Valentina Toneva | University of Plovdiv (original) (raw)

Papers by Valentina Toneva

International Journal of Molecular Sciences

Plant viruses have traditionally been studied as pathogens in the context of understanding the mo... more Plant viruses have traditionally been studied as pathogens in the context of understanding the molecular and cellular mechanisms of a particular disease affecting crops. In recent years, viruses have emerged as a new alternative for producing biological nanomaterials and chimeric vaccines. Plant viruses were also used to generate highly efficient expression vectors, revolutionizing plant molecular farming (PMF). Several biological products, including recombinant vaccines, monoclonal antibodies, diagnostic reagents, and other pharmaceutical products produced in plants, have passed their clinical trials and are in their market implementation stage. PMF offers opportunities for fast, adaptive, and low-cost technology to meet ever-growing and critical global health needs. In this review, we summarized the advancements in the virus-like particles-based (VLPs-based) nanotechnologies and the role they played in the production of advanced vaccines, drugs, diagnostic bio-nanomaterials, and o...

International Journal of Molecular Sciences

Plant small RNAs (sRNAs) are a heterogeneous group of noncoding RNAs with a length of 20–24 nucle... more Plant small RNAs (sRNAs) are a heterogeneous group of noncoding RNAs with a length of 20–24 nucleotides that are widely studied due to their importance as major regulators in various biological processes. sRNAs are divided into two main classes—microRNAs (miRNAs) and small interfering RNAs (siRNAs)—which differ in their biogenesis and functional pathways. Their identification and enrichment with new structural variants would not be possible without the use of various high-throughput sequencing (NGS) techniques, allowing for the detection of the total population of sRNAs in plants. Classifying sRNAs and predicting their functional role based on such high-performance datasets is a nontrivial bioinformatics task, as plants can generate millions of sRNAs from a variety of biosynthetic pathways. Over the years, many computing tools have been developed to meet this challenge. Here, we review more than 35 tools developed specifically for plant sRNAs over the past few years and explore some...

Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is doubtless ... more Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is doubtless and has increased recent years. Zoonotic spread of HEV, mainly due to genotype (gt) 3, has emerged in developed countries in the past decade. The current study has shown for the first time that domestic pigs in Bulgaria are widely infected with HEV. There is a need for a method for the cost-effective detection of the HEV infection and production of HEV immunogenic proteins that can be used as diagnostic antigens for the serological tests. Also, in this study we have compared the results from a commercial ELISA and an in-house ELISA test based on HEV open reading frame (ORF) 2 protein produced in planta, the sensitivity and the specify of the tests were evaluated. The development of a HEV diagnostic kit based on plant-derived ORF2 capsid protein is our goal and this will help to reduce the cost of such a kit and to enable diagnoses of HEV in developing countries.

Paulownia witches' broom (PaWB) is caused by phytoplasma of Aster Yellows group, and resulted... more Paulownia witches' broom (PaWB) is caused by phytoplasma of Aster Yellows group, and resulted in severe damage to the growth and premature death of the infected tree. In vitro cultures of meristems are important tools for solving problems with plant diseases. In recent study meristem cultures from phytoplasma infected <em>Paulownia</em><em> </em><em>tomentosa</em> have been created in an attempt to obtain healthy plants. The faster procedure with longer explants generated 95% regeneration but no healthy plants. The combination of smaller explants and thermotherapy resulted in the generation of plantlets with a healthy phenotype. The PCR analyzes showed the absence of phytoplasma only in one of the produced plants. Despite the low efficiency, expressed in one phytoplasma-free regenerant of a total of 480 explants, these results can serve to continue the work on optimizing the used strategy.

Life, 2022

Emerging and re-emerging zoonotic diseases cause serious illness with billions of cases, and mill... more Emerging and re-emerging zoonotic diseases cause serious illness with billions of cases, and millions of deaths. The most effective way to restrict the spread of zoonotic viruses among humans and animals and prevent disease is vaccination. Recombinant proteins produced in plants offer an alternative approach for the development of safe, effective, inexpensive candidate vaccines. Current strategies are focused on the production of highly immunogenic structural proteins, which mimic the organizations of the native virion but lack the viral genetic material. These include chimeric viral peptides, subunit virus proteins, and virus-like particles (VLPs). The latter, with their ability to self-assemble and thus resemble the form of virus particles, are gaining traction among plant-based candidate vaccines against many infectious diseases. In this review, we summarized the main zoonotic diseases and followed the progress in using plant expression systems for the production of recombinant p...

Comparative metabolic profiling of Haberlea rhodopensis,

Biotechnology & Biotechnological Equipment, 2012

The "resurrection plant" Ramonda serbica is included in the Albanian and Bulgarian list of endang... more The "resurrection plant" Ramonda serbica is included in the Albanian and Bulgarian list of endangered plants as a rare, Balkanic-endemic species and tertiary relic species, while R. nathaliae is characteristic in the FYROM area. Our research groups have established national in vitro collections of R. serbica in Albania and Bulgaria and R. nathaliae in Macedonia. The aim of this joint study is (1) the choice of successful micropropagation methods as the basis for the in vitro collections and (2) the evaluation of polymorphism of some natural populations. In both collections, seeds were used as a convenient starting point for micropropagation in the nutrient medium JG-B. In the Bulgarian in vitro collection, a dry sterilization of seeds was applied for a first time for Gesneriaceae family. The micropropagation and conservation of Ramonda seeds and plantlets were similar in Albanian and Bulgarian collections. The plantlets in vitro as an explant material were developed in JG-B medium with different phytohormones. The direct organogenesis of two Ramonda species is very similar models. The method of conservation in vitro with minimal growth method (modification of nutrient medium) was used. Meiosis and mitosis examinations of the natural populations and plantlets cultivated in vitro were carried out by standard Squash method. Cytogenetical study of natural populations showed polyploid forms as a mixture (2n=72) and (2n=96). Mitosis preparations in plantlets in vitro showed a predominance of the most frequent form (2n=96). The biodiversity in interspecific level of the natural populations of Ramonda serbica of Central Albania, R. nathaliae from Macedonia and cultivated plantlets in vitro from Albania and Bulgaria was evaluated via molecular markers (RAPDs). The micropropagation method is a convenient way for the international exchange of germplasm of rare and endemic plants. From the data obtained in this study it can be concluded that RAPD markers provided a useful technique to study genetic diversity in Ramonda serbica and R. nathaliae populations. This technology allows the identification of different populations as well as the assessment of the genetic similarity among different populations.

Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desi... more Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea\u27s remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4◦C) and subsequent return to optimal temperatures (21◦C) was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive Arabidopsis thaliana. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21◦C. H. rhodopensis had significantly more raffinose, melibiose, trehalose, rhamnose, myo-inositol, sorbitol, galactinol, erythronate, threonate, 2-oxoglutarate, citrate, and glycerol than the other two species. A. thaliana had the highest levels of putrescine and fumarate, while T. halophila had much higher levels of several amino ...

Plants, Dec 24, 2019

The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fe... more The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110–610) and chimeric M2 HEV 110–610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22–36 nm in diameter and specifically reacted with the anti-M2e antibody.

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particl... more The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform f...

Biologia plantarum

Small RNAs (sRNAs) are essential components of gene-regulatory networks, which guide plant develo... more Small RNAs (sRNAs) are essential components of gene-regulatory networks, which guide plant development and tune it to environmental challenges. Though the past years have witnessed evidences on sRNA importance for stress response, there is scarce data on their involvement in resurrection plant survival under severe drought. Haberlea rhodopensis (hrh) is an angiosperm resurrection species, whose vegetative tissues can tolerate desiccation and recover upon rehydration. In this study, high-throughput sequencing sRNAs indicated a higher complexity of the sRNA population, especially of a 24 nt sRNA category, in the desiccated vegetative tissue of H. rhodopensis compared to unstressed tissues. The cross-species discovery was performed to predict 77 mature microRNAs (miRNAs), most of which were assigned to 23 high-confidence conserved miRNA families in the leaf tissue. Several members of the miR156/157, miR166, and miR399 families were found to be desiccation-responsive. The miR156/157 family members were found up-regulated upon dehydration and down-regulated upon rehydration, while the miR166 and miR399 family members followed an opposite trend of expression. A probable miR156/157 target, orthologous to the SQUAMOSA promoter binding proteinlike, was reconstructed in H. rhodopensis based on genomic data available for this species and the closely related Boea hygrometrica. Reverse transcription quantittative PCR analysis confirmed the expression profile of hrh-miR156a-5p and hrh-miR157-5p established by sRNA sequencing and revealed an inverse expression pattern between these miRNAs and their targets in the desiccated tissue. Our study suggests that the miR156/157 and miR399 families are essential for plant survival under severe drought due to their ability to control plant development and growth by modulating transcription factor expression.

Vaccines

Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in develop... more Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous partic...

Animals

Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is undisputed... more Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is undisputed and has increased in recent years. The zoonotic spread of HEV, mainly due to genotype (gt) 3, emerged in developed countries in the past decade. In addition, transmission via contaminated meat from pigs and boars was also established. Detailed analysis of viral dynamics and distribution is needed in order to identify associated risk factors. The aim of the current study is to present new and additional data on the HEV distribution among pigs, and for the first-time, also among the wild boar population in Bulgaria. Abstract: (1) Background: Hepatitis E virus (HEV) is a causative agent of acute viral hepatitis, predominantly transmitted by the fecal-oral route. In developed countries, HEV is considered to be an emerging pathogen since the number of autochthonous cases is rising. Hepatitis E is a viral disease with a proven zoonotic potential for some of its genotypes. The main viral reservoirs are domestic pigs and wild boar. Consumption of undercooked meat, as well as occupational exposure, are key factors for the spread of HEV. In order to evaluate the risks of future viral evolution, a detailed examination of the ecology and distribution of the virus is needed. The aim of the present study is to investigate the prevalence of anti-HEV IgG Ab in domestic pigs and wild boar in Bulgaria; (2) Methods: In this study, during the period of three years between 2017 and 2019, 433 serum samples from 19 different pig farms and 1 slaughterhouse were collected and analyzed. In addition, 32 samples from wild boar were also collected and analyzed during the 2018-2019 hunting season. All samples were analyzed by commercial indirect ELISA; (3) Results: Overall, HEV seroprevalence was 60% (95% CI 42.7-77.1) in domestic pigs and 12.5% (4/32) in wild boar. The observed seroprevalence of the slaughter-aged pigs was 73.65% (95% Cl 58.7-87.3). Prevalence in domestic pigs was significantly higher in the samples collected during 2019 (98% (95% Cl 96.1-99.9)) compared to those collected during 2017 (45.33% (95% CI 2.7-87.3)) and 2018 (38.46% (95% CI 29.1-49.7.); (4) Conclusions: Our findings suggest that domesticated pigs and wild boar might be the reason for the increased HEV transmission across Bulgaria. The genotypic characterization of HEV found in pigs, wild boar and humans will give a more accurate view of the zoonotic transmission of this virus.

Plant physiology, Jan 22, 2018

The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any ... more The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any visible senescence. Here, we investigated the molecular mechanisms of this adaptation to prolonged (30 days) darkness and subsequent return to light. H. rhodopensis plants remained green and viable throughout the dark treatment. Transcriptomic analysis revealed that darkness regulated several transcription factor (TF) genes. Stress- and autophagy-related TFs such as ERF8, HSFA2b, RD26, TGA1, and WRKY33 were upregulated, while chloroplast- and flowering-related TFs such as ATH1, COL2, COL4, RL1, and PTAC7 were repressed. PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a negative regulator of photomorphogenesis and promoter of senescence, was also downregulated. In response to darkness, most of the photosynthesis- and photorespiratory-related genes were strongly downregulated, while genes related to autophagy were upregulated. This occurred concomitant with the induction of SUCROSE NON-FERMENTING...

RNA-dependent DNA methylation (RdDM) is an important regulatory event involved in repressive epig... more RNA-dependent DNA methylation (RdDM) is an important regulatory event involved in repressive epigenetic modifications that can trigger transcriptional gene silencing (TGS). The criteria we used to pick out promoter sequences targeted by RdDM in Arabidopsis thaliana were the main RdDM hallmark properties: 24nt siRNAs as inducers of DNA methylation and transposable elements (TE) as one of the major targets of RdDM. Those genes whose promoters comprised overlapping sites for 24nt siRNA hits, TE and DNA methylation (siRNA/TE/Methylation overlapping regions), were defined as candidates that might be silenced by RdDM. On this basis two gene sets were created which include abiotic and biotic stress-responsive genes whose promoters may be silenced by RdDM. The DNA methylation status of the At3g50770 (CML41) promoter e one of the selected candidates, was experimentally assayed, and it showed dependence on the RdDM-associated Polymerase IV and Polymerase V. A publicly available 24nt siRNA-centered database called starPRO was developed that allows users easily to discover whether a particular promoter sequence is related to RdDM-associated features such as 24nt siRNA-target sites, TE, tandem repeats and DNA methylation.

ABSTRACT Haberlea rhodopensis (Gesneriaceae) is a less explored endemic plant within a very small... more ABSTRACT Haberlea rhodopensis (Gesneriaceae) is a less explored endemic plant within a very small group of poikilohydric angiosperms that are able to survive in an almost completely dehydrated state. The purpose of the present study was to obtain extracts from Haberlea rhodopensis leaves using alternative technique- microwave-assisted extraction and to determine the total phenolic content and antioxidant activity of the extract by applying reliable methods: reducing power and ABTS radical cation decolorization assay. Different extraction conditions- microwave power and extraction times were investigated. Total phenolic content was determined using a spectrophotometric technique based on the Folin-Ciocalteu reagent and calculated as mgGAE/gDW (leaf dry weight). The results from the total phenolic assay and the antioxidant activity tests were significantly correlated. Major phenolic acids and some flavonoid- aglycones and - glycosides were analyzed by HPLC. Luteolin (3307.08 μg/gDW), myricetin (702.0 μg/gDW), ferulic acid (652.92 μg/gDW) and hesperidin (607.32 μg/gDW) were predominant in the H. rhodopensis microwave-assisted extracts and some of the effects of the plant could be attributed to the presence of these valuable constituents.

International Journal of Molecular Sciences

Plant viruses have traditionally been studied as pathogens in the context of understanding the mo... more Plant viruses have traditionally been studied as pathogens in the context of understanding the molecular and cellular mechanisms of a particular disease affecting crops. In recent years, viruses have emerged as a new alternative for producing biological nanomaterials and chimeric vaccines. Plant viruses were also used to generate highly efficient expression vectors, revolutionizing plant molecular farming (PMF). Several biological products, including recombinant vaccines, monoclonal antibodies, diagnostic reagents, and other pharmaceutical products produced in plants, have passed their clinical trials and are in their market implementation stage. PMF offers opportunities for fast, adaptive, and low-cost technology to meet ever-growing and critical global health needs. In this review, we summarized the advancements in the virus-like particles-based (VLPs-based) nanotechnologies and the role they played in the production of advanced vaccines, drugs, diagnostic bio-nanomaterials, and o...

International Journal of Molecular Sciences

Plant small RNAs (sRNAs) are a heterogeneous group of noncoding RNAs with a length of 20–24 nucle... more Plant small RNAs (sRNAs) are a heterogeneous group of noncoding RNAs with a length of 20–24 nucleotides that are widely studied due to their importance as major regulators in various biological processes. sRNAs are divided into two main classes—microRNAs (miRNAs) and small interfering RNAs (siRNAs)—which differ in their biogenesis and functional pathways. Their identification and enrichment with new structural variants would not be possible without the use of various high-throughput sequencing (NGS) techniques, allowing for the detection of the total population of sRNAs in plants. Classifying sRNAs and predicting their functional role based on such high-performance datasets is a nontrivial bioinformatics task, as plants can generate millions of sRNAs from a variety of biosynthetic pathways. Over the years, many computing tools have been developed to meet this challenge. Here, we review more than 35 tools developed specifically for plant sRNAs over the past few years and explore some...

Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is doubtless ... more Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is doubtless and has increased recent years. Zoonotic spread of HEV, mainly due to genotype (gt) 3, has emerged in developed countries in the past decade. The current study has shown for the first time that domestic pigs in Bulgaria are widely infected with HEV. There is a need for a method for the cost-effective detection of the HEV infection and production of HEV immunogenic proteins that can be used as diagnostic antigens for the serological tests. Also, in this study we have compared the results from a commercial ELISA and an in-house ELISA test based on HEV open reading frame (ORF) 2 protein produced in planta, the sensitivity and the specify of the tests were evaluated. The development of a HEV diagnostic kit based on plant-derived ORF2 capsid protein is our goal and this will help to reduce the cost of such a kit and to enable diagnoses of HEV in developing countries.

Paulownia witches' broom (PaWB) is caused by phytoplasma of Aster Yellows group, and resulted... more Paulownia witches' broom (PaWB) is caused by phytoplasma of Aster Yellows group, and resulted in severe damage to the growth and premature death of the infected tree. In vitro cultures of meristems are important tools for solving problems with plant diseases. In recent study meristem cultures from phytoplasma infected <em>Paulownia</em><em> </em><em>tomentosa</em> have been created in an attempt to obtain healthy plants. The faster procedure with longer explants generated 95% regeneration but no healthy plants. The combination of smaller explants and thermotherapy resulted in the generation of plantlets with a healthy phenotype. The PCR analyzes showed the absence of phytoplasma only in one of the produced plants. Despite the low efficiency, expressed in one phytoplasma-free regenerant of a total of 480 explants, these results can serve to continue the work on optimizing the used strategy.

Life, 2022

Emerging and re-emerging zoonotic diseases cause serious illness with billions of cases, and mill... more Emerging and re-emerging zoonotic diseases cause serious illness with billions of cases, and millions of deaths. The most effective way to restrict the spread of zoonotic viruses among humans and animals and prevent disease is vaccination. Recombinant proteins produced in plants offer an alternative approach for the development of safe, effective, inexpensive candidate vaccines. Current strategies are focused on the production of highly immunogenic structural proteins, which mimic the organizations of the native virion but lack the viral genetic material. These include chimeric viral peptides, subunit virus proteins, and virus-like particles (VLPs). The latter, with their ability to self-assemble and thus resemble the form of virus particles, are gaining traction among plant-based candidate vaccines against many infectious diseases. In this review, we summarized the main zoonotic diseases and followed the progress in using plant expression systems for the production of recombinant p...

Comparative metabolic profiling of Haberlea rhodopensis,

Biotechnology & Biotechnological Equipment, 2012

The "resurrection plant" Ramonda serbica is included in the Albanian and Bulgarian list of endang... more The "resurrection plant" Ramonda serbica is included in the Albanian and Bulgarian list of endangered plants as a rare, Balkanic-endemic species and tertiary relic species, while R. nathaliae is characteristic in the FYROM area. Our research groups have established national in vitro collections of R. serbica in Albania and Bulgaria and R. nathaliae in Macedonia. The aim of this joint study is (1) the choice of successful micropropagation methods as the basis for the in vitro collections and (2) the evaluation of polymorphism of some natural populations. In both collections, seeds were used as a convenient starting point for micropropagation in the nutrient medium JG-B. In the Bulgarian in vitro collection, a dry sterilization of seeds was applied for a first time for Gesneriaceae family. The micropropagation and conservation of Ramonda seeds and plantlets were similar in Albanian and Bulgarian collections. The plantlets in vitro as an explant material were developed in JG-B medium with different phytohormones. The direct organogenesis of two Ramonda species is very similar models. The method of conservation in vitro with minimal growth method (modification of nutrient medium) was used. Meiosis and mitosis examinations of the natural populations and plantlets cultivated in vitro were carried out by standard Squash method. Cytogenetical study of natural populations showed polyploid forms as a mixture (2n=72) and (2n=96). Mitosis preparations in plantlets in vitro showed a predominance of the most frequent form (2n=96). The biodiversity in interspecific level of the natural populations of Ramonda serbica of Central Albania, R. nathaliae from Macedonia and cultivated plantlets in vitro from Albania and Bulgaria was evaluated via molecular markers (RAPDs). The micropropagation method is a convenient way for the international exchange of germplasm of rare and endemic plants. From the data obtained in this study it can be concluded that RAPD markers provided a useful technique to study genetic diversity in Ramonda serbica and R. nathaliae populations. This technology allows the identification of different populations as well as the assessment of the genetic similarity among different populations.

Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desi... more Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea\u27s remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4◦C) and subsequent return to optimal temperatures (21◦C) was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive Arabidopsis thaliana. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21◦C. H. rhodopensis had significantly more raffinose, melibiose, trehalose, rhamnose, myo-inositol, sorbitol, galactinol, erythronate, threonate, 2-oxoglutarate, citrate, and glycerol than the other two species. A. thaliana had the highest levels of putrescine and fumarate, while T. halophila had much higher levels of several amino ...

Plants, Dec 24, 2019

The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fe... more The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110–610) and chimeric M2 HEV 110–610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22–36 nm in diameter and specifically reacted with the anti-M2e antibody.

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particl... more The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform f...

Biologia plantarum

Small RNAs (sRNAs) are essential components of gene-regulatory networks, which guide plant develo... more Small RNAs (sRNAs) are essential components of gene-regulatory networks, which guide plant development and tune it to environmental challenges. Though the past years have witnessed evidences on sRNA importance for stress response, there is scarce data on their involvement in resurrection plant survival under severe drought. Haberlea rhodopensis (hrh) is an angiosperm resurrection species, whose vegetative tissues can tolerate desiccation and recover upon rehydration. In this study, high-throughput sequencing sRNAs indicated a higher complexity of the sRNA population, especially of a 24 nt sRNA category, in the desiccated vegetative tissue of H. rhodopensis compared to unstressed tissues. The cross-species discovery was performed to predict 77 mature microRNAs (miRNAs), most of which were assigned to 23 high-confidence conserved miRNA families in the leaf tissue. Several members of the miR156/157, miR166, and miR399 families were found to be desiccation-responsive. The miR156/157 family members were found up-regulated upon dehydration and down-regulated upon rehydration, while the miR166 and miR399 family members followed an opposite trend of expression. A probable miR156/157 target, orthologous to the SQUAMOSA promoter binding proteinlike, was reconstructed in H. rhodopensis based on genomic data available for this species and the closely related Boea hygrometrica. Reverse transcription quantittative PCR analysis confirmed the expression profile of hrh-miR156a-5p and hrh-miR157-5p established by sRNA sequencing and revealed an inverse expression pattern between these miRNAs and their targets in the desiccated tissue. Our study suggests that the miR156/157 and miR399 families are essential for plant survival under severe drought due to their ability to control plant development and growth by modulating transcription factor expression.

Vaccines

Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in develop... more Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous partic...

Animals

Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is undisputed... more Hepatitis E virus (HEV) is a lesser-known hepatitis virus, but its worldwide spread is undisputed and has increased in recent years. The zoonotic spread of HEV, mainly due to genotype (gt) 3, emerged in developed countries in the past decade. In addition, transmission via contaminated meat from pigs and boars was also established. Detailed analysis of viral dynamics and distribution is needed in order to identify associated risk factors. The aim of the current study is to present new and additional data on the HEV distribution among pigs, and for the first-time, also among the wild boar population in Bulgaria. Abstract: (1) Background: Hepatitis E virus (HEV) is a causative agent of acute viral hepatitis, predominantly transmitted by the fecal-oral route. In developed countries, HEV is considered to be an emerging pathogen since the number of autochthonous cases is rising. Hepatitis E is a viral disease with a proven zoonotic potential for some of its genotypes. The main viral reservoirs are domestic pigs and wild boar. Consumption of undercooked meat, as well as occupational exposure, are key factors for the spread of HEV. In order to evaluate the risks of future viral evolution, a detailed examination of the ecology and distribution of the virus is needed. The aim of the present study is to investigate the prevalence of anti-HEV IgG Ab in domestic pigs and wild boar in Bulgaria; (2) Methods: In this study, during the period of three years between 2017 and 2019, 433 serum samples from 19 different pig farms and 1 slaughterhouse were collected and analyzed. In addition, 32 samples from wild boar were also collected and analyzed during the 2018-2019 hunting season. All samples were analyzed by commercial indirect ELISA; (3) Results: Overall, HEV seroprevalence was 60% (95% CI 42.7-77.1) in domestic pigs and 12.5% (4/32) in wild boar. The observed seroprevalence of the slaughter-aged pigs was 73.65% (95% Cl 58.7-87.3). Prevalence in domestic pigs was significantly higher in the samples collected during 2019 (98% (95% Cl 96.1-99.9)) compared to those collected during 2017 (45.33% (95% CI 2.7-87.3)) and 2018 (38.46% (95% CI 29.1-49.7.); (4) Conclusions: Our findings suggest that domesticated pigs and wild boar might be the reason for the increased HEV transmission across Bulgaria. The genotypic characterization of HEV found in pigs, wild boar and humans will give a more accurate view of the zoonotic transmission of this virus.

Plant physiology, Jan 22, 2018

The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any ... more The desiccation-tolerant plant Haberlea rhodopensis can withstand months of darkness without any visible senescence. Here, we investigated the molecular mechanisms of this adaptation to prolonged (30 days) darkness and subsequent return to light. H. rhodopensis plants remained green and viable throughout the dark treatment. Transcriptomic analysis revealed that darkness regulated several transcription factor (TF) genes. Stress- and autophagy-related TFs such as ERF8, HSFA2b, RD26, TGA1, and WRKY33 were upregulated, while chloroplast- and flowering-related TFs such as ATH1, COL2, COL4, RL1, and PTAC7 were repressed. PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a negative regulator of photomorphogenesis and promoter of senescence, was also downregulated. In response to darkness, most of the photosynthesis- and photorespiratory-related genes were strongly downregulated, while genes related to autophagy were upregulated. This occurred concomitant with the induction of SUCROSE NON-FERMENTING...

RNA-dependent DNA methylation (RdDM) is an important regulatory event involved in repressive epig... more RNA-dependent DNA methylation (RdDM) is an important regulatory event involved in repressive epigenetic modifications that can trigger transcriptional gene silencing (TGS). The criteria we used to pick out promoter sequences targeted by RdDM in Arabidopsis thaliana were the main RdDM hallmark properties: 24nt siRNAs as inducers of DNA methylation and transposable elements (TE) as one of the major targets of RdDM. Those genes whose promoters comprised overlapping sites for 24nt siRNA hits, TE and DNA methylation (siRNA/TE/Methylation overlapping regions), were defined as candidates that might be silenced by RdDM. On this basis two gene sets were created which include abiotic and biotic stress-responsive genes whose promoters may be silenced by RdDM. The DNA methylation status of the At3g50770 (CML41) promoter e one of the selected candidates, was experimentally assayed, and it showed dependence on the RdDM-associated Polymerase IV and Polymerase V. A publicly available 24nt siRNA-centered database called starPRO was developed that allows users easily to discover whether a particular promoter sequence is related to RdDM-associated features such as 24nt siRNA-target sites, TE, tandem repeats and DNA methylation.

ABSTRACT Haberlea rhodopensis (Gesneriaceae) is a less explored endemic plant within a very small... more ABSTRACT Haberlea rhodopensis (Gesneriaceae) is a less explored endemic plant within a very small group of poikilohydric angiosperms that are able to survive in an almost completely dehydrated state. The purpose of the present study was to obtain extracts from Haberlea rhodopensis leaves using alternative technique- microwave-assisted extraction and to determine the total phenolic content and antioxidant activity of the extract by applying reliable methods: reducing power and ABTS radical cation decolorization assay. Different extraction conditions- microwave power and extraction times were investigated. Total phenolic content was determined using a spectrophotometric technique based on the Folin-Ciocalteu reagent and calculated as mgGAE/gDW (leaf dry weight). The results from the total phenolic assay and the antioxidant activity tests were significantly correlated. Major phenolic acids and some flavonoid- aglycones and - glycosides were analyzed by HPLC. Luteolin (3307.08 μg/gDW), myricetin (702.0 μg/gDW), ferulic acid (652.92 μg/gDW) and hesperidin (607.32 μg/gDW) were predominant in the H. rhodopensis microwave-assisted extracts and some of the effects of the plant could be attributed to the presence of these valuable constituents.