidin B2, both lacking in any galloyl function, showed only comparatively low binding activity and, most interestingly, quercetin-3-O-α-l-rhamnopyranosyl-(1 rightward arrow 6)-β-d-glucopyranoside and 3-carboxymethyl-indole-1-N-β-d-glucopyranoside did not show any protein binding at all, although the later N- and O-glycosides exhibited extraordinarily low sensory threshold concentrations of less than 0.001 and 0.0003 μmol/L, respectively. The data give some first evidence that the quantity of the non-bound, “free” astringent stimulus in the saliva liquid might be more closely related to the sensory perception of astringency than the amount complexed or precipitated by proteins. It is therefore questionable as to whether oral perception of astringency is related to the complexation and/or precipitation of salivary proteins.
A competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed to detect and to quantitate the RNA of group A rotaviruses. In the assay, a 433 bp fragment is amplified by a ...one-tube RT-PCR protocol using primers with binding sites located in a highly conserved region of segment 6 of the rotavirus genome. An in vitro synthesized RNA with a 43-base deletion with respect to the wild-type sequence of this fragment was used as an internal control. Using these transcripts as templates, 10 RNA molecules were amplified reproducibly and detected in ethidium bromide-stained agarose gels or by fluorimetry using the SYBR Green I dye in a real-time RT-PCR assay. The efficiency of the protocol was confirmed by the detection of small amounts of viral RNA of group A rotaviruses in clinical samples obtained from various animal species and man.
Sequential application of solvent extraction, gel permeation chromatography, and HPLC in combination with taste dilution analyses, followed by LC-MS and 1D/2D NMR experiments, led to the discovery of ...seven velvety mouth-coating molecules in cured beans of Vanilla planifolia. Among these, 5-(4-hydroxybenzyl)vanillin, 4-(4-hydroxybenzyl)-2-methoxyphenol, 4-hydroxy-3-(4-hydroxy-3-methoxybenzyl)-5-methoxybenzaldehyde, (1-O-vanilloyl)-(6-O-feruloyl)-β-d-glucopyranoside, americanin A, and 4′,6′-dihydroxy-3′,5-dimethoxy-1,1′-biphenyl-3-carboxaldehyde were previously not reported in vanilla beans. Sensory studies revealed human recognition thresholds for the velvety mouth-coating sensation between 1.0 and 5.0 μmol/kg (water). Interestingly, the biphenyl derivatives were found to enhance the perception of creaminess and fatty body of sweetened skim milk, among which 4′,6′-dihydroxy-3′,5-dimethoxy-1,1′-biphenyl-3-carboxaldehyde showed the lowest threshold level of 5 μmol/kg. Quantitative analysis of these compounds in cured vanilla beans from different origins as well as in noncured beans revealed that, with the exception of americanin A, all of the other taste compounds are not present in the green vanilla beans and are formed during the bean curing process.
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