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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.
In real life, because there are differences in climate, economy, education, and medical treatment, the population is not divided by rules like cellular automata. For example, in China, the population density in the southeast coastal areas is greater than that in the northwest. In addition, because of the different distribution of business districts, schools, and hospitals, the distribution of population in the same city is not uniform. In areas with a large population density, the distance between individuals is shorter and the spread range of individuals is wider. Individuals in the population have higher contact frequency and more neighbors around them, so their infectivity and the probability of being infected also increase.
In order to study and analyze the influence of population density on infectious disease spread, each individual is mapped into a cell in the cellular automata model. When there is no individual and the individual is in dead state in a cell, they are not infectious. In order to simulate the difference in population density, the population density can be simulated by setting the value of D(t) in the initial state. At this time, D(t) does not represent the number of dead individuals, but represents that there is no individual in the cell. In this paper, a sparse matrix was used to simulate the random distribution of population and the infectious disease spread, and then the trend of infectious disease spread under different population densities as well as the influence of different population densities on infectious disease spread were analyzed.
Due to the influence of economic development and other factors, the population ratio and age structure in different regions are also different. For example, young and middle-aged people in remote mountainous areas go to work in big cities, resulting in a large number of old and young people in the original area. In areas where labor is scarce, such as coal mines and crude oil mining areas, there is an imbalance in the proportion of men to women. Therefore, it is of great practical significance to study the influence of sex ratio and age structure on infectious disease spread.
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.
Due to factors such as mobility and spatial environment, age structure of the population presents different distributions. The age structure of a city can be divided into three types: young, adult, and aged according to the proportion of children, adolescents, youth, middle-aged people, and elderly people.
According to related materials, the influence coefficient of pandemic influenza A (H1N1) on children, adolescents, youth, middle-aged people, and elderly people is very different. In simulation experiments, the influence coefficients of pandemic influenza A (H1N1) on children, adolescents, youth, middle-aged people, and elderly people were set to f1=0.65, f2=0.58, f3=0.46, f4=0.37, f5=0.68, respectively.
All things being equal, the parameters of three simulations for the SLIRDS model were set as follows:
(1) Young: children: adolescents: youth: middle age: old age = 0.025:0.05:0.5:0.4:0.025.
(2) Adult: children: adolescents: youth: middle age: old age = 0.2:0.2:0.3:0.25:0.15.
(3) Aged: children: adolescents: youth: middle age: old age = 0.1:0.05:0.05:0.2:0.6.
Three simulation results are shown in Figure 7.
The death, susceptibility, infection, and immunization curves are shown in Figure 7a–d, respectively. It can be seen that the number of deaths in the aged cities is the largest. The number of young urban deaths is only inferior to that of the aged cities, whereas the number of deaths in the adult cities is the least. However, the overall trend of change is gradually stable after rising for all types of cities. The difference in age structure of the population has little influence on the susceptible population, and the number of members of the susceptible population in adult cities decreases first and then reaches a stable value. The difference in age structure of the population has little influence on the infected population. The number of members of the infected population in the aged cities is the highest, but the general trend is rising first and then decreasing for all types of cities. The difference in age structure of the population has little influence on the immunization population, and the number of immune individuals in the adult city rises first to then reach a stable level.
According to the above analysis, it is known that infectious diseases spread more slowly in adult cities than in aged and young cities, but the resistance of young cities to infectious diseases is slightly greater than that of aged cities.
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.
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.
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).
Tannins are known bactericides because they react with proteins irreversibly, thus complexing within bacterial membranes, neutralizing their activity. As a consequence, tannin-based pharmaceuticals to cure intestine infections have long-time been marketed. They have effective anticaries properties. Tannins have also many applications for other pharmaceutical/medical uses but all these are targeted for future use rather than the present.
Several experimental studies on the pharmaceutical use of tannins have been published with antitumor and anti-oncogenic activities particularly well documented. Their antiviral effectiveness is also well documented by in vitro screening for a variety of 12 different hydrolysable and condensed tannins. The tannin’s minimal inhibitory concentration (MIC) needed for reducing by 50% the cytopathogenicity induced by a number of viruses was used as an evaluation of their effectiveness. The lower MIC values yielded the best antiviral behavior. The different tannin’s minimum cytotoxic concentration (MCC) needed to detect microscopic alteration of normal cell morphology was also determined. Less toxic is the tannin tested in the patient’s cells, thus the higher is its MCC value, the more acceptable it is as an antiviral compound. The ideal antiviral compound is then the one presenting a combination of the lowest MIC and the highest MCC. The effectiveness of different tannins due to their polyphenolic nature can be very high against a number of different viruses. This is due to their irreversible reaction and combination with the viruses capsid proteins. It is the same reaction used in leather tannins and in their association with carbohydrates.
Thus, a number of commercially available tannins have been tested, namely mimosa bark tannin extract and its derivatives, chestnut tannin extract, tara tannin, quebracho wood tannin extract both sulphited and natural, pecan nut tannin extract, pine bark tannin extract, sumach tannin extract, and spruce tannin extract. The viruses against which all these have been tested are highly varied, such as HIV-1 and HIV2, Herpes simplex 1 and 2, Vaccinia virus, vesicular stomatitis virus, Coxsackie virus B4, respiratory syncytial virus, Influenza A H1-N1, Influenza A H3-N2, Influenza B, Human Corona virus, Reovirus-1, Feline Corona virus, Sindbis virus, para-influenza 3 virus, and Punta Toro virus.
The inhibitory effects of these tannins have also been tested on proliferation of murine leukemia cells, murine mammary carcinoma cells, and human T-cells.
Acutissimin A is a bound flavonoid with an ellagi-tannin. It is formed by the interaction of a wine flavonoid with the vescalagin generated by the barrel’s oak tannin. Acutissimin A has been found to present an effectiveness 250 times higher to stop tumors growth than the drug Etoposide.
While many studies have been conducted on a variety of tannins derived from a wide variety of plants as an anticancer treatment, some studies on the possibility of using tannin for other medical applications have also been highlighted. Condensed tannins are traditionally used for the treatment of intestinal problems. This is due to their complexation ability with other molecules and their antioxidant behavior. The extract of Stryphnodendron rotundifolium and of other tannins has proven their effectiveness against ulcers by functioning as a protective coating of the gastrointestinal tract. Other possible mechanisms of action of phenolic plant extracts as herbal medicines against ulcers and gastritis have also been described.
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.
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 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.
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.
Astroviruses are small non-enveloped, positive sense RNA viruses with a wide host range. Astroviruses are classified into two genera; the Mamastrovirus genera includes astroviruses isolated from humans, pigs, cattle, cats and dogs, whereas astrovirus isolates from birds are categorised into the Avastrovirus genera (King et al., 2011). Astroviruses were first identified in 1975 in the stools of children with diarrhoea (Appleton and Higgins, 1975; Madeley and Cosgrove, 1975), and are now estimated to cause up to 10% of gastroenteritis cases in children worldwide (Moser and Schultz-Cherry, 2005).
Canine astrovirus (CaAstV) was first described in the USA, following the identification of star-shaped particles in diarrhoeic stools from a litter of beagles (Williams, 1980). Later studies have identified CaAstV in Italy, France, China, Korea and Brazil (Castro et al., 2013; Choi et al., 2014; Grellet et al., 2012; Martella et al., 2011; Zhu et al., 2011).
The genome of astroviruses is typically 6.4–7.3 kb and divided into three open reading frames (ORFs), ORF1a, ORF1b and ORF2 with a 5′ untranslated region and a 3′ poly-A tail (King et al., 2011). ORF1 codes for the non-structural proteins and ORF2 encodes the capsid precursor protein. The complete coding sequence of ORF2, and a partial sequence of ORF1b has previously been determined for a number of CaAstV isolates (Toffan et al., 2009; Zhu et al., 2011), but to date, no full-length genome sequence of CaAstV has been reported.
The purpose of this study was to determine the prevalence of CaAstV in the UK dog population and to obtain where possible the complete genetic sequence of circulating strains. Four CaAstV strains were identified in dogs showing clinical signs of gastroenteritis and sequencing of ORF2 found significant genetic diversity between strains. The first full-length sequences of two CaAstV strains was subsequently determined.
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.
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.
CaAstV has previously been detected sporadically in dogs across the world, but the association with disease, prevalence levels and genetic diversity is largely unknown. This study presents the first identification and molecular characterisation of CaAstV cases in dogs in the UK. Sequencing of the viral capsid for all four strains revealed extensive genetic diversity and the first full-length sequences for CaAstV were determined for two strains.
The prevalence of CaAstV in gastroenteritis cases in this study was shown to be 6.0%. This prevalence was unexpectedly high given that CaAstV has not previously been reported in the UK. It may be predicted that this is an underestimation of prevalence based on the population of dogs surveyed. The majority of previous CaAstV epidemiological studies have focused on dogs less than 6 months old, whereas this study included dogs of any age. Serological studies have shown that exposure to CaAstV typically occurs in young animals, with dogs older than 3 months significantly more likely to be seropositive than younger dogs (Martella et al., 2011). This suggests that studies focusing only on young dogs will identify more positive CaAStV cases. However, the decision to survey dogs of any age in this study enabled detection of a CaAstV case in a 7-year-old dog. This is oldest case of CaAstV reported to date and highlights the need to have an index of suspicion for infectious causes of gastroenteritis in dogs of any age. Indeed although human astrovirus is more common in paediatric populations, infections in the elderly are reported (Fernández et al., 2011).
The pathology caused by CaAstV in dogs is uncertain as CaAstV has previously been detected in the stools of both healthy and diseased dogs (Grellet et al., 2012; Martella et al., 2011). However, our study shows a clear relationship between the presence of CaAstV RNA in stool samples, and the presence of clinical signs of gastroenteritis (p < 0.001). This finding is in agreement with two previous studies from Italy and China (Martella et al., 2011; Zhu et al., 2011), but is at odds with a French study which found no significant difference in CaAstV identification between diarrhoeic (27%) or healthy puppies (19%) (Grellet et al., 2012). Determination of the specific pathology induced by CaAstV infection in dogs is often confounded in a clinical setting by co-infections with other gastroenteric pathogens (e.g. CPV in dogs 1 and 4 in this study). Experimental studies will be required to confirm or refute the association of CaAstV with gastroenteritis, but this study does suggest CaAstV can cause disease.
Sequencing of the capsid region of each CaAstV strain identified in this study revealed significant sequence variation. This mirrors the variation previously identified within human astrovirus isolates (De Benedictis et al., 2011). At present, astroviruses are named according to the species in which they are isolated, and subsequent classification is based upon serotypes; these are defined if there is a 20-fold or greater two-way cross neutralisation titre (King et al., 2011). Sequence analysis has verified this classification, with human astroviruses 1–8 having 86–100% nucleotide identity within a serotype, based on capsid sequences (Noel et al., 1995). The nucleotide variation within the capsid region of the four CaAstV strains was shown to be 77.1–81.1%, which strongly suggests these strains are also different serotypes. Confirmation of this requires serological analysis, but unfortunately repeated attempts to grow the CaAstV isolates identified in this study in cell culture failed (data not shown).
Identification of four possible CaAstV serotypes circulating in the UK alone raises questions regarding the possible origins of these strains. Phylogenetic analysis of the UK capsid strains alongside the limited number of CaAstV sequences previously listed in GenBank was unexpected. There was no clustering of the UK strains, unlike the grouping of all Chinese strains. Instead UK strains each grouped with a different CaAstV isolate from either China or Italy. With such limited sequences available it is not possible to determine whether CaAstV strains have spread globally, or independent evolution has occurred. Clearly a high rate of evolution does occur within all astroviruses however, as their RNA genome facilitates introduction of point mutations and recombination events.
Given the strain diversity identified, it has been suggested that some CaAstV strains may be more pathogenic than others. This has previously been reported for astroviruses of mink, which show variation in their ability to invade the central nervous system in a strain related manner (Martella et al., 2012). Assessment of this risk will require wider epidemiological and clinical studies. Another concern raised by the existence of multiple circulating CaAstV strains is regarding future disease control. Management of viral causes of gastroenteritis in dogs is best achieved by widespread vaccination, exemplified by the widely used canine parvovirus vaccine. However, if CaAstV vaccine development is considered, the presence of multiple strains will make vaccine design challenging.
Full genome sequencing of two CaAstV isolates revealed them to be closely related and possess a typical astrovirus organisation. The first full length sequence of an astrovirus was for human astrovirus in 1994 (Willcocks et al., 1994), and relatively few full length sequences have since been determined. Sequence analysis of the CaAstV strains identifies the presence of a serine protease and VPg within ORF1a as for other astroviruses, which is separated from and conserved RdRp of ORF1b by a −1 frameshift.
In summary, this study has not only identified CaAstV circulating in the UK dog population, but also found significant genetic diversity within the CaAstV strains. Furthermore, full genome sequencing of two CaAstV isolates has enabled detailed molecular characterisation of this astrovirus species, and provides the astrovirus field with further examples of genome variation.
The increasing preference for camels on higher land in northern Kenya can be understood as a climate adaptation strategy, as it is, in part at least, a response to experiences of lower rainfall and more frequent drought. The case challenges many portrayals of pastoralists in dryland areas of Africa as conservative and as having ‘low adaptive capacity.’ Here, adaptation strategies are ongoing, and it is the herders who are initiating change. The adaptation practices are relatively straightforward, are spontaneous and autonomous, and are being eagerly embraced. They show that adaptation strategies do not have to be costly and ‘painful’ (Adger et al.
2009a: 2). These camel-based adaptation strategies may also be sustainable (in the sense of being continued) as they are ‘owned by’ and not imposed on the people themselves.
The account also challenges portrayals of pastoralists as vulnerable to climate change shocks and stresses because of their isolation and lack of access to robust markets (Boko et al.
2007). The camel market in northern Kenya – so far – is alive and well, and the increase in camels is as much an adaptation to new economic conditions: to the rising price of camels; to the increasing demand for milk in the growing urban centres; and to the reduction of grassland pasture on the mountain and the increase in euphorbia fences. Some of these economic changes are also related to the changing political situation: at an international level, protracted conflicts elsewhere have created an opportunity for northern Kenya; at a local level, new Borana practices are linked to strained relations with the Gabra since the 1990s.
The increasing preference for camels can be said to have improved resilience in northern Kenya in its literal sense. As hardy, drought-resistant animals, camels provide the basis of livelihoods that are better able to cope with some of the unexpected and variable shocks and stresses that climate change may bring. Camels have been shown to contribute to household incomes and livelihoods (agriculture, firewood, nutrition), to lead to further forms of diversification, and some interviewees even claimed that camels could lead to more collaborative gender relations. But our research also identified four vital concerns and risks that require attention.
First, the above conclusions are true under a scenario in which the climate becomes warmer and drier, but climate change predictions also point to more rain and bouts of heavy flooding. Under the latter scenario, camels on the mountain may prove a more risky rather than a more resilient strategy.
Secondly, insufficient understanding of, and provision for, camel health means that there are high losses associated with camels and there may also be new camel-related risks to human health.
Thirdly, the market-based and commodified nature of the new camel economy means that there may be new risks attached to participation; in adapting to the vagaries of the climate, the new camel herders may become further exposed to the vagaries of the market (O’Brien and Leichenko 2000).
Fourthly, and relatedly, the case demonstrates the continuing significance of equity issues (Thomas and Twyman 2005; Nelson et al.
2007). In 2012, the switch to camels was a form of diversification that improved resilience for those who had resources; many questions have been raised about the extent to which it represented a successful form of resilience building for the poor, the most vulnerable to climate shocks.
These concerns and risks should not detract too much from the idea that camels can be the basis of a more resilient ‘adaptation pathway.’ On the contrary, they demonstrate that with appropriate and necessary support from government and development organizations for mobility, animal health, adequate nutrition and marketing, camels could improve resilience further and for more people. More detailed research is required to see whether or not these developments result in increasingly resilient livelihoods over time, but certain findings are already clear: the Borana interviewee’s assertion that ‘[i]f the whole world took to camels, there wouldn’t be any poverty’ is too simplistic. There are multiple risks and potential new inequalities linked to the shift to camels. The most important finding from our research, for state bodies and NGOs who would further the trend, is that wider support is needed for livestock keeping in general, and for camels in particular. It is not enough just to give an animal.
Transgenic carrots (Daucus carota) expressing the B subunit from E. coli thermolabile toxin induced IgA and IgG production, and occurred at the intestinal and systemic levels in rats. In 2010, the UreB subunit of Helicobacter pylori in transgenic carrots was reported to have potential use as a possible vaccine. Carrots, along with A. thaliana, were also utilized in experimental edible vaccines for surface HIV antigen expression, and studies performed in rats showed more positive effects in treated animals compared to non-treated animals. The utilization of carrots to treat HIV appears promising not only because carrots are healthy and delicious but also because the consumption of carrot-derived carotenoids increases lymphocytes, monocytes and other immune defenders in rats. Thus, people with weakened immune systems might benefit from consuming this potentially edible anti-HIV vaccine. Studies in humans must be conducted to confirm the potential of these vaccines.
The complete genome sequences of both viruses FS03 and BM100 have been deposited in GenBank under accession no. KM820744 to KM820763.
Summary statistics were calculated to assess the overall quality of the data. Analysis of variance (ANOVA) was used for assessment of the mean clinical score and microscopic lesion scores. The significance level was set for a P value of <0.01 and a 95% confidence interval. Statistical analysis was performed using GraphPad Prism software (version 6.0; Graph Pad Software, Inc., San Diego, CA).
Synonymous codon usage is biased and the bias seems to be different in different organisms[1,2]. Many factors are concerned to be the reasons for this bias, such as degree and timing of gene expression, codon-anticodon interactions, transcription and translation rate and fidelity, codon context, and global and local (C+G) content[3,4]. Understanding the extent and causes of biases in codon usage is essential to the understanding of viral evolution, particularly the interplay between viruses and the immune response. More recent studies have revealed that patterns of codon usage bias and nucleotide composition within many cellular genomes are far more complex than previously imagined, and the factors shaping their evolution are still not entirely understood. In general, natural selection and/or mutation pressure for accurate and efficient translation in various organisms are the main reasons to this bias. In addition, compared with natural selection, mutation pressure plays an important role in synonymous codon usage pattern in some RNA viruses.
Picornaviruses are positive single-stranded RNA viruses that cause a variety of important disease states in humans and animals, such as foot-and-mouth disease. The Cardiovirus genus of the family Picornaviridae consists two distinct species: Encephalomyocarditis virus (EMCV) and Theilovirus ThV. The EMCVs comprise a single serotype and have a wide host range, while the ThV species, probably includes four serotypes: Theiler's murine encephalomyelitis virus (TMEV), Vilyuisk human encephalomyelitis virus (VHEV), Thera virus (TRV; isolated from rats) and Saffold virus (SAFV; isolated from humans) 1-8., which appear to have much narrower host ranges than EMCV. Like the other virus within Picornaviruses family, the strains in Cardiovirus also consist a open-read-frame (ORF), 5'-untranslate region (5'-UTR) and 3'-untranslate region (3'-UTR). However there are still many complete nucleotide sequences of this type are not reported especially, such as SAFV, therefore there is much more work to study this type virus.
Nevertheless, little information about codon usage pattern of Cardiovirus genus genome including the relative synonymous codon usage (RSCU) and codon usage bias (CUB) in the process of its evolution is available. In this study, the key genetic determinants of codon usage index in Cardiovirus genus were examined.
Understanding host-pathogen interactions is important for the development and assessment of medical countermeasures to infectious agents. The advent of new imaging and “omic” technologies has provided the ability to follow these interactions from whole animal to cellular and molecular levels, enabling a greater understanding of the mechanisms involved; this facilitates the development and refinement of new and existing vaccines and therapeutics. For example, advances in bioimaging provide a noninvasive means of identifying the internal systemic spread of infection in animal models and the impact of a prophylaxis or a therapy on the disease process. This can be combined with the analysis of responses at a cellular level using flow cytometry and microscopy techniques. The use of microarrays has also enhanced our understanding of the host response to infection and provides supportive information to help elucidate the innate and adaptive immune mechanisms essential for protection against pathogens, as well as the virulence mechanisms deployed by the pathogen. Although in its infancy, next generation sequencing also holds great potential for defining host-pathogen interactions. This review will assess the impact of these technologies on the ability to assess the host response and how this has been applied to help progress the development of vaccines and immunotherapies against biodefence agents described in the Centers for Disease Control and prevention (CDC) Select Agent list (http://www.selectagents.gov/). Biodefence agents are dangerous pathogens that require high levels of biocontainment and are relatively less-studied (compared with the majority of public health pathogens) and cases are relatively rare. Therefore, studies to test the efficacy of therapeutics in a healthy population from an endemic area are often not feasible and the use of animal models is essential. This review focuses on the use of these new techniques to help us understand host responses in animal models as well as humans. In this context, both “omic” and imaging technologies hold great promise for important breakthroughs in the rational development of vaccines and therapies.
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.
Edible vaccines represent a valuable solution to treating certain diseases whose control and prevention is restricted by the inherent limitations of traditional vaccines, such as their production costs, storage requirements, and expensive logistics. Sixteen foods are already producing antigens to counter human and animal diseases. However, some challenges remain, such as the development of edible vaccines using plants whose genetic transformation is difficult to attain or is unexplored, whose cultivars can be developed on all continents with low water and nutritional requirements, and whose consumption may be accomplished in a raw form or with minimal boiling. Because vaccine legal regulations are devoid of bylaws, many uncertainties would arise if such distribution were to gain acceptance. For example, who will be in charge of assigning the correct dose? As a drug that is contained in a plant or its fruit, should it be evaluated, authorized, and supervised by Public Health Institutes or a similar Human Health Organization in each country? These vaccines have undeniable potential to counter hundreds of diseases, particularly in countries where traditional vaccines are difficult to obtain or where the frequency of outbreaks of certain diseases makes their control and prevention more difficult. Beyond the pros and cons of edible vaccines, one of the most complex problems to address is the establishment of collaborations for the development of a stable vaccine that can actually be used in human medicine. Advances in the development of transgenic plants and antigen expression for stimulation of the immune system associated with the mucosa have been in the botanical field and not in immunology. As explained in the previous sections, it is very difficult to establish a stable antigenic protein concentration in plant tissues, and there is no certainty that the expressed antigen will produce an immune response. Efforts by immunologists and conventional vaccine developers could be of great value to advance this alternative to current vaccines. In addition to their possible benefits, edible vaccines will decrease the costs of vaccination and allow minimally invasive vaccine administration. Furthermore, reiterating the need to increase vaccine performance and stability, developments in the generation of transient vaccines using viruses do not obviate the development of transgenic plants as a long-term and longer-lasting measure. The potential opposing role of oral tolerance might be beneficial for the treatment of autoimmune diseases in which dendritic cells play a fundamental role in regulating and maintaining the balance between immunity and tolerance. However, it is also necessary to discuss the potential global consequences of the inappropriate use of edible vaccines, particularly with respect to ecosystem imbalance (pollen and seed flight) and disturbances of worldwide peace and safety.
In summary, to reduce outbreaks of infectious diseases worldwide, the implementation of control and prevention measures on a massive scale is required. In this scenario, edible vaccines represent a valuable alternative to mitigate and prevent infectious outbreaks in countries where the conventional vaccination is difficult. In addition, in countries where the prevalence of infectious diseases is controlled, edible vaccines may support public health programs to reduce the risk of disease outbreaks, analogous to the use of prebiotics and probiotics as a complement to food. As shown in this work, the current production of edible vaccines is focused on a small group of plants, some of which are consumed globally. However, promoting the genetic transformation of plants with higher impact on the consumption chain in specific countries remains challenging. In addition, increasing the agricultural products of each country must be based on a consideration of country-specific policies with respect to the production or commercialization of genetically modified plants as well as ecological and cultural regulations, especially in those countries considered centers of origin of some important crops.