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Deep Learning Technology: Sebastian Arnold, Betty van Aken, Paul Grundmann, Felix A. Gers and Alexander Löser. Learning Contextualized Document Representations for Healthcare Answer Retrieval. The Web Conference 2020 (WWW'20)
Funded by The Federal Ministry for Economic Affairs and Energy; Grant: 01MD19013D, Smart-MD Project, Digital Technologies
The results of curve fitting (Fig. 4) showed that Model 1 fitted well to the data (χ2 = 0.00015, P > 0.999). Calculated by the model, the mean value of b was 0.0898 (95% CI: 0.0851–0.0946) and bw was 1.1264 × 10− 9 (95% CI: 4.1123 × 10− 10–1.8416 × 10− 9) from 2005 to 2017 in the province. The difference between the two parameters was as much as more than seven orders of magnitude in each year (Table 3). Results of the “knock-out” simulation (Fig. 5) showed that the number of cases simulated by scenario A (b = 0 and bw = 0) was almost the same as that simulated by scenario B (b = 0), and that the number of cases simulated by scenario C (bw = 0) was the almost same as that simulated by scenario D (control), which means that parameter bw had almost no contribution to the transmission.
The effective reproduction number Reff of the disease was calculated with mean value of 1.19 (95% CI: 1.13–1.25) using Model 2 (Fig. 6). The mean value of Reff was 1.08 (95% CI: 0.83–1.34) in the year of 2005. It reached a peak value of 1.39 (95% CI: 1.14–1.65) in 2006. Although it had a lowest value in 2010, it decreased slightly yearly (Table 4). By fitting the 11 equations with Reff calculated by the SEIAR model with the reported data, the three most fitting models were Cubic, Linear, and Quadratic (Table 5). The fitted results were shown in Fig. 7. The Linear and Quadratic models forecasted that the mean value of Reff would decrease yearly. Based on the Linear model, Reff would reach a low value of 1.00 (95% CI: 0.67–1.34) in the year of 2028 and be down to an epidemic threshold of 0.99 (95% CI: 0.65–1.34) in 2029. Based on the Quadratic model, Reff would reach a low value of 1.00 (95% CI: 0.00–2.93) in the year of 2031 and be down to an epidemic threshold of 0.999 (95% CI: 0.00–3.12) in 2032. However, the Cubic model predicted an increasing trend after the year 2018.
From January 1, 2005 to December 31, 2017, 130,770 Shigellosis cases were reported in the province. Among them 13 cases were dead with a case fatality rate of 0.01%. The median yearly reported incidence was 19.96 per 100,000 persons (range: 5.99 per 100,000 persons – 29.47 per 100,000 persons). Figure 1a showed that the number of reported cases and reported incidence yearly were both decreased significantly (trend χ2 = 25,470.27, P < 0.001).
Of all the cases, male cases were account for 56.57% (73,981/130770) which was significantly higher than that of female cases (χ2 = 2255.19, P < 0.001). This incidence difference between male and female was observed in each year (Fig. 1b). Children cases with an age of 10 years and younger had the highest case proportion of 39.30%, with the “0 – 1” years old children or new-born cases were account for 61.33% (Table 1). The second age group with highest case proportion was “11 – 20” years (15.20%) followed by “61 –” years (14.51%). The distribution of incidence of age group was similar in different years (Fig. 1c). The median duration from illness onset date to diagnosed date (DID) of all the cases was 1.7 days (inter-quartile range [IQR]: 2.3 days). Each year had a similar distribution of DID (Fig. 2). The DID of 71.24% cases were lower than 4 days, especially lower 1 days (24.43%) and 2 days (30.87%).
Among the 17 sub-regions of Hubei Province, Wuhan City had the most reported cases (39.88%) and the highest cumulative incidence during the past 13 years. The second highest number of cases was reported in Yichang City (8.66%) and Jingzhou City (8.24%). However, the rank of reported incidence was very different. Xiantao City and Yichang City had the second and third highest incidence, respectively. Shennongjia, which is a forest region, had the least reported cases (0.05%) and the lowest reported incidence (Table 2).
The mean reported incidence of shigellosis was 58.53 per 100,000 in urban areas (95%CI: 51.91 per 100,000–65.15 per 100,000) and 14.10 per 100,000 in rural areas (95% CI: 11.34 per 100,000–16.86 per 100,000) (Fig. 3). The difference of the incidences was statistically significant between urban and rural areas (t = 12.1345, P < 0.001).
Plant pests and pathogens have an important role in global food crops causing significant economic losses in the agricultural industry and threatening food security [,,]. Yam (Dioscorea spp.) is one of the most important staple food crops worldwide and plays a major role in food security and income generation for more than 60 million people in West Africa, with this region contributing over 95% of the world's total yam production. Yams are generally propagated vegetatively through their tubers, which facilitates the spread and accumulation of pathogens, particularly viruses. To date, several virus species belonging to different genera (Potyvirus, Badnavirus, Cucumovirus, Aureusvirus, Potexvirus, Macluravirus, and Carlavirus) [,,,,,] have been reported and characterized in yams. These viral infections restrict the international exchange of yam germplasm and have a significant impact on tuber yields and quality. For example, reports from the Ivory Coast and western Nigeria have described average annual yield losses of 30–50% due to virus infections. Additional constraints to increase yam production and productivity are the unavailability and associated high costs of high-quality virus-free (termed ‘clean’) seed yams and the absence of a formal seed yam certification system.
Infections by potyviruses (genus Potyvirus; family Potyviridae) cause the most economically important diseases of yams and are widespread across the numerous yam growing regions worldwide. The best described potyvirus infecting yam is Yam mosaic virus (YMV), known to infect several species of yam, particularly the most widely cultivated D. rotundata, D. cayenensis and D. alata, while the second most described yam potyvirus, Yam mild mosaic virus (YMMV) is more commonly found on D. alata.
Historic data suggest a strong influence of human activity on the dissemination of viruses through trade and transportation of infected plant material [1,,,]. Applying full phytosanitary surveillance in plant quarantine and certification facilities is unrealistic due to high costs associated with increasing inspection rates. Therefore, there is an urgent need to develop improved detection methods for yam viruses to help make timely decisions on the health status of yam planting material. Several serological and PCR-based methods have been developed and applied for the detection of YMV and YMMV. Some considerations must be taken into account when choosing the detection method, such as sensitivity, specificity, cost and time to obtain results. Although PCR-based assays are often preferred for their sensitivity and specificity, they require specific technical expertise and sophisticated equipment. In addition, PCR-based methods usually require the extraction of high-quality DNA/RNA from the sample material, which is time-consuming, generally involves hazardous chemicals and cannot be done in the field.
An isothermal amplification method called recombinase polymerase amplification (RPA), overcomes the disadvantages of PCR-based assays as it reduces the need for expensive apparatus to control reaction temperature as well as providing rapid and reliable results with sensitivity and specificity comparable to conventional PCR assays. RPA has been successfully used in the detection of several animal [,,], human and plant [,,] pathogens. Recently, we developed a sensitive and robust reverse transcription-recombinase polymerase amplification (RT-RPA) assay for the specific detection of YMV. To develop further this promising diagnostic method and bring it closer to a format suitable for on-site detection of the two most important yam potyviruses, the time-consuming RNA purification step needs to be removed. In this study, we report a RT-RPA method for the detection of YMV and YMMV directly from the crude extract of infected plant material using a simple and inexpensive extraction method. Yam and potyviruses form an excellent combination as a general working model of wide applicability to other plant virus systems as: (1) potyviruses comprise the largest genus of plant RNA viruses causing significant losses in different crops worldwide and (2) yams represent particularly recalcitrant leaf tissue that contain high levels of PCR-inhibitory compounds such as polyphenols and polysaccharides, and hence the technique should be suitable for application to a diverse range of plant species.
The method developed in this study, termed ‘Direct RT-RPA’, thus has the potential to be adapted to any recalcitrant plant species and be used to obtain rapid responses in certification laboratories, reducing costs by minimising quarantine time. In addition, this method will specifically strengthen current efforts in West Africa to multiply and deliver ‘clean’ certified yam planting material to smallholder farmers and thereby improve food and income security.
The presence of YMV and YMMV was confirmed by RT-PCR using the primer pairs YMV-F/-R and YMMV-F/-R, which amplify a 586 bp and a 249 bp region comprising the coat protein (CP) gene and the 3′ UTR region of the YMV and YMMV genomes, respectively. An assay for detection of the yam actin gene was used as an internal control as described by Silva et al.. RT-PCR amplifications were set up in 20 μL reactions containing either 40 ng RNA or 2 μL of crude extract, 0.2 μM of each primer, 0.25 mM of each dNTP, 2.5 U AMV Reverse Transcriptase (Promega, Southampton, UK), 1 U DreamTaq DNA polymerase and 1x DreamTaq Green buffer (Thermo Scientific, Loughborough, UK) containing 2 mM MgCl2. The following cycle conditions were used: 50 °C for 10 min for reverse transcription, 95 °C for 4.5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and one final extension of 72 °C for 5 min. Amplification products were analysed by agarose gel electrophoresis using 1.5% (w/v) agarose gels containing 1x RedSafe nucleic acid stain (iNtRON Biotechnology, Seongnam, South Korea) in 0.5x Tris-Boric acid-EDTA (TBE) buffer.
The many uses and application of tannins described above need to be put into perspective with regards to possible further advances, existing drawbacks, and future potential. Of all the applications described above, leather tanning is still the main industrial use of vegetable tannins. While their use for this application has been progressively decreasing and limited to heavy duty leathers, as displaced for finer applications by chrome tanning, the real or perceived toxicity of chrome has spurred considerable research on feasible alternatives. Not all of these have gone in the direction of vegetable tanning, as synthetic resins such as melamine-based ones have been considered. However, vegetable tanning has regained some interest for soft leathers either in combination with oils or with synthetic resins. Although further development in this application for vegetable tannins cannot be considered as static, nonetheless the potential for future expansion is rather limited.
The second most important application of vegetable tannins is in wood adhesives. The strong shift away from synthetic resins based on formaldehyde has favored the interest in the use of tannins as well as of other natural raw materials for this application. The tannin adhesive technology is definitely more advanced, and more used, than other bio-sourced materials, having proved itself industrially in several countries over a number of decades. Its drawback at present is that their present supply is limited. The potential world supply of tannins is really huge, what is lacking, however, is a marked increase in the factories extracting them. A few new tannin extraction factories have been created in the last decade, but competition with other bio-sourced materials already industrially available either as waste or obtained by other already existing production sources is rather intense at present.
Medical and pharmaceutical applications are one of the more interesting and active fields of research at present for the evaluation of vegetable tannins. While some pharmaceutical applications already exist, for further progress the results being developed in this field need to be proven in vivo, this being an important phase of development. It is difficult to say for what specific pharmaceutical uses tannin might be successfully adopted. The main drawback here is that the balance of properties favorable and unfavorable to each application have to be evaluated. It is nonetheless an application that is likely to further flourish in the future.
More established and fully functional is the use of tannins in the beverage industry, be it wine, beer, or fruit juices. Their use will expand with the expansion of these markets due to the expansion of the population, but not for different applications in the field.
Tannin based foams, be it the more developed phenolic–furanic type, isocyanate-derived polyurethanes, or the newer, less developed non-isocyanate poly(hydroxy)urethanes, is a fast moving research field for thermal and/or acoustic insulation, for hydroponics, and a number of other applications. A considerable amount of research is still going on in this field, and some industrial trials too, but all this has not as yet materialized in an industrial application.
Tannin-based antipollution flocculants and corrosion inhibitors have been developed quite a long time ago, in the late 1960′s and early 1970′s and used industrially for some time. After a period of having practically disappeared from the market they are regaining favor, both in research and industrially, due to the interest in substituting bio-sourced material for somewhat toxic or oil derived synthetic materials.
As regards the other applications, foundry sand binders is used but it is unlikely to increase in market share due to the competition of other more performant materials. The same is valid for drilling fluids. Corrugated cardboard adhesives are used in a few developing countries for a niche industrial market, namely the moisture proofing of starch-bonded corrugated cardboard boxes for fruit exports having to pass through humid conditions, such as in the tropics.
Very new is the development of adhesives to bind teflon to steel and aluminum. While the technology exists and has proven itself it seems that the moment for a bio-sourced adhesive for this application has not yet come, although patents on the subject have been created. The writer supposes that this technology might eventually be used industrially once environmental protection awareness becomes stronger, and stricter environmental protection rules may force its application.
The situation is the same for the hard plastics used as matrices for abrasive angle grinders, discs, and automotive brake pads. Only time will tell if these developments, some of them patented, will ever reach industrial use.
Epoxy resins based on tannins have been developed by a few groups, one of which is in direct contact with interested industries. It is likely that some industrial development will eventually arise from this line of research, although none is known to date.
All the other applications are all in the purely experimental phase, and it is difficult to see if they will ever develop further or not.
Finally, ferric inks, the main source of writing inks for several past centuries, is definitely out of interest as more performant materials exist today, and no further interest in them is apparent.
There are a number of detailed reviews on the use of tannins for wood adhesives. The reader is referred to these detailed studies. However, here existing technologies and industrial use of wood tannin adhesives are presented.
As extensive studies already exist, and this application of tannin is now the second most important after leather manufacturing, only a few of the main achievements of tannin-based adhesives for wood products will be highlighted. (1) The development, optimization, and industrialization of non-fortified but chemically modified thermosetting tannins for particleboard, other particle products, and plywood. (2) The technology for rapidly pressing tannin adhesives for particle board, which is also industrial. (3) The development and industrialization of tannin–urea–formaldehyde adhesives for plywood and in particular as impregnators for corrugated board starch binders. (4) The development and industrialization of cold-setting tannin–resorcinol–formaldehyde adhesives for glulam and fingerjointing. (5) The large-scale development and industrialization of fast-setting “honeymoon” separate application cold-setting adhesives for tannin-bonded glulam and fingerjoints (Figure 4).
(6) The development and industrialization of zinc salts to accelerate the hardening of non-fortified tannin adhesives for plywood. (7) Successful formulation, development, and industrialization in Chile of pine bark tannin adhesives for particle boards and for glulam and fingerjointing. (8) The development of isocyanate/tannin copolymers as difficult-to-bond hardwood adhesives and for plywood and other applications. (9) The development of very low formaldehyde tannin adhesives for particle boards and other wood panels. (10) The development of the use of hardeners other than formaldehyde for thermosetting tannin adhesives. (11) The discovery and development of self-condensation of tannin for adhesives.
All industrialized technologies today are based on paraformaldehyde or hexamethylene tetramine (hexamine). The latter is much more user and environmentally friendly.
As regards wood adhesives, a number of experimental improvements have been studied, dictated by the new environment in which wood adhesives must operate. First of all, the relative scarcity of tannins produced in the world, compared to the tonnage of synthetic adhesives used in the panel industry, has led to a great deal of research on the extension of the tannin resource in order to have larger tonnage. As the potential material for tannin extraction shows that millions of tons of this material can be extracted each year worldwide, some companies have started to build additional extraction plants. This movement is still relatively small, but it is ongoing. The second approach, to extend the tannin with another abundant and natural material, has led to the preparation of adhesives based on in situ copolymers of tannins and lignin or copolymers of tannin and protein or soy flour, and the use of tannin–furfuryl alcohol adhesive formulations, furfuryl alcohol being also a bio-based material.
The second new constraint is the demand of most companies to eliminate formaldehyde emissions from tannin adhesive. This quest has taken two approaches: (1) total elimination of formaldehyde by substituting it with aldehydes, which are less or non-toxic, and non-volatile, such as glyoxal, glutaraldehyde, or vanillin, the latter giving a fully bio-based tannin adhesive, and even aldehydes generated by the action of sodium periodate on glucose, sucrose and even oligomeric carbohydrates, (2) the use of non-aldehyde hardeners such as trishydroxymethylnitromethane and trishydroxymethylaminomethane or even by combination with furfuryl alcohol, the latter functioning both as a hardener and a contributor to a tannin/furan copolymer. (3) The use of hexamine with the formation of –CH2–NH–CH2– bridges between the tannin molecules, where the secondary amine is capable of absorbing any emission of formaldehyde from the heating of the wood itself or any other emission of formaldehyde to produce truly zero-formaldehyde emission panels. (4) Lastly, the hardening of the tannins by autocondensation without the addition of a hardener, autocondensation catalysed by the wood substrate itself in the case of fast-acting procyanidin tannins, such as pine bark tannins, and for slower tannins by addition of silica or silicate or other accelerators allowing the preparation of wood particleboard of indoor quality.
The present report has adhered to systematic review guidelines. The search of each of the different parts in PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) identified a total of 214 hits from 1976 to 2017.
The maintenance and care of experimental animals was carried out in accordance with the European Directive 2010/63/EU and Czech law (246/1992 and 359/2012) for biomedical research involving animals. Experiments have been performed under legal consent of the Expert Commission of the Section of Biology, Faculty of Science, Charles University in Prague and the Ministry of Education, Youth and Sports of the Czech Republic (ref. no. MSMT-31114/2013-9).
There are several ways by which therapeutic compounds interfere with viral replication. The antiviral effects can either be through prevention of viral attachment to host cell, binding to enzymes responsible for transcription, and prevention of cleavage of viral particles. Viruses mutate over time and develop resistance to antiviral drugs and therapeutic compounds. Thus, there is a need to discover and develop antiviral agents that do not become ineffective over time owing to development of resistance by the virus. But the pipeline of new drugs is drying up. There would be a tremendous benefit by integrating combinations of modern drugs with traditional medicinal plant extracts that have been used as folk medicine to broaden the curing spectrum via generating synergistic effects.
Traditional medicinal trees are evergreen, abundant and available year round in tropical regions. Local communities used various parts of these trees in their traditional practice because of their high nutritive values but yet some of their detailed medicinal properties remain unknown. The plant studied, Garcinia parvifolia produces cherry-like fruit which is locally known as “asam kandis” or “asam kundong”, whilst the young leaves are sometimes eaten as a vegetable. The leaf extracts of this plant were screened against pseudorabies virus (PrV). It is a broad host range herpesvirus, causes fatal encephalitis in a wide variety of animal species except its natural host, the adult pig [4–7]. Since PrV is not a human pathogen, it is safe to be used in a laboratory set-up. The virus can easily be grown in the laboratory thus it is practical and convenient to be used in the screening and development of antiviral drugs or compounds.
G. parvifolia which belongs to the family of Clusiaceae (Guttiferae), is native in tropical and subtropical countries of South East Asia such as here in Malaysia, Thailand, Brunei, and Indonesia [8, 9]. Garcinia is known to produce xanthones and benzophenones [9, 10] and many of these compounds show interesting biological activities including anti-human immunodeficiency virus activity [9, 10]. There are at least 300 distinct Garcinia species and many contains bioactive compounds to include flavonoids, xanthones, triterpernoids, and benzophenones with beneficial biological activities [11–14]. The crude extracts of some parts of G. parvifolia have shown antiplasmodial, antioxidant, cytotoxic and antibacterial activities. However, the antiviral properties of the G. parvifolia extract are not known. Since G. parvifolia has rather similar properties with other Garcinia sp, it potentially has antiviral activities and hence is of great interest to test in the current study. In this study, their leaf extracts were obtained by using either ethyl acetate, ethanol, or hexane and screened for the efficiency to inhibit PRV.
Bornaviruses are enveloped, 80 to 100 nm in diameter with a non-segmented genome of single-stranded negative sense RNA of around 8900 nucleotides in length, belonging to the order Mononegavirales. Bornaviruses replicate in the nucleus of the nerve cells of various organs and establish persistent, non-cytolytic infections by exploiting the cellular splicing mechanisms to efficiently use its genome, organized into six open reading frames (ORFs) (Figure 1). Alternative transcription start and stop sites and splicing produce mRNAs that are translated to produce the viral-encoded proteins (Figure 1). The first transcription unit contains an ORF for the nucleoprotein (N), the second transcription unit contains two overlapping ORFs for the phosphoprotein (P), and the X protein (X) (Figure 1). The third transcription unit is spliced differently, and also has different transcription initiation and termination signals, enabling polymerase read-through during transcription, which results in expression of the matrix protein (M), the glycoprotein (G), and the RNA-dependent RNA-polymerase (L) (Figure 1). The P and X ORFs overlap; as well as, the M and G ORFs (Figure 1). The immunogenicity of phosphoprotein, as well as, the degree of conservation of the phosphoprotein and its gene, within and between bornavirus species, make them good candidates as universal targets in laboratory diagnosis. Once the family Bornaviridae is expanding speedily, producing knowledge about highly conserved regions within phosphoprotein will be useful for the development of sensitive laboratory diagnostic tools. So far, genus Carbovirus, Cultervirus and Orthobornavirus have been identified, which comprise 11 species. From those, 10 species are associated with the development of severe neurological and/or gastrointestinal disease and death of its hosts [5–15] (Figure 2). The disease has been reported in humans, several species of pets, production and wild animals [5–15]. Namely, two species infect mammals (Mammalian 1 to 2 orthobornavirus), five infect birds (Passeriform 1 to 2 orthobornavirus, Psittaciform 1 to 2 orthobornavirus and Waterbird 1 orthobornavirus) and three infect reptiles (Queensland carbovirus, Southwest carbovirus and Elapid 1 orthobornavirus) (Figure 2). However, some Mammalian 1 orthobornavirus showed the ability to also infect farmed ostriches and wild birds (mallards and jackdaws). Wild birds are hosts of avian bornaviruses (e.g. strains of Waterbird 1 orthobornavirus and Psittaciform 1 orthobornavirus) [13–15,17] and mammalian bornaviruses (e.g. genotypes of Mammalian 1 orthobornavirus). Therefore, co-infection may play a role in the emergence of new pathogenic and zoonotic bornavirus species; once X and P proteins of PaBV-4 look like to have different ancestors.
In Psittaciformes, parrot bornavirus 1 to 8 (PaBV-1 to 8) can cause proventricular dilatation disease (PDD), characterized by a flaccid and distended proventriculus impacted with feed, as a result of the inability of seeds’ digestion (however throughout the gastrointestinal tract can occur variable distention). Psittaciformes can also show uncoordinated movements, postural disorders, apathy, blindness, and behavioural disorders, such as loss of appetite and self-mutilation (resulting from lesions in the central nervous system). Within captive birds, the virus has become relevant for Psittaciformes housed in reserves, in breeding projects of rare species, in private collections and zoos, because of the severe effects it may cause for bird welfare, economy, and biodiversity levels. Analyses of molecular epidemiology suggested that a world trade of psittacines without biosafety measures has been carried out. There is, to the best of our knowledge, no publications identifying and characterizing the avian bornaviruses infecting pet parrots in Portugal. Moreover, there are still unresolved questions on the epidemiology of bornaviruses, such as the role of waterbirds in the emergence and dissemination of new pathogenic and zoonotic species, and the localization of highly conserved regions within P gene inter- and intra-species of bornaviruses.
The aim of this study was to identify and phylogenetically characterize the etiologic agent associated with clinical signs and necropsy findings consistent with avian bornavirus infection in two pet parrots in Portugal, as well as to produce molecular epidemiologic knowledge on bornaviruses.
AIDS – Acquired Immunodeficiency Syndrome
CV – Coefficient of variation
HIV – Human Immunodeficiency Virus
pdf – Probability density function
SARS – Severe Acute Respiratory Syndrome
vCJD – variant Creutzfeldt-Jakob disease
Rapid, sensitive and specific detection of plant pathogens is an important aspect of disease management. Polymerase chain reaction (PCR) has become one of the most commonly-used nucleic acid based methods for the detection of plant pathogens due to its speed, specificity and sensitivity. PCR and reverse-transcriptase PCR (RT-PCR) can be designed to detect a narrow or broad range of targets through the use of specific or degenerate primers and amplified products can be viewed in electrophoretic gels, sequenced directly, and/or cloned. [2–4]. The disadvantages of PCR/RT-PCR for pathogen detection are the dependence on a thermocycler, inhibitory effects of co-extracted host plant inhibitors on amplification, and the time investment per sample [5–7]. While PCR/RT-PCR assays are used in the detection of plant viruses in research, the cost and time required are too great for many plant disease clinics. Real-time PCR (qPCR), reduces the time required for detection compared to PCR/RT-PCR, but has high start-up and instrument maintenance costs which has discouraged its adoption by many plant disease clinics.
Some of the disadvantages of PCR/RT-PCR have been avoided by the use of loop mediated isothermal amplification (LAMP) assays, which have been developed for over 100 animal and plant pathogens [8–10]. LAMP is an isothermal reaction that has a sensitivity and specificity similar to that of PCR. LAMP does not require a thermocycler, uses a shorter amplification time than PCR, but like PCR/RT-PCR requires a RNA or DNA template mostly free of host contaminants. However, downstream applications of LAMP products such as direct sequencing, cloning and restriction analysis are more complicated than PCR/RT-PCR, as LAMP generates multimeric products.
Another type of isothermal amplification methodology is Recombinase Polymerase Amplification (RPA). RPA was first developed in 2006 and relies on the extension of primers induced by recombination proteins. DNA binding proteins (gp32 single-strand DNA binding protein and two ATP-dependent recombinases, usvX and usvY) bind the primers and scan for the homologous sequence (target). The primers recombine with the target, and a mesophilic polymerase (Bacillus subtilis DNA polymerase I) extend the 3’ end of the invading primer using the opposite strand as a template. RPA reactions have been performed at constant low temperatures (25 °C to 42 °C), achieving amplification in as little as 15 min. PCR purification columns remove DNA-binding proteins from the amplicons to allow visualization of results. The use of columns adds to the expense and time required to complete the assay. The most commonly used methods to obtain results of RPA assays are visualization of amplified DNA by gel electrophoresis or amplicon sequencing although alternatives such as fluorescence and/or hybridization have been reported [12–14]. In addition to end-point detection of DNA targets, RPA formats have been developed for detection of RNA templates (RT-RPA), target quantification and chip-based detection [13–16], which demonstrate the flexibility of this type of assay for rapid pathogen diagnostics. RPA is widely used in the detection of animal and human pathogens [13, 17]. Its use in plant pathogen detection has been limited to four plant pathogens: two viruses, (Little cherry virus 2 (LChV2) and Plum pox virus (PPV)), a bacteria (Candidatus Liberibacter asiaticus (HLB - Las)) and a fungus (Fusarium oxysporum f.sp. vasinfectum) ([18, 19] AgDia, Inc. Elkhart, IN).
There is a real need in diagnostic laboratories for technology that provides rapid and affordable diagnosis of viruses and has the flexibility for downstream applications or applied disease management. This is especially true for emerging viruses such as species in the genus Begomovirus (Family Geminiviridae) [20–23]. Species in the genus have been emerging over the last three decades to become plant pathogens that threaten crop production in tropical and subtropical regions around the world. One of these viruses, Tomato yellow leaf curl virus (TYLCV), emerged in the eastern Mediterranean in the 1960s. TYLCV has since spread via the plant trade to virtually all tomato production areas wherever its vector, Bemisia tabaci species complex, is endemic. Current methods for the detection of TYLCV and other begomoviruses are primarily PCR, but other methods include enzyme-linked immunosorbent assay (ELISA), lateral flow immunochromatographic assays, dot blot hybridization, rolling circle amplification (RCA) and LAMP [25–33].
This manuscript describes the development and diagnostic laboratory evaluation of RPA for the detection of three begomoviruses. We modified RPA assays to reduce expense and time, and compared these with RPA assays using manufacture’s recommended protocols. We then compared RPA with the better known PCR in terms of sensitivity, specificity and adaptation to downstream applications in the detection of TYLCV.
Amplicons could be clearly visualized in agarose gels after incubation at 65 °C or 95 °C for 10 min, or by the addition of either 5 or 10 % SDS in the loading buffer (Fig. 2b). Use of formamide gave unacceptable results. Amplicons treated with heat or SDS resulted in thicker bands in the agarose gels than those cleaned with PCR purification columns. Both heat and SDS treatment caused some lower molecular weight (LMW) bands to appear at 100–150 bp in gels (Figs. 1 and 2). These LMW bands were not present in those samples cleaned with PCR purification columns (Fig. 2b, lane 2). LMW bands were more obvious in those amplifications using crude extracts (Fig. 2b, lanes 3–8) than purified DNA (Fig. 1). LMW bands did not present a problem for interpretation because the amplicon bands (325–464 bp) were distinctly larger.
The present study revisited previous works concerned with models of the incubation period of acute infectious diseases. In particular, the following were highlighted: (i) the earliest modeling effort conducted using incomplete data of a pandemic influenza, (ii) the explicit distribution of the incubation period, (iii) the application of a lognormal assumption to estimations of the time of exposure during a point source outbreak, and (iv) the validity of assuming lognormal distribution for the incubation period. Although it was not highlighted in the present paper, Norman T. J. Bailey also formed a framework using a chain binomial model, which is useful for household transmission data. This method estimates the incubation period as the sum of the mean latent period, which follows normal distribution, and a further fixed infectious period; however, the estimated period does not precisely imply the incubation period, but rather is closer to what is presently referred to as the serial interval. That is, the incubation period that can be extracted from household transmission data remains to be clarified.
The lessons that can be learnt from the presented discussion are as follows: (I) although it is historically remarkable that the incubation period of pandemic influenza was assessed based on an explicit understanding of an unknown time of exposure, the assumed periods of exposure were too long and equal probability of exposure was assumed for each possible date. Well-defined short periods of exposure are needed to decipher the incubation period distribution using appropriate statistical methods. Taking this point into account will be critically important in estimating the incubation period of newly emerging diseases in the future. (II) The epidemiologic usefulness of the lognormal assumption was highlighted with respect to the basic characteristics of lognormal distribution, but this assumption is likely to remain unwarrantable until details of disease mechanisms are fully clarified; thus, this assumption may be merely an approximation of the right-skewed distribution. For example, considering the mechanisms of disease development, the lognormal assumption does not hold for HIV/AIDS and prion diseases. However, this limitation of the lognormal assumption does not imply that such approximation of the incubation period distribution is meaningless. Rather, it suggests that when parametric models are assumed, it is at least necessary to compare the goodness-of-fit for several distributions in order to overcome some of the uncertainty. Various datasets on the same disease would also help assess the uncertainty. Further, it would be informative if the determinants could be clarified even by simple stratifications (e.g., with respect to sex, age and genetic factors). Ideally, assumptions in the future should be supported by a detailed understanding of the underlying disease mechanisms provided by observations of within-host dynamics. Since the incubation period of infectious diseases is directly relevant to prevention and control, and because such knowledge can enhance our theoretical understanding of the spread of disease, further clarifications of the above points are deemed necessary.
The avian schistosome Trichobilharzia regenti Horák, Kolářová et Dvořák, 1998 is a neurotropic nasal parasite of waterfowl, especially ducks. Although birds serve as suitable definitive hosts, cercariae of T. regenti have been identified as the aetiological agent of cercarial dermatitis in man. Experimental infections of mice showed that cercariae readily penetrate also mammalian skin and transform to the subsequent stage, schistosomulum, which is able to persist in mice for several days. Schistosomula of the species migrate through peripheral nerves to the spinal cord and brain of both bird and mammalian hosts, and feed on the nervous tissue; damage to the central nervous system (CNS) can give rise to various neuromotor disorders [2–4]. In mammals, however, T. regenti is unable to mature and complete its life cycle [2, 5].
Repeated infections of mammals including man lead to an inflammatory reaction known as cercarial dermatitis, which develops after destruction of cercariae in the skin. In sensitive individuals, intensive itching may be accompanied by fever and local lymph node swelling [6–8].
Antibody response of bird hosts against avian schistosome antigens has not yet been studied in detail. Just some antigenic structures recognized by antibodies from infected ducks or mice were shown by immunohistochemistry. In particular, cercarial penetration glands as well as glycocalyx of T. regenti cercariae contained antigens triggering immune response. The latter reacted also with antibodies from human patients with known history of cercarial dermatitis and from mice experimentally infected with T. regenti; a cross-reaction of antibodies has also been observed with heterologous antigens of the related species T. szidati and the human blood fluke Schistosoma mansoni [9, 10]. The sera of compatible hosts recognised schistosomular and adult gut associated antigens. Antibodies of the classes IgG and IgE from sera of repeatedly infected mice and patients with confirmed cercarial dermatitis recognized 25 kDa and 34 kDa protein bands on Western blots of T. regenti cercarial homogenates. These antigens were identified by mass spectrometry as triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. Indeed, host antibody response is also directed towards other developmental stages that are in contact with the host. In addition to the antigens in glycocalyx, penetration glands and tegument of cercariae, also the tegument of schistosomula and adults is recognized by antibodies from infected mice.
Here we show the antibody response during the infection by T. regenti in specific hosts (ducks). Bird humoral immunity has some specifics compared to mammals. Since the divergence of birds and mammals ca. 300 million years ago, some differences in the antibody responses of those two vertebrate groups have evolved. The most significant departures from mammals include a partly different set of antibody classes, lower variability of Ig binding sites and maturation of B lymphocytes in a specialized immune organ, bursa of Fabricius. Also, mammals generate new antigen-binding sites throughout their life; in contrast, bird antibody diversity is generated only during a brief period of the embryonic development.
There are 3 classes of immunoglobulins in birds: IgA, IgM and IgY. Avian IgA has a similar function and structure as in mammals - it is predominant in body secretions and participates in mucosal immunity. IgM is the first class of immunoglobulins being expressed during the embryonic development. It is the predominant isotype produced after initial exposure to a new antigen in primary antibody response. Production of IgY is stimulated in subsequent stages of infection. The IgY molecule is an evolutionary precursor of mammalian antibody classes IgG and IgE [16, 17]. There are two isoforms of IgY in ducks: complete IgY and a smaller version of IgY, called IgYΔFc, which lacks the Fc part of the heavy chain. IgYΔFc is produced during the alternate splicing of mRNA of the heavy chain. This truncated form cannot mediate effector cell functions, therefore it is not very clear why it is produced - maybe it can participate in agglutination of antigenic particles.
We aimed to describe the dynamics of antibody response of experimentally infected domestic ducks and wild mallards Anas platyrhynchos to various antigens of Trichobilharzia regenti, and to identify particular antigens with diagnostic potential. So far, diagnosis at necropsy of infections by schistosomes in birds has been the method of choice. In living hosts, the adult worms of T. regenti lay the eggs in the nasal mucosa of birds and, therefore, no eggs are released with faeces to enable coprological examination [8, 19]. As a consequence, immunological methods seem to be a useful alternative in immunoecological studies concerning the influence of pathogens on birds. In addition to the infections of birds, highly antigenic proteins (subsequently produced in a recombinant form) may potentially be used for confirmation of cercarial dermatitis in human patients.
How to cite this article: Plazzotta, G. et al. Effects of memory on the shapes of simple outbreak trees. Sci. Rep.
6, 21159; doi: 10.1038/srep21159 (2016).
Bats were deeply anesthetized and maintained with 3% isoflurane and an oxygen flow rate of 1.5 L/min. Deep pain was assessed by firmly pinching skin and toes with forceps and assessed for any response. A thoracotomy was then performed with sterile standard scissors to puncture through the skin, muscle and diaphragm just caudal to the sternum and cut through the wall of the chest cavity caudally to cranially—removing and preventing negative pressure from building in the thorax.
Cardiac blood was collected with a 21 gauge sterile needle inserted into the apex of the heart. A maximum blood volume of between 1 and 1.5mls is collected in a syringe and transferred to a red top tube (RTT). RTTs sat at room temperature for one hour to allow a clot to form and then centrifuged at 1000 x g for 10 min at room temperature. Serum was removed from the clot, placed in a new microcentrifuge tube and stored at -20°C.
Serum from bats at 2 and 5 dpi were used to assess for viremia. Serum from 10 dpi and the 28 dpi pilot study bats were used to determine antibody titers. Because blood draws yield a small volume of blood (50 μl whole blood for a non-terminal blood draw, 500 μl whole blood for terminal blood draw) it was necessary to prioritize samples to optimize data retrieved. In order to assay the serum for viral RNA and perform serology, earlier time points were used to assess for viremia and later time points for seroconversion. Along with sample partitioning for data maximization, the small blood volume led to concerns that there would be an undetectably small viral load. To circumvent this issue, neat serum and 1:10 diluted serum were inoculated onto Vero cells to amplify any virus that may have been present at low levels. One blind passage on Vero cells was done and cell supernatants assayed by qRT-PCR. The remaining serum from three of the four bats was assayed directly for ZIKV RNA.
Necropsies were performed immediately after euthanasia. Bats were assessed for gross pathology. The following tissues were collected for both experiments: heart, lung, liver, spleen, kidney, urinary bladder, prostate, testes, and brain. A portion of tissues were collected and kept at -80°C for RNA extraction, and a portion placed in 10% buffered formalin for histology at a 1:10 weight to volume ratio for histology.
For a negative control animal a male bat was trapped from the colony and euthanized under the same protocol as the experimental infection bats.
Here we analyze the behavioral decision model used by the agents to decide whether or not to engage in prophylactic behavior. In particular, we are interested in identifying the level of disease prevalence above which agents would switch behavior, i.e., a switching point. A switching point is defined as the proportion of infectious agents beyond which it would be advantageous for an agent to switch from non-prophylactic to prophylactic behavior or vice-versa. We can visualize switching points by plotting the expected utility for the susceptible and prophylactic states as a function of the proportion of infectious agents. A switching point is where the expected utilities cross, if they cross. Figure 2A illustrates the situation in which the utilities do not cross, thus there is no switching point. Figure 2B illustrates the situation in which there is a single switching point; below the switching point the susceptible state has the higher utility, whereas above that point the prophylactic state has the higher utility. Figure 2C shows the situation in which the utilities cross twice, thus there are two switching points; between the switching points the prophylactic state has the higher utility, whereas the susceptible state has higher utility on the margins.
Figure 2D shows a heat map of switching points for Disease 1 (see Fig. S1 for a heat map for Disease 2). The figure is divided into three regions—A, B, and C—that correspond to the three different utility situations illustrated in Figs. 2A, 2B, and 2C respectively. Region A corresponds to the situation in which agents never engage in prophylactic behavior because the utility of being in the susceptible state is never less than the prophylactic state regardless of disease prevalence (Fig. 2A). This situation occurs for low protection efficacy or short planning horizons. In the case of low protection efficacy, agents do not have an incentive to adopt prophylactic behavior because they expect to get infected regardless of their behavior. Thus, their best strategy is to become infected and then recover in order to collect the recovered payoff as quickly as possible (i.e., “get it over with”). In the case of short planning horizons, the relative contributions of the expected times of being in the susceptible or prophylactic state dominate the utility calculations, as shown in Fig. 3. The figure illustrates how, when the planning horizon is short, the expected percentage of time spent in the susceptible or prophylactic states are much greater than the expected percentage of time spent in the infectious or recovered state. Given that the susceptible payoff is greater than the prophylactic payoff, agents never adopt prophylactic behavior. The figure also shows how increasing the planning horizon changes the distribution of time spent in each state, which reduces the influence of the difference between the susceptible and prophylactic payoffs on behavioral decision.
Returning our focus to Fig. 2D, region B corresponds to the situation in which agents will adopt non-prophylactic or prophylactic behavior depending on the prevalence of the disease (Fig. 2B). If the disease prevalence is smaller than the switching point, the agent opts for the susceptible behavior; otherwise it adopts the prophylactic behavior. Region C corresponds to the situation in which two switching points exist instead of a single one (Fig. 2C). When the proportion of infectious agents is between these switching points, agents adopt prophylactic behavior, while values outside this range drives agents to adopt non-prophylactic behavior. This situation is of particular interest because it shows that the adoption of prophylactic behavior is not always monotonically associated with the prevalence of the disease.
The utility calculations that agents use to decide whether to adopt a behavior are complex (see Eqs. (9) and (10)); an exhaustive exploration of the parameter space is not undertaken here. We instead investigate several paradigm cases related to the payoff ordering. We assume that the payoff for the infectious state (uI) relies upon biological parameters of the disease and always corresponds to the lowest payoff, thus we need only consider the relationship between the other three payoffs. In particular, we are interested in looking at situations where the recovery payoff ranges from complete recovery (case 1) to less than the prophylactic state (case 4).
Because our model consists of a constant population of N agents (i.e., no mortality), cases in which uS > uR represent situations where an individual suffers chronic harm from the disease.
Figure 4 displays the switching point heat maps for these different ordering cases of Disease 1 (Figs. 4A–4D) and Disease 2 (Figs. 4E–4H). The most dramatic difference between the two diseases is that changing the payoff for being recovered has a large effect on the agents’ behavioral change in the cases of Disease 2, but little effect in the case of Disease 1. The reason for this has to do with the biological parameters of the model, in particular, the disease recovery time for Disease 1 is large (Recovery Time = 65), but small for Disease 2 (Recovery Time = 8). Consequently, an agent expects to spend more time in the recovered state when considering Disease 2 than Disease 1. When weighting these expected times with different payoffs for calculating the utilities, there will be less variation in Disease 1 compared with Disease 2.
The effects of progressively reducing the recovered payoff are more evident for Disease 2. Reducing the recovered payoff means that lower levels of prevalence will be sufficient for agents to change their behavior. In the case of equal value for recovered and susceptible payoffs, agents consider changing behavior only in narrow parameter range of protection efficacy and planning horizon values (Fig. 4E). Progressively reducing the recovered payoff, i.e., moving from case 1 (Fig. 4E) to case 4 (Fig. 4H), the range of parameter values that induce agents to change their behavior expands (i.e., there are large areas of the parameter space in which the agents would consider changing behavior) and the disease prevalence necessary for such change to occur decreases (i.e., gradual change of the color towards blue).
In addition to this numerical analysis, we have also obtained analytical results for case 2 (payoff ordering uS > uR > uP > uI) that are available in Sec. S4 Supplemental Information 1.
We turn now to understand how the above conditions for behavioral change may influence epidemic dynamics. Here we are particularly interested in analyzing the effects of the planning horizon H and the decision frequency δ on the dynamics of Disease 1 and Disease 2. Because we assume that the interactions among the population are well-mixed, we execute the simulations using the ODE model for a population of 100, 000 agents (initially 99, 900 agents in the susceptible state and 100 in the infectious state) with a decision frequency of δ = 0.01.
Figure 5 shows the effects of different planning horizons on the epidemic dynamics for both Disease 1 (Fig. 5A) and Disease 2 (Fig. 5B). For short planning horizons (i.e., H = 1), agents do not ever consider changing behavior in either disease. This corresponds to the situation in Region A in Fig. 2A in which being prophylactic is never worth the cost, hence the epidemic dynamics are not affected. Similarly, in the cases of H = 30 for Disease 1 and H = 45 for Disease 2, we notice that neither of the two epidemic dynamics change. The dynamics are not affected because the disease prevalence does not reach the switching point (the switching points are indicated by the dashed lines in Fig. 5).
In the cases of H = 45 and 90 for Disease 1 and H = 30 for Disease 2, however, agents change behavior and thereby affect epidemic dynamics. For Disease 1, the effect is characterized by the decrease on the epidemic peak size and a prolonged duration of the epidemic. Although the dynamics of Disease 2 are also affected, the effect is small because a lower portion of the population crosses the switching point.
In other cases, increasing the planning horizon further may cause agents to never contemplate a change in their behavior. This occurs, for example, when H = 90 for Disease 2. This means that agents willingly assume the risk of getting infected and then recover, which is intuitive given the short recovery time and mild severity of the disease.
To assess the effect of the frequency that agents make behavioral decisions on the epidemic dynamics, we fix the value of the planning horizon for Disease 1 (H = 90) and Disease 2 (H = 30), and vary the decision rate. Figure 6 shows the effects of different decision frequencies on the epidemic dynamics. This figure illustrates how increasing the decision frequency reduces the epidemic peak size while prolonging the epidemic. It additionally may generate multiple waves of infection for Disease 1. These waves are generated because raising the decision frequency means individuals react faster to an increase in prevalence and adopt the prophylactic behavior. This bends the trajectory of disease incidence downward, but the reduction in prevalence causes the pendulum to swing back and individuals return back to their non-prophylactic behavior, thus creating an environment for the resurgence of the epidemic.
For the pilot study, bats were visually monitored twice daily for fourteen days, and then monitored once a day for an additional fourteen days. For the time course study, bats were monitored twice a day throughout the experiment. For both studies, energy levels, behavior, ability to ambulate, respirations, presence of oral or nasal discharge, and fecal consistency were all assessed.
Vegetables and fruits are an important source for therapeutic products which can prevent, relieve or cure numerous illnesses as they are an important source of phytochemicals and other bioactive compounds. Reactive Oxygen Species (ROS) are implicated in a large number of illnesses, especially chronic ones. Nitrogenous species and free radicals start chain reactions which can favour the initiation and progression of many complications in diseases. Oxidative stress may be defined as an imbalance between ROS levels in the organism and the capacity of antioxidant mechanisms. A free radical is a species which contains one or more unpaired electrons, which makes them very reactive as they need another electron to fill the orbital and become more stable. Free radicals are formed in different ways: (i) many organic molecules (glyceraldehydes, adrenaline, l-dopa, dopamine, cysteine etc.) oxidize in the presence of O2 to form the superoxide radical; (ii) many of these radicals in vivo are produced by an incomplete transfer of electrons on O2 just before the terminal cytochrome oxidase step. The superoxide radical (O2•−), which is very active, is formed when a single electron is added to the ground state O2 molecule. Sometimes the presence of ROS in the organism is beneficial as they are used in the immune response to kill ingested or extra-cellular bacteria. Unfortunately, ROS are not limited to this action and they can also contribute to undesired effects as they induce oxidation processes. The term “antioxidant paradox” is often used to refer that ROS are implicated in several human diseases but there is no good evidence, in human population, that large doses of dietary antioxidants have always preventive or therapeutic effects. In addition, there are some environmental factors which contribute to the production of free radicals such as exposure to ultraviolet radiation, pollution and cigarette smoke. Nitrogen dioxide, one of the major oxidants in smog, is also found in cigarette smoke. Two free radicals have been found in tobacco smoke, the main radical NO•, found in tar, is capable of reducing oxygen to the superoxide radical.
The antioxidants family include a series of molecules with low oxidation potential which act by donating electrons to deactivate ROS and other free radicals that produce DNA damage and consequently can provoke tumorigenesis. Antioxidants scavenge free radicals through different mechanisms like Hydrogen Atom Transfer (HAT), Single Electron Transfer followed by Proton Transfer (SET or ET-PT) and the Sequential Proton Loss Electron Transfer (SPLET) mechanism. Each one of these mechanisms presents different kinetics. When the free radicals are generated in vivo, many antioxidants act in order to defend the organism from oxidative damage. In the organism there is a first line of defence made up of peroxidases and metal chelating proteins which serve as a preventive barrier as they inhibit the formation of ROS and free radicals by capturing metal ions, reducing hydroperoxides, hydrogen peroxide and quenching superoxide and singlet oxygen. A second line of defence is formed by vitamin C and vitamin E which scavenge radicals and so prevent any propagation reactions. The third line of defence of the organism repairs lipids, proteins, sugars and DNA with oxidative damage. This also includes proteases, lipases, DNA repair enzymes and transferases. The antioxidants used could be endogenous such as catalases, which transform H2O2 to O2 and H2O, and superoxide dismutases, which convert superoxide radical (O2•−) to H2O2 and O2, or likewise they could be exogenous, coming from one’s diet. Diet is very important as this provides the antioxidants that intervene in the second line of defence such as vitamin C and E and other antioxidants such as β-carotene, phenols including flavonoids and essential minerals, which participate in the formation of the antioxidant enzymes. Natural antioxidants are found, above all, in vegetables, herbs, berries, spices, tea, coffee and cocoa. For all these reasons, different epidemiological studies have reached the conclusion that consumption of these products, of vegetable origin, are associated with a lower risk of suffering chronic diseases as well as with lower mortality.
Likewise, antioxidants are a type of additive which is used in the food-processing industry with the aim of preventing an oxidizing deterioration of the lipids as well as preventing loss of nutrition values and the development of odours in the food. The antioxidants additives allowed in food industry may be synthetic or natural, although currently natural antioxidants are more readily acceptable than synthetic antioxidants. The most used synthetic antioxidants in the food processing industry are: butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), propyl gallate (PG) and di-tert-butylhydroquinone (TBHQ), all of which are phenolic synthetic antioxidants. However, since the late XX century the use of synthetic antioxidants additives has become restricted because of their possible toxic and carcinogenic effects. This question has caught the attention of both the scientific community as well as among the general public, and currently there is a lot of interest in developing methods that could provide information and means of isolating both individual antioxidants or those present in extracts coming from different natural sources. Thus, among the positive list of additives permitted by the EU can be found rosemary extract (E392) and different types of tocopherols (E306 an extract rich in tocopherols, E307 α-tocopherol, E308 γ-tocopherol, E309 δ-tocopherol). Given that rose hips are rich in vitamins, especially, vitamin C, as well as phenolic compounds, carotenoids, tocopherol, bioflavonoids, tannins, volatile oils and pectins, these pseudo-fruits could constitute an alternative source of antioxidants for the food industry as well as serving for therapeutic use.
Astroviruses (AstV) are non-enveloped, single-stranded positive-sense RNA viruses with an icosahedral virion structure, appearing as a star-like shape in electron microscopy (Caul & Appleton, 1982). The AstV genome is 6.2–7.8 kb in size and polyadenylated at the 3′ end. It presents at least three open reading frames (ORF): ORF1a, ORF1ab, and ORF2. ORF1a and ORF1ab encode nonstructural precursor proteins, nsp1a, and nsp1ab. The latter is translated via a ribosomal frameshift mechanism, where ORF1b is translated together with ORF1a (Marczinke et al., 1994). ORF2 encodes the capsid precursor protein, which is then intra- and extracellularly further processed to mature structural proteins (Willcocks et al., 1994).
According to the affected host class, two AstV genera were established: Mamastroviruses (MAstV) representing genotype species affecting mammalian species and Avastroviruses containing those viruses found in avian species. Due to the availability of high throughput next-generation-sequencing (NGS) technologies and the use of broadly reactive Pan-AstV RT-PCR protocols, there has been a remarkable increase in the number of AstV discovered in diverse species during the last years (Boujon, Koch & Seuberlich, 2017). AstV were first described in 1975 in a human stool sample (Appleton & Higgins, 1975; Madeley & Cosgrove, 1975). In humans, AstV are best known as a major source of outbreaks of gastroenteritis, especially in infants, young children and immunocompromised people (De Benedictis et al., 2011; Fischer, Pinho Dos Reis & Balkema-Buschmann, 2017). However, intestinal tissue infected with AstV shows only minor histological changes such as a mild intestinal inflammatory response (Sebire et al., 2004) and the knowledge on the pathogenesis of gastroenteric disease associated with AstV is still limited (Moser & Schultz-Cherry, 2005).
In 2010, AstV were found for the first time in association with encephalitis in a child with immunodeficiency (Quan et al., 2010). Thereafter, several novel AstV genotype species were detected in other human encephalitis cases (Brown et al., 2015; Lum et al., 2016). Encephalitis-associated AstV could be detected in stool samples, as well as in other body fluids, such as cerebrospinal fluid and plasma, suggesting that in these patients the infection spreads from the gastrointestinal tract to the brain (Cordey et al., 2016).
In animals, the state of knowledge about the tissue tropism of AstV is even more limited. Even though the presence of ovine astroviruses (OvAstV) in fecal sheep samples constituted the first report of AstV in animals (Snodgrass & Gray, 1977), still little is known about AstV infections in small ruminants, their transmission within and across species as well as their association with disease. In recent years a wide variety of mammalian domestic animal species were found positive for AstV in their feces; for example, cattle (Woode & Bridger, 1978), sheep (Snodgrass & Gray, 1977), red deer (Tzipori, Menzies & Gray, 1981), takins (Guan et al., 2018) and also domestic carnivores (Hoshino et al., 1981; Williams, 1980), mice (Kjeldsberg & Hem, 1985), and pigs (Bridger, 1980), but their role in the context of disease remained largely unclear. Remarkably, almost at the same time as the discovery of the first AstV-associated encephalitis in humans, the so-called shaking mink syndrome was described, which could be traced back to neurovirulent AstV infection (Blomstrom et al., 2010). One year later, in 2011, a neurovirulent porcine AstV type 3 could be identified as the cause of disease in outbreaks of meningoencephalomyelitis in piglets (Arruda et al., 2017; Boros et al., 2017).
Since 2013, different novel AstV genotype species were found as a plausible cause of non-suppurative encephalitis in cattle (Bouzalas et al., 2014; Li et al., 2013; Schlottau et al., 2016) and a few years later also in sheep (Pfaff et al., 2017). In Switzerland, three neurotropic AstV were identified in brain-tissue of ruminants; bovine astrovirus CH13 (BoAstV-CH13) and bovine astrovirus CH15 (BoAstV-CH15) in cattle (Bouzalas et al., 2014; Seuberlich et al., 2016), as well as ovine astrovirus CH16 (OvAstV-CH16) in sheep. The capsid protein as well as the non-structural proteins of this encephalitis-associated AstV in sheep (OvAstV-CH16) show a high similarity—around 99% on both the nucleotide and the amino acid level—to BoAstV-CH15, suggesting interspecies transmission of this genotype species between sheep and cattle (Boujon et al., 2017).
To date, there are 19 genotype species of Mamastrovirus (MAstV 1–19) recognized by the International Committee on Taxonomy of Viruses (ICTV). In particular, in OvAstV, little is known about their diversity. OvAstV-1 belongs to MAstV 13 and is the only enterotropic AstV closely related to neurotropic strains, but their exact taxonomy is still pending (Boujon, Koch & Seuberlich, 2017). Based on phylogenetic analyses of different viral strains of bovine, ovine and porcine origin, further evidence of possible interspecies transmission could be found (Donato & Vijaykrishna, 2017). The close clustering of farmed animals’ AstV strains reinforce the assumption of probable interspecies transmission events.
The aims of the present study included the assessment of a potential shedding of neurotropic AstV in fecal samples, the investigation of diverse AstV in different ruminant species and the examination of a potential interspecies transmission. Fecal samples of sheep, goats, deer, alpaca, and llamas were tested for BoAstV-CH13 as well as BoAstV-CH15/OvAstV-CH16 and screened for other AstV using a Pan-AstV RT-PCR. NGS and bioinformatics were used to recover viral genome sequences and to perform a phylogenetic comparison as well as a recombination analysis with other known AstV.
The question whether enterotropic AstV can cause disease in small ruminants remains so far unresolved. Due to the mainly unknown health status of the tested animals, the importance of AstV occurring in small ruminants’ feces remains so far unclear and needs to be further investigated. Still, this study describes five novel AstV discovered in small ruminants, including the first description of an AstV in goats and gives new insights into the frequency and diversity of AstV in ruminant species.
Our practice of excluding cellular endosymbionts was interpreted as avoidance of genome size attraction artifacts (Harish et al., 2016), when in reality our intention was to exclude organisms with ill-defined hologenomes of holobiont collectives (the host and its associated organismal communities), which are known to complicate definitions of taxa (Zilber-Rosenberg and Rosenberg, 2008; Keeling, 2011). No such exclusion was extended to the viral supergroup since one hallmark of viruses is harboring a life cycle with strict dependence on a cellular host (see below). We previously confirmed that cellular endosymbionts and obligate parasites harbor an FSF domain repertoire that is distinct from the other members of their respective superkingdoms (Nasir et al., 2011). Cellular organisms committed to obligate parasitism show an increase in informational domains that is sometimes offset by loss of metabolic domains. This unique signature is conserved among nearly all known endosymbionts (Nasir et al., 2011) and distinguishes these organisms from other members of their respective superkingdom. The existence of two unique signature FSF repertoires in cellular organisms (i.e., of free-living organisms and endosymbionts) creates conflict when the two lifestyles are considered together in genome-composition phylogenies. It leads to distortions when endosymbionts from different superkingdoms cluster together irrespective of their taxonomic affiliation). In turn, there are no “free-living” viruses and this conflict does not exist in the virosphere.
Viruses are also different from cellular endosymbionts in their FSF composition profile (Figure 6) and hence do not cause any distortions to the cellular subtrees (Figure 3). Harish et al. (2016) disregarded the rationale and added questionable taxa to their data matrices. These taxa were likely “cherry-picked” from extreme proteomic outliers and sometimes even outside our initial sampling (e.g., Cand. Nausia deltocephalinicola). For example, Cand. Tremblaya princeps included in their trees (Figure 2 in Harish et al., 2016) is part of a three-pronged endosymbiotic organismal system (McCutcheon and von Dohlen, 2011). Its genome encodes only 55 universal FSFs. It is not considered an independent organism since it depends on its host (Planococcus citri) and its endosymbiont (Cand. Moranella endobia) to synthesize essential metabolites (López-Madrigal et al., 2011). Similarly, Cand. N. deltocephalinicola is an obligate endosymbiont of leafhoppers, which harbors the smallest known bacterial genome (Bennett and Moran, 2013) and encodes only 53 universal FSFs. These extreme proteomic outliers do not bias tree reconstructions because of their genome size nor induce “grossly erroneous rootings,” as suggested by Harish et al. (2016). Instead, their hologenomes arise from relatively modern genomic exchanges and recruitments likely resulting from complex trade-off relationships that complicate the dissection of their evolutionary origin and their definition as single valid taxon in the phylogenetic data matrices. Phylogenetically, they represent problematic taxa that should be excluded from analysis pending further understanding of their genetic makeup. The intentional inclusion of problematic taxa is expected to generate biased reconstructions (e.g., see Wilkinson et al., 2000 for a dinosaur phylogeny example and the detection of problematic taxa with double decay analysis).
In the absence of tree statistics, it is impossible to evaluate the effect of progressive inclusion of extremely-reduced obligate parasitic taxa on the reconstructions of Harish et al. (2016). We therefore performed a series of tests to determine if “rogue” taxon addition affected the support of unrooted phylogenies (Figure 7, Table S3). In unrooted trees, the smallest phylogenetic statement is the relationship of a quartet of leaves. When examining BS-resampled phylogenies, the frequency of alternative resolved quartets provides measures of support for the position of each leaf and the accuracy of the tree (Thorley and Wilkinson, 1999). These BS-based leaf stability (LS) indices describe phylogenetic instabilities that often result from either insufficient samplings or conflicting data. Since the genomic census is exhaustive, the culprit of LS varying scores can be character incongruence imposed by problems in the definition of taxa and characters. An unstable leaf can lower the LS scores of the other leaves and affect the overall LS of the taxon set by either occurring in unstable quartets (direct effects) or by lowering the stability of quartets in which it does not occur (indirect effects) when there is character conflict. Figure 7A shows a 20-taxon strict consensus tree with equal representation of supergroup taxa from 2,000 BS replicates used as a control (C). BS replicates were also generated for all 5 possible permutations of the free-living Acidobacterium capsulatum control and the obligate endoparasite R. prowazekii with the taxon set of the corresponding bacterial supergroup. These replicates were used to evaluate LS measures (Figure 7B, Table S3). Remarkably, LS indices from R. prowazekii permutations were significantly more variable and globally lower than those of A. capsulatum, explaining the reduced support of phylogenetic relationships we observed at the base of our ToL when the obligate parasites were added (Figure 3B). Similar results were obtained when alternative tree statistics such as LS difference and LS entropy were compared (Table S3) indicating the potentially “rogue” R. prowazekii taxon could be excluded from tree reconstructions for better and reliable recovery of evolutionary relationships. Explicitly Agree (EA) similarity, the proportion of quartets including the leaf that are resolved and of the same type in the trees, describe the similarity of the position of leaves (Estabrook, 1992). EA values increase with the putatively rogue R. prowazekii taxon (Table S3). Thus, their addition decreases leaf stability while at the same time resulting in similar leaf positions. Finally, the RogueNaRok algorithm (Aberer et al., 2013) also indicated that the R. prowazekii taxon was rogue and was a candidate for pruning.
Given that the persistence of viruses as a supergroup depends on viral interactions with cellular hosts, considerations of lifestyle and taxon definition alone cannot be used to exclude viruses in phylogenomic reconstructions. Cellular dependency is a necessary condition for the propagation of all viruses (with no exceptions), which generally occurs through lysis, exocytosis and transport (Nasir et al., 2017). Viruses can also engage in host-specific dependency and dormancy interactions via symbiosis and latency (e.g., polydnaviruses and wasps behaving as holobionts; Federici and Bigot, 2003). However, cellular dependencies could result in viruses acting as rogue taxa in phylogenetic reconstructions. We therefore tested the impact of including viruses on the stability of ToL topologies. Figure 8 shows that the reconstruction of 24-taxon unrooted BS trees with 8 taxa each for Archaea, Bacteria and Eukarya, but no viruses (the dataset 8880, Figure 8A) had LS indices that were not significantly different (LSmaximum, P = 0.98 LSdifference, P = 0.61; LSentropy, P = 0.60) from those where the most “stable” cellular organisms were replaced by 6 viral taxa to produce a balanced 4-supergroup BS set (dataset 6666, Figure 8B). Thus, LS distributions show that viruses and cellular organisms are equally stable in ToLs (Figure 8C). To further inspect the two BS tree sets, we measured taxon instability indices (TII), which compute the variation of pair-wise patristic distances between taxon pairs across all trees (Maddison and Maddison, 2001). TII also evaluates leaf stabilities and the impact of rogue taxa (Aberer et al., 2013). Figure 8D shows that the 8880 unrooted BS trees gain a 37% significant decrease (P < 0.01) in taxonomic instability by replacements with the balanced 6666 BS set (Table 2). In addition, none of the viruses that were added were considered rogue taxa and candidates for pruning by the RogueNaRok algorithm (Aberer et al., 2013). Therefore, and contrary to the claims of Harish et al. (2016), phylogenetic stability provides one more reason to include viruses in ToLs.
Vero cells (1.6 × 105 cells) in RPMI 1640 containing 2% FBS were seeded in each well of a 24-well plate and incubated under 5% CO2 humidified atmosphere at 37 °C in for 24 h. The medium was discarded, replaced with fresh medium and plates were again incubated under 5% CO2 humidified atmosphere at 37 °C for 48 h. The virus was diluted with fresh RPMI 1640 with 2% FBS to obtain a working virus solution. One hundred microliters of virus suspension containing 1X107 PFU PrV was added to each containing Vero cells in 1 mL of 1% methylcellulose and 2% FBS and the plate incubated rocking for 1 h. Infected cells were fixed with methanol and stained with 0.5% crystal violet solution for 30 min. The number of plaques formed were counted and expressed as PFU/mL.