Studies on the genetic diversity of arthropod-borne viruses circulating in rural regions can provide critical early indications on new emerging viruses essential for global epidemic preparedness. In this study, we describe the discovery of four phleboviruses in sandflies from the Kenyan Rift Valley. The novel viruses are related to the two medically important serocomplexes, sandfly fever Naples and sandfly fever Sicilian, that are associated with febrile illness and neuroinvasive infections and which were previously not known to occur in sub-Saharan Africa. Knowledge on the occurrence of sandfly-borne phleboviruses in Kenya and elsewhere in Africa can help to decipher their contributions in the etiologies of fevers of unknown origin in patients. Our findings on five genetically diverse phleboviruses detected in Kenya suggest that the common ancestor of Old World phleboviruses existed in sub-Saharan Africa, a hot spot for emerging arboviruses. Phenuiviridae, order Bunyavirales) are associated with febrile illness and infections of the nervous system in humans. These viruses are almost exclusively found in tropical areas of the New World and restricted to semiarid and temperate zones in the Old World. Here, we discovered seven strains of four previously unknown phleboviruses, named Bogoria virus (BOGV), Embossos virus (EMRV), Kiborgoch virus (KBGV), and Perkerra virus (PERV), as well as the recently discovered Ntepes virus, in sandflies collected in the Kenyan Rift Valley. The genomes have a tripartite organization with conserved termini typical of phleboviruses. LOBV, PERV, and EMBV showed low similarity to known phleboviruses, with less than 55% pairwise amino acid identities in the RNA-directed RNA polymerase (RdRp) proteins, and defined a highly diversified monophyletic clade in sister relationship to the sandfly fever Sicilian serocomplex. All three viruses failed to react with sandfly fever Sicilian virus antisera in recombinant immunofluorescence assays (rIFA), suggesting that the viruses belong to a yet-unknown serogroup. In contrast, KBGV was closely related to Toscana virus (84% identity of RdRp proteins) and shared a most recent common ancestor with the clade comprising sandfly fever Naples and Toscana viruses. KBGV reacted with sandfly fever Naples and Toscana virus antisera in rIFA. The genetic diversity of the detected viruses and their phylogenetic positions implies that the Old World sandfly-borne phleboviruses originated from sub-Saharan Africa. Importantly, our findings suggest that diseases associated with sandfly-borne phlebovirus infections may also affect the Kenyan population.Sandfly-transmitted phleboviruses (family IMPORTANCE Studies on the genetic diversity of arthropod-borne viruses circulating in rural regions can provide critical early indications on new emerging viruses essential for global epidemic preparedness. In this study, we describe the discovery of four phleboviruses in sandflies from the Kenyan Rift Valley. The novel viruses are related to the two medically important serocomplexes, sandfly fever Naples and sandfly fever Sicilian, that are associated with febrile illness and neuroinvasive infections and which were previously not known to occur in sub-Saharan Africa. Knowledge on the occurrence of sandfly-borne phleboviruses in Kenya and elsewhere in Africa can help to decipher their contributions in the etiologies of fevers of unknown origin in patients. Our findings on five genetically diverse phleboviruses detected in Kenya suggest that the common ancestor of Old World phleboviruses existed in sub-Saharan Africa, a hot spot for emerging arboviruses. Phenuiviridae, order Bunyavirales) of the Old World have mainly been reported in the Mediterranean area, in North Africa, and in Western and Central Asia  and sandfly fever Sicilian serocomplex (comprising sandfly fever Sicilian virus and related viruses). Toscana virus is the only sandfly-borne phlebovirus which has a neurotropism; it typically causes meningitis and encephalitis, but other neuroinvasive manifestations, such as Guillain-Barré syndrome, hydrocephalus, myositis, fasciitis, polymyeloradiculopathy, deafness, and facial paralysis, have also been reported . Toscana10–Gordil phlebovirus, Massilia phlebovirus, Naples phlebovirus, Punique phlebovirus, Saint Floris phlebovirus, Tehran phlebovirus, Toscana phlebovirus, and Zerdali phlebovirus by the International Committee on Taxonomy of Viruses (ICTV). The sandfly fever Sicilian serocomplex comprises the ICTV-classified species Dashli phlebovirus, Toros phlebovirus, and Sicilian phlebovirus. Several additional phleboviruses that group in phylogenetic analyses together with Toscana virus and sandfly fever Naples virus have been described. Whereas the majority of these viruses were detected in sandflies from the Mediterranean, e.g., Massilia virus in Portugal and France or Punique virus in Tunisia, two distantly related viruses, namely, Gordil virus and Saint Floris virus, were isolated from rodents in the Central African Republic in 1971  in sandflies from Kenya . NeutralIn 2015 and 2016, 3,958 phlebotomine sandflies were collected in and around households in the villages of Ntepes and Kapkuikui from dusk until dawn . SpecimeCOI gene was amplified, and 10 clones were sequenced from each virus-positive sandfly pool . Of note, sequences for which no clear species association was possible were also detected in the PERV-positive pools SP162 and SP166, the EMBV-positive pool SP288, the NPV-positive pool SP375 and the KBGV-positive pool SP381. In addition to S. schwetzi, Sergentomyia inermis sandflies were identified in the EMBV-positive pool SP288 and Sergentomyia dreyfussi in the NPV-positive pool SP375.To estimate which type of sandflies were present in the virus-positive pools, a fragment of the invertebrate fly pool . SergentCOI gene. Sequencing of the amplicons of sandfly pools SP105 (positive for BGRV) and SP166 (positive for PERV) targeting the COI gene resulted in high similarities to the mitochondrial genome of humans  and sequencing of SP381 (positive for KBGV) showed high similarity to cattle (98.7% Bos taurus) in GenBank database searches. No amplicon was obtained from the remaining virus-positive samples  (We further sought to identify the vertebrate sources on which the virus-positive sandflies had fed using a PCR targeting the vertebrate d SP394) .https://pfam.xfam.org) . Sequence gaps were closed using seminested RT-PCR, resulting in complete coding sequences (CDS) and almost complete noncoding regions. All viruses showed a tripartite genome organization comprising a large (L), a medium (M), and a small (S) segment . The L sfam.org) . The S sPhlebovirus. A related taxonomic proposal has been submitted to the ICTV.According to the phlebovirus species demarcation criteria of the ICTV, unique species show more than 5% distance in their RdRp protein sequences . The det233Glu to 233Leu) and various synonymous nucleotide substitutions randomly distributed across the N genes: four for EMBV and five for PERV.To test for intraspecies diversity, we sequenced the N genes of EMBV and PERV based on virus-specific primers flanking the N ORF. We observed a single nonsynonymous substitution for EMBV (508Ser to 508Asp), GPC (433Lys to 433Arg), and NSs  genes of the newly sequenced NPV strain SP375-KE-2016 derived directly from sandflies. No nonsynonymous nucleotide substitution was detected in the N gene.In addition, the entire CDS of NPV was directly sequenced from the sandfly homogenate and compared to the strain initially sequenced from infectious cell culture supernatant originating from sandflies collected in the same geographic region in 2014 . For thePhylogenetic analyses based on the RdRp proteins consistently showed that BGRV, EMBV, and PERV form a diversified monophyletic clade in sister relationship to the clade comprising the sandfly fever Sicilian viruses, Dashli virus, and Toros virus , whereasAttempts to isolate the viruses in cell culture using cell lines derived from sandflies, mosquitoes, and nonhuman primates failed. Thus, we used synthesized N-gene constructs of EMBV, BGRV, KBGV, and PERV to establish recombinant immunofluorescent assays (rIFA) to test for cross-reactivity of the newly detected viruses with related serogroups. Incubation of the Toscana virus N antiserum with overexpressed KBGV N showed prominent reactivity, whereas BGRV N, EMBV N, and PERV N did not react with the antiserum . These rn = 244) collected from patients from Marigat subcounty with fevers of unknown origin were investigated by specific real-time RT-PCR to probe for direct evidence of the virus in human blood. All serum samples tested negative for BGRV, EMBV, PERV, and KBGV. Unfortunately, testing for the presence of antibodies against the four viruses was not carried out due to the paucity of material.Extracted RNA of individual human serum samples , as well as the detection of NPV in sandflies from an area with tropical savanna climate in sub-Saharan Africa. The viruses are distantly related to the medically important sandfly fever Sicilian serocomplex , sandfly fever Naples serocomplex (KBGV), and Karimabad serocomplex (NPV), indicating the circulation of taxonomically highly diverse sandfly-borne phleboviruses in Kenya. We recently described the discovery and isolation of NPV from sandflies collected in Ntepes village, Marigat district, in 2014 . The repSergentomyia schwetzi being the most abundant species reported to feed on humans, cows, goats, and rabbits (–n = 10) were used in these analyses, the blood meal sources may also stem from a sandfly that was not infected with any of the viruses. Further studies aiming at the identification of the vertebrate host and sandfly species involved in maintenance of BGRV, EMBV, PERV, and KBGV will be key to understanding the ecology of these viruses. In addition, studies involving testing of human sera are needed to identify if humans can be infected with the newly detected viruses and whether infections are associated with symptoms of disease.BGRV, EMBV, PERV, and KBGV were exclusively detected in sandflies collected in one of the two sampling locations, in the village Kapkuikui . BGRV, E rabbits –25. HoweIn the absence of data on virus isolation, preliminary serological investigations were conducted using rIFA based on expressed N proteins. None of the N proteins of BGRV, EMBV, and PERV reacted with antisera against the phylogenetic related group of sandfly fever Sicilian viruses or with antisera against the sandfly fever Naples serocomplex and Toscana virus N protein, suggesting that the three viruses belong to a previously unknown serogroup. The absence of serological cross-reactivity of BGRV, EMBV, and PERV with the known sandfly-borne serogroups may have prevented earlier detection of sandfly-borne phleboviruses in Kenya. Although KBGV showed reactivity with antisera against the sandfly fever Naples serocomplex and Toscana virus N protein, antibodies against the latter two have so far not been identified in Kenya. Antibodies against the sandfly fever Naples serocomplex were found in Ethiopia and Djibouti but not in Somalia, Senegal, Liberia, Kenya, and Sudan in the 1970s , 9.Sergentomyia have been suggested to be involved in the transmission of NPV  originating from a restricted ecology of the Kenyan Rift Valley, the presence of additional sandfly-borne phleboviruses in Kenya and elsewhere in sub-Saharan Africa is highly likely. Taken together with our previous discovery of NPV, our findings represent the southernmost detection of sandfly-associated phleboviruses of potential public health significance in the Old World. Beside mosquitoes and ticks, sandflies should be included in arbovirus surveillance programs focused on epidemic preparedness in Kenya and beyond.The detection of four highly diverse novel phleboviruses distantly related to the sandfly fever Sicilian and sandfly fever Naples serocomplexes implies that sandfly-borne infections and associated diseases contribute to the health burden in Kenya. Since these viruses were found in a relatively small number of sandflies  CDC light traps emitting different wavelengths of light . Traps were placed about 1 m aboveground in and around households and operated for 12 h from dusk until dawn. Adult sandflies were recovered from the field immediately after sunrise, immobilized using triethylamine, transported in liquid nitrogen to the laboratory at the International Centre of Insect Physiology and Ecology (ICIPE), and stored at −80°C until further processing.www.ncbi.nlm.nih.gov/genbank/).Sandflies were organized into pools of 10 individuals each, according to collection date and location. Pools were homogenized in 500 μl phosphate-buffered saline (PBS)  using ceramic beads and a SpeedMill Plus . A 50-μl portion of cleared supernatant from each of 10 pools was used to generate superpools, of which 140 μl was used for RNA extraction using the QIAamp viral RNA minikit . Random hexamer-primed cDNA was synthesized using the SuperScript III RT system  according to the manufacturer’s instructions, and superpools were tested for phleboviruses as described earlier . ObtaineVirus-specific quantitative real-time RT-PCR assays were established for BGRV , EMBV , KBGV , and PERV  and used to test individual pools of virus-positive superpools and cell culture supernatants. The assay used for the detection of NPV was described earlier .Aedes albopictus using bwa mem 0.7.15-r1140 (http://www.cbs.dtu.dk/services/NetNGlyc/).Sample libraries were prepared from RNA of phlebovirus-positive sandfly pools using the KAPA HyperPlus kit  and sequenced using the Illumina MiSeq HTS platform as described earlier . After d15-r1140 . For the15-r1140  search w15-r1140  (downloa15-r1140  (downloa15-r1140 . Genome 15-r1140 . PCR pro15-r1140 . Geneiou15-r1140  was usedc oxidase subunit I (COI) gene as described earlier  as suggested by Valinsky and colleagues  in coeluted DNA from RNA extracts  genes were amplified from each detected virus strain by RT-PCR using gene-specific primers. PCR products were sequenced by Sanger sequencing.Amino acid sequences of the L, Gn, Gc, and N proteins were aligned using the MAFFT E-INS-I algorithm . Phyloge4 cells were spotted onto multitest cover slides. Cells were fixed using ice-cold acetone-methanol (1:1 ratio). In addition, commercial multitest cover slides from Euroimmun  containing cells infected with sandfly fever Sicilian virus (SFSV), sandfly fever Naples virus (SFNV), and Toscana virus (TOSV) were used. Humanized rabbit anti-TOSV N monoclonal antibodies, rabbit anti-FLAG antibodies, and human serum samples reactive against SFSV, SFNV, and TOSV were diluted in sample buffer  and applied on the multitest cover slides coated with transfected VeroE6 cells. Secondary fluorophore-labeled goat anti-rabbit IgG–Alexa Fluor 488 and goat anti-human IgG–Cy2 antibodies  were applied after washing steps to the corresponding samples, and multitest cover slides were examined using a fluorescence microscope.Synthesized FLAG-tagged full-length N genes of BGRV, EMBV, KBGV, and PERV were purchased from Integrated DNA Technologies  and cloned into the pCG1 vector. VeroE6 cells were transfected with the respective construct, and 1.25 × 10Chlorocebus aethiops) VeroE6 cells, mosquito  C6/36 cells, rhesus macaque (Macaca mulatta) LLC-MK2 cells, sandfly  LL-5 cells, sandfly (Phlebotomus papatasi) PP-9 cells, and Syrian hamster (Mesocricetus auratus) BHK-21 cells were used to perform virus isolation trials from diluted sandfly homogenates (1:10 and 1:100) as described previously (Grivet (eviously . Cells wn = 244) were collected from patients with fever of unknown origin hospitalized in Marigat subcounty. A 5-μl portion of individual sera was used for RNA extraction using the QIAamp viral RNA minikit . cDNA was synthesized using random hexamer primer and the SuperScript III RT system . Virus-specific quantitative real-time RT-PCR assays as mentioned above were used to test individual extracted RNA for direct evidence of the viruses in human blood.Human serum samples .MT270825 to MT270836 and those of NPV under accession numbers MT625964 to MT625966. Partial N-gene sequences of additional strains of Embossos virus and Perkerra virus are deposited under accession numbers MT625967 and MT625968. The COI sequences of sandflies and blood meal sources have also been deposited in GenBank under accession numbers SAMN15848018, SAMN15848019, SAMN15848020, and SAMN15848021. HTS data were deposited in the Sequence Read Archive (SRA) under accession no. PRJNA657829.Coding-complete genomes of Bogoria virus, Kiborgoch virus, Perkerra virus, and Embossos virus have been deposited in GenBank under accession numbers


Capsicum annuum L.) is an important vegetable and spice crop of tropical and sub-tropical regions. Chili plants showing upward leaf curling, leaf crinkling, and leaf yellowing symptoms, collected from Sikar district of Rajasthan, India, were found to be associated with begomovirus and satellite molecules. The presence of virus was confirmed by PCR using virus-specific primer. The full-length genomic DNA-A of three begomovirus  and two satellites (MM-2 and MM-3) were cloned which was identified from single symptomatic chili plant. The genome organization of isolated three viruses is similar to those of other Old World monopartite begomoviruses. The comparison of the sequences and closest phylogenetic relationships for the begomoviruses, betasatellite and alphasatellite DNAs revealed that MM-1 was designated as DNA-A of Chili leaf curl virus (ChiLCV), CS-1 is considered to be a new distinct species of Tomato leaf curl Gujrat virus (ToLCGV) whereas RV-1 as a new strain of Cotton leaf curl Multan virus (CLCuMuV). The DNA-A component of ChiLCV showed 8.6%, ToLCGV of 16.6% and CLCuMuV of 7.7% average evolutionary divergence, concomitantly, the betasatellite and alphasatellite molecule had 9.9% and 5.9% overall sequence divergence, respectively. Interestingly, most of the begomoviruses were found to be intra-species recombinants. The dN/dS ratio and Tajima D value of all viral DNA-A component and their associated betasatellite showed their selective control on evolutionary relationships. The nucleotide substitution rates were determined for the DNA-A genomes of ChiLCV (7.22 × 10–4 substitutions site–1 year–1), CLCuMuV (1.49 × 10–4 substitutions site–1 year–1), ToLCGV (7.47 × 10–4 substitutions site–1 year–1), the genome of associated ChiLCB (4.20 × 10–4 substitutions site–1 year–1) and CLCuMuA (1.49 × 10–4 substitutions site–1 year–1). Agro-inoculation studies indicate that the presence of DNA betasatellite induce severe symptoms in N. benthamiana and chili, suggesting prerequisite association for typical disease development.Chili ( Geminivirade into nine genera: Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus  and movement protein (MP) respectively, complementary sense strand has AC1, AC2, AC3, and AC4 ORFs encoding replication associated protein (Rep), transcriptional activator protein (TrAP), replication enhancer protein (REn) and AC4 protein, respectively . DNA satlication . Likewislication  via its lication . The SCRlication .Cucumber mosaic virus (CMV), Pepper venial mottle virus (PVMV), Tobacco leaf curl virus (TLCV), Potato virus X (PVX), Potato virus Y (PVY), Tobacco ring spot virus (TRSV), Pepper vein bending virus (PVBV), Tomato leaf curl New Delhi virus (ToLCNDV), Chili mosaic virus and Capsicum chlorosis virus.Chili is known due to its active ingredient Capsaicin. It is an excellent source of Calcium and Vitamin C, while studies showed its anti-proliferative effect against cancer sinusitis and bronchitis etc. In India, Chili is cultivated almost in every state, including Andhra Pradesh, Karnataka, Rajasthan, Tamil Nadu, Maharashtra, Orissa, and West Bengal. In India, during 2017-18, green chilies were cultivated in 309000 hectares with a total production of 3592000 metric tonnes . Leaf CuTomato leaf curl virus and ChiLCV in chili plant was also previously reported in India (Tomato leaf curl Gujrat virus (ToLCGV) and ChiLCV in Chili.Mixed infection of in India , howeverEmergence of new variants of viruses due to recombination and mutations in the genomes, introduction of susceptible plant varieties and change in climatic conditions are responsible aggravating the increasing incidence and disease severity problems of begomovirus during the last two decades . In IndiIn this study, we have identified and characterized three distinct begomoviruses and association of alphasatellite and betasatellite with chili plant showing leaf curl symptoms from Sikar, India. We further, analyzed the sequence variability, phylogenetic relationship, and recombination breakpoints in the genome sequences of ChiLCD.During the routine survey (2017-18) for begomovirus infection, chili plants showing upward leaf curling, leaf crinkling, and leaf yellowing symptoms  were colHindIII (for ChiLCV and ToLCGV), BamHI (CLCuMuV and CLCuMuA) and KpnI (for ChiLCB). The resultant products of ∼2.7 and∼1.3 kb, were eluted and cloned into pUC19 vector  and designated as M1 (ChiLCV), CS-1(ToLCGV), RV-1(CLCuMuV), MM2 (ChiLCB) and MM3 (CLCuMuA)  was carried out using CP gene-specific degenerate primers AC-1048 (5′GGRTTDGARGCATGHGTACATG3′) and AV-494 (5′GCC YATRTAYAGRAAG CCMAG3′) . Beta01 CLCuMuA) . To veriCLCuMuA) –5. The oT v.1.21 .We performed Model Test to identify the best nucleotide substitution model by means of the lowest BIC score and subsequently Maximum-likelihood (ML) phylogenetic tree for each data set was constructed by using MEGA X  with 1007 chain length in the Bayesian Markov Chain Carlo method in BEAST v.1.8.4 . The full-length insert of both the clone, released using HindIII, ligated into the binary vector to Chi-A and Tom-A infectious clone. Similar strategy was used for the infectious clone of RV-1 in pCAMBIA1304 using a ∼1000 bp BamHI-SalI fragment and full-length insert using BamHI to produce Cot-A. For, infectious clone of MM2, KpnI-XbaI is used for the release of ∼600 bp from the ChiLCB clone and ligated into the binary vector followed by full length insert using KpnI named as Chi-β. Further, a fragment of ∼550 kb was cut from MM3 clone using BamHI and EcoRI and cloned into pCAMBIA1304. Then, the complete genome of CLCuMuA was excised from MM3 insert using BamHI and ligated to binary vector to get a binary plasmid Cot-α. All the infectious clone contains the IR to ensure successful infection.Infectivity of the three cloned begomovirus and associated satellites was studied in n system . DimericAgrobacterium tumefaciens LB 4401 by electroporation with a Gene Pulser apparatus . Agro-inoculation was performed as described by N. benthimiana plants. The inoculated plants were incubated in a growth chamber with 16h day light at 25 ± 2°C. For confirmation, DNA blots were hybridized with α32P-labeled dCTP coat protein gene (AV1) of previously mentioned begomoviruses and betasatellite and alphasatellite by nick translation. Viral DNA blots were detected using a phosphor image analyzer.The insertion and orientation were confirmed by restriction digestion with appropriate enzymes. Recombinant plasmids and empty vector were introduced into MF737343, MF737345, and MF737344). The genome organizations of these viruses were like those of Old World begomoviruses constructed by using PlasMapper tool .Based on nucleotide sequence identity (91%) as the species demarcation threshold for DNA-A , these iChili leaf curl betasatellite (ChiLCB) and MM3 isolate sowed similarity with AR4 isolate  from Pakistan therefore, designated as Cotton leaf curl Multan alphasatellite (CLCuMuA)  therefore, designated as CLCuMuA) . The nucCLCuMuA) .Papaya leaf curl virus (PaLCuV). The coding nucleotide sequence of the CLCuMuV (MF737345) shared 88.12– 100% similarity with various isolates of Okra enation leaf curl virus (GU111996), Pepper leaf curl Lahore virus (AM691745), Ageratum enation virus (KJ488991) and CLCuMuV (KC412251)  from Pakistan. Based on the highest percentage identity, our result showed that there is a frequent recombination and mutation in the sequence of present study.The predicted nucleotide sequences of the ORFs  of all the begomovirus isolates and associated satellite molecules were also compared with other begomoviruses . The ORFC412251) . The maxdN) and synonymous (dS) nucleotide positions because the dN/dS ratio can be used as an index representing the pattern of selective constraint in evolutionary relationships  as compared to the ChiLCB  was 0.94. In addition, ToLCGV transition rates ranged from 9.75 to 19.22 and the transversion rates range from 4.79 to 7.19 and the transition/transversion bias (R) was calculated to be 1.08, likewise, the transition rates and transversion rates of CLCuMuV ranged from 13.43 to 15.42, 5.01 to 7.30, respectively and the transition/transversion bias (R) was 1.04. Sequence comparison of DNA-A isolates with other geographical isolates showed average evolutionary divergence ranged from 2.1% to 10.3% . Whereasionships . The dN/e ChiLCB .To analyze the phylogenetic relationship, evolutionary history was inferred using the ML Tree based on the best fit nucleotide substitution model . In a phTo determine the incidence of recombination, RDP analysis was conducted based on alignments with full-length sequences of the top 25 hit retrieved from the NCBI database. We used several algorithms  implemented in RDP 4.2 and perceived DNA-A genomic region of CLCuMuA and ToLCGV which has high number of recombination events . The βC1–4) than ChiLCV and ToLCGV and suggests rapid evolution. Since mutation plays a significant role in selection process leading to genetic variation, we measured the rate of mutation of all the three codon position and found that ChiLCV isolate MM1, CLCuMuV isolate RV1 had higher mutation rate in codon position 1 and high mutation rate was observed in codon position 2 in ToLCGV isolate CS1  i.e., 0.0914  and showlate CS1 .Additionally, haplotype sequence polymorphisms and diversity are also studied for ChiLCV based on host . RegardiN. benthamiana and Chili plants after 21 dpi , Chili leaf curl India virus (ChiLCINV), Chili leaf curl Kanpur virus (ChiLCKaV), Chili Leaf curl Vellanad virus (ChiLCVV), Pepper leaf curl Bangladesh virus (PepLCBV), Tomato leaf curl New Delhi virus (ToLCNDV), Papaya leaf curl virus (PaLCuV), Radish leaf curl virus (RaLCuV), Tomato leaf curl virus (ToLCV) and Tomato leaf curl Joydebpur virus (ToLCJV) have been reported in chili crop, thereby causing leaf curl disease  and Pepper golden mosaic virus (PepGMV) on pepper in Mexico (Tomato leaf curl New Delhi virus, Squash leaf curl China virus and Tomato leaf curl Palampur virus infecting pumpkins in India (Several reports such as geminiviruses infecting cassava in Africa , suggestn Mexico , an assoin India . In Indiin India . These rThe diverse Indian climate supports the year-round cultivation of various crops and the survival of whiteflies. This is the reason a huge number of begomoviruses have been reported in India. The polyphagous nature of vector whitefly and mixed cropping system of India is responsible for its overlapping host range. Earlier studies have demonstrated the ChiLCV infecting variety of crops like tobacco, tomato, papaya, eggplant, petunia . CLCMuV MF737343, MF737344, MF737345, MF737346, and MF737349.The datasets generated for this study can be found in NCBI, accession numbers RG and PS designed and analyzed the data and wrote and edited the first draft. MM and RV contributed equally to the experiments and computational work. AM helped in computational analysis. All authors read and approved the manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heterothermy allows organisms to cope with fluctuating environmental conditions. The use of regulated hypometabolism allows seasonal heterothermic species to cope with annual resource shortages and thus to maximize survival during the unfavorable season. This comes with deep physiological remodeling at each seasonal transition to allow the organism to adjust to the changing environment. In the wild, this adaptation is highly beneficial and largely overcomes potential costs. However, researchers recently proposed that it might also generate both ecological and physiological costs for the organism. Here, we propose new perspectives to be considered when analyzing adaptation to seasonality, in particular considering these costs. We propose a list of putative costs, including DNA damage, inflammatory response to fat load, brain and cognitive defects, digestive malfunction and immunodeficiency, that should receive more attention in future research on physiological seasonality. These costs may only be marginal at each transition event but accumulate over time and therefore emerge with age. In this context, studies in captivity, where we have access to aging individuals with limited extrinsic mortality , could be highly valuable to experimentally assess the costs of physiological flexibility. Finally, we offer new perspectives, which should be included in demographic models, on how the adaptive value of physiological flexibility could be altered in the future in the context of global warming. In seasonal environments, resource availability fluctuates over the year, mainly between a phase of resource abundance and a phase of resource shortage. Heterothermy is an adaptation to these seasonal changes, which are mainly periodic and therefore predictable, in environmental conditions. Heterothermy is defined as “the pattern of temperature regulation” in an endothermic species  “in which the variation in core temperature, either nychthemerally or seasonally, exceeds that which defines homeothermy” . To copeHeterothermy comes with both ecological and physiological benefits . OverallIn this paper, we propose new perspectives on the marginal costs of seasonal flexibility. We do not intend to provide an extensive review of the literature on the regulation of seasonal heterothermy but rather to shed light on putative physiological costs that have been overlooked so far and should be considered in future research. We propose that these costs should be better supported by experimental evidence to be further included in studies modeling the interactions between heterothermy and environmental changes .Heterothermy brings ecological benefits. First, matching metabolic activity with resource availability maximizes survival, as the organism is less constrained by energy and water shortages. The match of metabolic activity to resource fluctuations in SH also enhances reproductive success. Second, heterothermy promotes metabolic flexibility, i.e., the ability to adjust the origin of substrates used for oxidative metabolism, switching from carbohydrate to lipid usage. Metabolic flexibility is thought to enhance the control of energy balance and has even been proposed to favor longevity . RegulatHowever, seasonal heterothermy may also come with ecological costs. In the wild, these costs are compensated by the benefits of heterothermy and are therefore hidden (not observable). However, several recent studies revealed such costs. For example, some species avoid using torpor when environmental conditions are good enough e.g., . This suThere is an apparent paradox between the fact that seasonal heterothermy is an adaptation to predictable environmental changes and the recent hypothesis that the associated circannual physiological changes are detrimental to the organism in terms of increased mortality and early emergence of aging phenotypes . Indeed,We discuss here several possible ways in which frequencies and magnitudes of seasonal physiological transitions may shape mortality over age.Microcebus murinus) by h’(t) of mortality at a given age was increased). This supports accelerated aging in line with empirical observation that accelerated individuals exhibit early appearance of phenotypes and pathologies associated with aging (h’(t)/h(t)  was constant.th aging . Howeverb of the Gompertz function. For instance, 1 year of time of reference individuals was equivalent to 2 years for accelerated individuals in h’(t) and h’(t)/h(t) were larger in accelerated individuals. This model would have been a perfect fit for the expectation that increasing transition frequency accelerates biological aging. This was, however, not supported by the data in An alternative effect of seasonal transitions could have been an accelerated failure time effect AFT, . In thisLet us now focus on changes in the magnitude of mortality fluctuations with age. Microcebus murinus; Increased frequency of seasonal transitions has been linked to increased mortality in captive gray mouse lemurs . Hence, the physiological costs that damage the organism over the long term  may actually also operate on a daily basis . If the costs remain unchanged, the cost/benefit imbalance should reduce the net selective advantage of seasonally flexible genotypes, with a reduction in net immediate survival, reproduction, or growth, or an increase in the aging rate. Costs that are negligible compared to benefits in current environments may become crucial in determining future population dynamics in the case of environmental changes. In this respect, opportunistic heterotherms but not SH should be advantaged by environmental changes . Indeed,The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.JL, SP, P-YH, and JT contributed to the conception and writing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vitis vinifera L.). While both have been shown to be useful to eliminate the main grapevine viruses, their efficiency differs depending on the virus and grapevine variety. In our work, we investigated the efficiency of these two virus elimination methods using small RNA high-throughput sequencing (HTS) and RT-PCR as virus diagnostics. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids, were selected for elimination via somatic embryogenesis (SE) and meristem culture (ME). Our results show for the first time that using SE, elimination in mother plants was effective for all viruses, i.e., grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), Grapevine virus T (GVT) and grapevine Pinot gris virus (GPGV). This study also confirms previous studies showing that SE is a possible strategy for the elimination of GFkV, GRSPaV, HSVd, and GYSVd-1. Our results demonstrate that the efficacy of virus elimination via SE is relatively high while the purging of viroids is lower. Our work provides evidence that the efficiency of SE is comparable to that of the technically difficult ME technique, and that SE will offer a more effective strategy for the production of virus-free grapevine in the future.Meristem culture and somatic embryogenesis are effective tools for virus elimination of vegetatively propagated crops including grapevine ( Vitis vinifera is a recalcitrant species for in vitro culture, and different cultivars exhibit different responses [Vineyards represent investments that often persist for many decades. To ensure their health, it is essential to use high quality propagating material (rootstocks and scions), free from pathogens, during their establishment. The lack of efficient methods to produce and verify clean planting stock is especially important in case of phytoplasmas and viruses. The elimination of phytoplasmas may be accomplished by a simple hot water treatment of the canes, while virus elimination requires more complex and specialized methods that may vary among different pathogen species. More than 80 viruses and many viroids can infest grapevine, including some that cause important and economically-significant diseases , and recesponses . The effesponses ,5. The mesponses .Somatic embryogenesis (SE) is an alternative method to overcome these challenges ,8, and hThe efficiency of the virus elimination is generally monitored by virus diagnostic methods, mostly by DAS-ELISA, RT-PCR, RT-qPCR alone or in different combinations , ‘Trilla’, and ‘Sziren’ , were selected for virus elimination. Virus detection in these four mother plants was done via small RNA HTS see .The mother plants were infected with four to six viruses, including GFkV, GRVFV, GSyV-1, GRSPaV, GVT, and GPGV, as well as two viroids, HSVd and GYSVd-1. The results of the small RNA HTS and RT-PCR were very similar for GFkV, GRVFV, GSyV-1, and GPGV, as well as for the viroids .In contrast, and in agreement with our previous observations, small RNA HTS failed to detect GRSPaV infection ,27; we oEmbryogenic callus cultures from all four mother plants were established with different success rate, depending on the genotype  were collected in May 2017. In vitro cultures were sampled after virus elimination. Whole plants of in vitro growing vines, representing independent lines, were used for virus diagnostics. RNA was extracted from the sampled plant materials via the CTAB method . RNA pooShoot tips and inflorescences were surface-sterilized by submersion in ethyl alcohol (70%) for 30 s, followed by rinsing in a sodium hypochlorite solution  for 10 min; then, the samples were washed three times in sterile distilled water.w/v) KOH. The plant materials were transferred monthly to a fresh medium.The medium prepared for the in vitro cultures contained Murashige and Skoog macroelements in half concentration; MS microelements and vitamins in full concentration; and folic acid (0.002 mg/L), biotin (0.002 mg/L), calcium-pantothenate (0.4 mg/L), p-aminobensoic acid (0.2 mg/L), riboflavin (0.2 mg/L), L-arginine (10 mg/L), and L-glutamine (100 mg/L) as a supplement. The pH was adjusted to 5.80 with 3%  wooden canes were cut into single budded nodes and rooted in granulated perlite moistened with tap water. Then, 0.5–1 cm-long tips of the shoots formed by these buds were surface-sterilized and placed on the above-mentioned basal medium containing 30 g/L sucrose and 0.5 mg/L meta-topolin (mT). The developing shoots were micro-propagated on the same medium without hormone.Meristems of the in vitro plants (with sizes of 0.2–0.5 mm) were carefully excised under a microscope and placed on a medium in Petri dishes including 30 g/L sucrose and 1 mg/L mT and solidified with 6 g/L agar. Meristems were kept in the dark for three days by wrapping the Petri dishes in aluminium foil to promote shoot formation and elongation rather than cotyledon or leaf growing .Developing plantlets without roots were passed into the medium in vials with the same media supplemented with 30 g/L sucrose, 0.2 mg/L mT and 6 g/L agar.Well-developed individual meristematic lines with roots were micro-propagated on the same hormone-free medium in jars supplemented with 10 g/L sucrose and 3 g/L gelrite. Plantlets showing poor growth or yellowing were first transferred to the MSAc medium  before tSE was carried out according to the previously described method . BrieflyCalli with embryogenic morphologies were passed into the basal medium supplemented with 1 g/L activated charcoal (MSAc), 10 g/L sucrose, and 3 g/L gelrite (Duchefa). The same medium was used during embryo development, germination, plant regeneration, and rooting.Individually regenerated plants were micro propagated on the same medium without activated charcoal.de novo assembly to build longer contigs from the non-redundant reads by employing an assembler of CLC (de novo assembly) using the default options: word size 20, bubble size 50, and simple contig sequences with a minimum 35nt length (see We used the CLC Genomics Workbench (Qiagen) for bioinformatics analysis. After trimming and quality control, the reads were used for V. vinifera actin gene. The result of the PCR was evaluated via gel electrophoresis of the PCR products.Pooled RNA extracts representing each mother plant and pools of the independent lines prepared with the specified method was used as templates for cDNA synthesis with a RevertAid First Strand cDNA Synthesis Kit  using random primers according to the manufacturer’s instructions. cDNA from all of the independent in vitro lines was also generated by the same method. The generated cDNA was used for the PCR analysis (the sequences and important parameters of the primers used to amplify the viral parts, together with the applied annealing temperatures, are provided in Vineyards that have been planted for decades are continuously endangered by several viruses and viroids. The best way to keep the infection risk low is prevention, which mainly involves the usage of virus-, viroid-, and phytoplasma-free propagation materials. To maintain the valuable traits of the cultivars, grapevine is propagated vegetatively. To prevent the distribution of viruses, virus diagnostics of the mother plants at the certified virus-free vineyards have key importance. Mother plants of the new breeds must be proven to be virus free, and, if not, must be sanitized. In our work, in line with the previous results, we demonstrated that the SE method is efficient not only for eliminating most viruses, but also for eliminating viroids . The art


The oil encapsulation efficiency was almost complete at 90.13% ± 0.93% in dried beads. The maximum bead swelling rate showed 982.23  in pH 6.8 and the drug release exceeded 90% in simulated gastrointestinal fluid (pH 6.8). Moreover, the beads were well distributed throughout various parts of the intestine. This designed formulation could possibly be advantageous in terms of increased bioavailability and targeted drug delivery to the intestine region and thus may find applications in some diseases like irritable bowel syndrome. Black seed oil (BSO) has been used for various therapeutic purposes around the world since ancient eras. It is one of the most prominent oils used in nutraceutical formulations and daily consumption for its significant therapeutic value is common phenomena. The main aim of this study was to develop alginate-BSO beads as a controlled release system designed to control drug release in the gastrointestinal tract (GIT). Electrospray technology facilitates formulation of small and uniform beads with higher diffusion and swelling rates resulting in process performance improvement. The effect of different formulation and process variables was evaluated on the internal and external bead morphology, size, shape, encapsulation efficiency, swelling rate, in vitro drug release, release mechanism, ex vivo mucoadhesive strength and gastrointestinal tract qualitative and quantitative distribution. All the formulated beads showed small sizes of 0.58 ± 0.01 mm (F8) and spherical shape of 0.03 ± 0.00 mm. The coefficient of weight variation (%) ranged from 1.37 (F8) to 3.93 (F5) ng. All formulations (F1–F9) were studied in vitro for release characteristics and swelling behaviour, then the release data were fitted to various equations to determine the exponent (ns), swelling kinetic constant (ks), swelling rate (%/h), correlation coefficient (r Black seed (Nigella sativa), and specifically black seed oil (BSO), is known to have high therapeutic value. For centuries, it has been used to treat various ailments. Perhaps one of the most significant phytochemical ingredients in BSO is thymoquinone (TQ) . In receMicroencapsulation is a process where relatively thin coatings are applied to small droplets of liquids and dispersions, or particles of solid material. Microencapsulated oil tends to have a high economic value where certain types of oils, including BSO, are considered as healthy foods and have therapeutic benefits. A microcapsule, or microsphere, protects oils from adverse environmental factors ,8. This The microencapsulation of oils can be challenging whereby sometimes there is a need for proper temperature control to avoid the degradation or the volatilisation of bioactive components. Oils can be encapsulated in several ways. Researchers have widely examined electrohydrodynamic atomisation (EHDA) to produce micro- and nano-sized structures ,12. In tIn this study, BSO was encapsulated using alginate, a natural polysaccharide suitable for oral consumption. Alginate is a naturally occurring anionic polymer and can be extracted from brown seaweeds. It is composed of two monomeric structures, 1-4bD-mannuronic (M) acid and a-L-guluronic (G) . It is n2+, Ca2+, or Zn2+, resulting in a cross-linked structure among the G groups of different alginate chains. The egg-box model aptly explains the gelation process where the divalent cation is bounded to two carboxyl groups on the alginate molecules located side-by-side [Alginate is preferred for encapsulation by ionic gelation as it can form ionic bonds with divalent cations such as Sr-by-side . It is p-by-side . In this study, the electro-hydrodynamic technique was adopted to examine the feasibility for encapsulating commercial oils through emulsification. Here, alginate-BSO beads were formulated and characterised in different aspects like yield percentage (Y%), physical appearance (SEM), encapsulation efficiency (EE%), TQ (EE%) (HPLC), particle size distribution, shape, weight uniformity, ex vivo mucoadhesive properties, in vitro drug release profile, and gastrointestinal tract (GIT) distribution. Prior to that, the compatibility was tested using Fourier-transform infrared spectroscopy (ATR-FTIR) and differential scanning calorimetry (DSC).Nigella sativa oil (NSO) (black seed oil) capsules at the dose of 450 mg have reported a potential therapeutic effect on inflammation in the gastrointestinal system [H. pylori activity in the gastrointestinal system [H. pylori infection in human clinical trials [There are several clinical trials that have reported the therapeutic benefits of BSO related to the gastrointestinal and immune systems. For example, daily twice administration of l system . In addil system . Furtherl system . Furtherl trials . Anotherl trials . Moreovel trials . Anotherl trials . HoweverThis influences the therapeutic efficacy because the oil may degrade in a stomach environment and lose its bioactivities. Therefore, it was necessary to develop a pH-sensitive BSO carrier to treat gastrointestinal diseases. There is a lack of information about the absorption of the oil or thymoquinone from the oil. There is also a lack of information on the stability of thymoquinone in the oil used in the formulations. This study aimed to provide sufficient information on developing a standard carrier of BSO with assured thymoquinone content. Therefore, this information is highly needed to conduct further in vivo research to optimize the therapeutic human dose, in order to treat various diseases with a standard pharmaceutical dosage form.2·2H2O, was purchased from Sigma-Aldrich . BSO was purchased from Blessed Seed Sdn. Bhd. . Lecithin was purchased from Fisher Scientific .Sodium alginate with medium viscosity (40–100 mPa s) and high guluronic acid content was obtained from Manugel DMB, ISP, . Calcium chloride dihydrate, CaClw/v relative to the total emulsion volume) in a solution of 1% w/v sodium alginate containing lecithin . The nano-emulsion was obtained by an ultrasonic homogenizer  for 55 s at 20% power. The emulsion was prepared by dispersing BSO  was used to determine the emulsion’s microstructure. Subsequently, for determination of particle size distribution and zeta potential, 0.1 mL of the freshly prepared emulsion was diluted with 9.9 mL of distilled water and analysed using a dynamic light scattering instrument  ,27.n = 3) were centrifuged at 4000 rpm for 5 min. The emulsion stability (ES) was calculated according to the phase separation interface position using the following equation:To estimate the emulsion stability, the emulsions (w/v). The emulsion was loaded into a 10 mL plastic syringe and pumped through a 22G needle using syringe pump  to control the flow rate. The positive electrode of high voltage power supply  was connected to the needle tip. The collector was a grounded beaker filled with 50 mL of 1% w/w calcium chloride (gelling bath). The dripping flow rate was varied at 1, 2 and 3 mL/min with the voltage of 3, 5 and 7 kV with 10 cm fixed distance above the surface of the calcium chloride solution. To ensure the complete gelation, the beads were kept in the gelling bath for 30 min for curing with stirring. Then, the beads were separated from the gelling bath using a stainless steel sieve. Then, the collected beads were washed by ultrapure distilled water and after that the beads were dried in a laboratory bench at room temperature for 16 h. The beads yield percentage was determined according to the following equation (Equation (2)):BSO alginate beads were prepared using an electrospray technique called electrohydrodynamic atomization (EHDA) . BSO emuw/v sodium alginate solution containing lecithin 1, 3 and 5% w/v.BSO-free alginate beads, referred to as blank beads, were similarly prepared using 1% max is the maximum diameter passing through a bead centroid (mm), and Dper is the diameter perpendicular to the Dmax passing through the bead centroid (mm). A zero SF represents a perfect sphere, while a higher SF indicates a higher shape distortion. Moreover, a bead with SF ≤ 0.05 is considered as a spherical bead.A Sigma ScanPro 5 image analyser was used to determine the bead size and shape while the images of the wet and dry beads were captured using a digital camera. Here, the roundness of the beads was determined using sphericity factor (SF) . The SF To determine the amount of BSO encapsulated in the beads, the beads were first disintegrated in phosphate buffered saline (PBS). This process returned the beads into emulsion. Then the absorbance was measured at 600 nm using UV-vis spectrophotometer  which represents the turbidity of the resulting emulsion. This method is based on previous reports ,30 that To evaluate the weight uniformity of the beads, 30 beads were taken and accurately weighed using an analytical balance . The weiA Carl Zeiss EvoV R 50 scanning electron microscope  was used to observe the microstructure and surface morphology of the BSO alginate beads. Some of the dried beads were cut to examine their inner surface before being spotted onto the aluminium stubs and pasted using carbon adhesive tapes. Subsequently, the beads were sputter-coated with carbon sputter module with the thickness of 100 and 50 Å, respectively, in a vacuum evaporator in an argon atmosphere . Lastly,DSC  was used to examine the compatibility and thermal characteristics of BSO-loaded and BSO-free alginate beads. About 3.20 mg of beads were weighed and sealed in a standard aluminium pan. The samples were analysed under a temperature range from 50–350 °C and heated under a stream of nitrogen flow rate of 20 L/min at a heating rate of 10 °C/min . −1, with a resolution of 1 cm−1 [ATR-FTIR spectra of sodium alginate, BSO, and beads were recorded between 4000–400 cmf 1 cm−1  with ambA 100 µg/mL TQ standard stock solution was prepared by dissolving 5 mg of TQ in 50 mL of methanol. The serial dilution of TQ stock solution was performed to get different concentrations up to 20 µg/mL. The solution was then filtered using a 0.2 µm syringe filter and 2 mL of the solution was placed in HPLC vials for further analysis. TQ was first quantified in the BSO used to prepare the beads. Fifty milligrams of BSO was carefully weighed then 10 mL methanol was added and mixed thoroughly using a vortex mixer and centrifuged at 4000 rpm for 5 min. Then, the upper layer (methanol) containing the TQ was collected and filtered using a 0.2 µm syringe filter. For the extraction of TQ from alginate beads, 0.3 g of dried alginate-BSO beads was broken by adding 1 mL of mixture of 0.5 M EDTA, phosphate buffer solution, pH of 6.8, with a volume ratio of 1:1. Subsequently, the mixture was placed for 30 min in an incubator shaker at 150 rpm. After the broken beads were completely transformed into an emulsion, 10 mL of methanol was added and mixed thoroughly with the emulsion. The mixture was immediately centrifuged at 4000 rpm for 5 min. The supernatant layer (methanol) was then collected and filtered using a 0.2 µm syringe filter.A HPLC method was used to quantify TQ as described in a previous study . The anaThe swelling behaviour of the optimised BSO-loaded bead formulations (F1–F9) was measured according to the weight gain percentage in two different aqueous media: the simulated gastric fluid  and simulated intestinal fluid . The conditions were maintained for 2 h at 37 ± 0.5 °C while 100 mg of dried alginate-BSO beads was immersed in 50 mL of both SGF and SIF fluids media . o is the dried beads’ initial weight and Wt is the weight of swollen beads at time t.At each predetermined time interval , the swollen beads were removed and filtered using a metal mesh. The swollen beads were dried with a paper towel to eliminate the excess fluid. The following equation was used to determine the percentage of swelling index,In addition, pictures of the beads were taken using a digital camera at the same time intervals after the weights of the swollen beads were measured. The bead samples were then characterised based on their thickness and morphological appearance. The dissolution was carried out at 37 ± 1 °C and 50 rpm using an orbital incubator shaker under continuous stirring . BSO beads (0.1 g) were added to 200 mL of SGF (pH 1.2) and the test was carried out for 2 h, and afterwards it was carried out in SIF (pH 6.8) for another 2 h. Five mL of aliquots were collected at regular time intervals and were replaced with a similar amount of fresh dissolution medium. The collected aliquots were filtered, suitably diluted using the dissolution medium, and the absorbance was measured with a UV–vis spectrophotometer  at 600 nm.The in vitro drug release data were then fitted to mathematical models like Korsmeyer–Peppas, zero order, first order, Higuchi, and Hixson–Crowell, to identify the drug release mechanism . 2) value [n < 1.0. Besides that, n = 1.0 indicates case II transport and n > 1 indicates super case II transport [These models were compared by calculating the squared correlation coefficient (r2) value . Meanwhiransport .The formulation’s mucoadhesive properties were evaluated using the modified wash-off method . A fluorThe study used Sprague–Dawley (SD) rats and the protocol for these tests has been approved from IIUM animal ethics committee dated 14/05/2018 ). Twelve rats were included in this study and all were between 6–8 weeks old and weighed between 200–220 g. The rats were not provided any food for 20 h before the experiment, except normal tap water that was provided ad libitum. The rats were divided into 3 groups with 4 rats in each. A fluorescent dye (6-Coumarin) was added to prepare the microsphere beads. Each rat was orally given 100 beads suspended in distilled water using 16G oral gavage under non-anaesthetic conditions before they were sacrificed at 1, 3 and 6 h post administration. After being sacrificed, the rats were immediately dissected where their stomach and intestine were removed and cut into pieces. The stomach was opened and washed with normal saline while the intestine was subdivided into six 14 cm sections  . These s2) of all kinetic models. Each measurement was done in triplicate (n = 3) and the measured data are expressed as the mean ± standard deviation (SD). Data were compared by t-test or one-way ANOVA followed by Tukey’s test considering p < 0.05 as indication of significant difference. Two software programs, Minitab and Design-Expert 8.0.6.1 software  were used to perform the statistical optimisation, while the KinetDS 3.0 Rev. 2010 software was used to ascertain the squared correlation coefficients  and was observed with 5% w/v lecithin concentration. Since a PDI value of 0.300 and below indicates homogenous particle size distribution [Lecithin concentration has an influence on the distribution of particle size and zeta potential . The higribution , the emuZeta potential has a direct effect on the stability of the colloidal structure. The emulsion droplets practically had high negative charges due to the chain of alginate being also negatively charged because of the presence of hydroxyl and carboxyl groups in the structure of the alginate molecule . In shorOn average, the percentage of yield was 94.87 ± 2.11% . The losThe size of the wet beads ranged from 4.76 ± 0.01 mm (F3) to 1.50 ± 0.02 mm (F8) and dry beads were from 1.86 ± 0.02 mm (F2 & F3) to 0.58 ± 0.01 mm (F8) . Few facThe percentage of EE of wet and dry bead formulations ranged from 67.20 ± 1.46% (F1) to 104.50 ± 4.04% (F8) and 59.92 ± 0.90% (F1) to 90.13 ± 0.93% (F8), respectively . The oveOne of the main objectives of this experiment was to achieve the targeted size, sphericity factor, EE% and drug release of BSO-loaded beads. In this sense, high electrical voltage (kV) was employed to achieve the goals. However, to adjust and optimize the emulsion flow rate along with a high electrical voltage was the big challenge. To optimize the formulation, different flow rates  and voltages  were applied. It is well understood from this experiment that flow rate and voltage have a significant impact on the size of BSO-loaded beads. The main effect clearly showed that as the voltage increases, bead size decreases from 1.86 ± 0.02 mm to 0.58 ± 0.01 mm. However, the bead size increased from 0.72 ± 0.01 0.93 ± 0.01 mm when the flow rate increased from 1 mL/min to 3 mL/min at 7 kV. The formulation (F8) achieved the lowest size of 0.58 ± 0.01 mm with the flow rate of 2 mL/min and voltage 7 kV A. MoreovPiornos et al. (2017), reported that the EE depended on the strength of the encapsulating matrix . MoreoveIn any pharmaceutical dosage formulation, it is necessary to maintain good manufacturing practices for the manufacturer. Its dose serves as an indicator of the quality of dosage formulation as well as quantity of active pharmaceutical ingredient (API) filled or encapsulate in the formulation. The coefficients of weight variation of BSO-loaded alginate beads varied from 1.37% to 3.93%. The recent findings exhibited that the mean weight of all the BSO-loaded beads did not deviate more than 5% . Accordi−1 for O–H stretching and at 1595 and 1408 cm−1 for C=O and C–C groups resulting from stretching vibration. A previous study also reported separate bands at 1600 and 1405 cm−1. These two bands were attributed to asymmetric and symmetric stretching vibration of –COOH groups [−1 are due to the C–H group. The lecithin spectra showed three bands at 2922, 2853 and 1465 cm−1 which are attributed to the C=O, C–H and C–C groups, respectively. The noticeable peaks for BSO were at 2953 and 2853 cm−1 (C–H in CH2 and C=C–H respectively). Moreover, the bands that appeared at 1753, 1653 and 1453 cm−1 indicated C=O, C=C and C–H, respectively due to predominance of carbon chains in the fatty acids. In general, fatty acids compose more than 98% of BSO. The present findings are very similar to previous reports [−1 is due to the symmetrical stretching bond of Ca–Cl. The broad peak at 3441 cm−1 is because of the O–H stretching vibration, probably originating from the residual water.ATR-FTIR spectra of sodium alginate, lecithin, CaCl2, BSO, alginate-BSO beads, blank beads, and physical mixture are shown in H groups . Moreove reports ,43. In c−1 remained in the physical mixture. Similar observation was noticed for the peaks of 1753 and 1408 cm−1 of lecithin. In the alginate-BSO bead spectra, most of the peaks in BSO were not seen, which indicates the encapsulation of BSO inside the beads and absence of the oil on the bead surface. In addition, the peaks of the carboxyl group in sodium alginate shifted to higher values, from 3400 to 3424 cm−1 in the blank and BSO-loaded alginate beads [−1 also confirms the complex formation. The above results confirm the complexation between sodium alginate and calcium chloride and the encapsulation of BSO into the alginate bead nanocavities. This complex occurred only in the solution but not in the dry physical mixture.The physical mixture spectrum of alginate, lecithin, CaCl2 and BSO seems to be an overlay of the individual spectra whereby the peaks appeared in their respective locations. For example, the peak from sodium alginate at 1595 and 1408 cmte beads . This isDSC is employed for the analysis of thermal behaviour of the active drug molecules and the excipients of the formulation, as well as their interactions throughout the preparation development process ,43. In tOn the other hand, the above-mentioned thermal events disappeared in the DSC thermogram of BSO-loaded beads. This might be attributed to the lower moisture content since the oil is dispersed in the bead matrix, making it less hygroscopic. The low moisture content and the absence of interaction between the oil and the gel matrix might also increase the stability of the beads whereby the degradation peaks were not observed within the test temperature and time. The scanning electron microphotographs of the optimized BSO-loaded dried beads revealed the surface and internal structure containing a large number of pores or channels in the beads . The porw/w). However, the percentage of TQ in the oil of BSO-loaded alginate dried beads was 1.589 ± 0.158% (w/w). This means that the EE of TQ in the optimized BSO-loaded alginate beads (F8) is 81.07 ± 0.23%. This highlights that even though the EE of the oil was 90.13 ± 0.93%, some TQ from the oil degraded or leaked to the gelling bath. This is attributed to the solubility of the small amount of TQ in water and the low stability of TQ in aqueous solutions. The HPLC chromatograms showed well resolved peaks of TQ with no interference. Chromatograms of the blank, standard TQ, BSO-loaded beads and BSO are shown in Bead swelling is a significant behaviour that influences the drug release pattern of polymeric materials . The sweThe swelling parameters such as swelling exponent (ns), kinetic constant (ks), swelling rate (%) and water penetration velocity (up to 1 h) were determined to understand the swelling mechanism . The per2+ and Na+ reversible ion-exchange reaction [+ ions reached in sea water media [The hydration of the alginate hydrophilic groups causes the dry beads to swell, due to differences in osmotic pressure of the fluid inside and outside the beads . The swereaction ,51,52. Rer media . Here, l2+ complex alginate. The presence of carboxyl groups in the alginate network helps to retain the beads without swelling.Moreover, erosion and dissolution of swollen cross-linked BSO-loaded alginate beads occurred within 100–110 min. All formulations were completely eroded at 120 min. One of the main purposes of this research is to release the drug in the intestine where drugs are usually absorbed. In acidic pH 1.2 (SGF), all formulations (F1–F9) retained their original shape or shrank, which reduces their disintegration tendency . The mai2+ than alginate ions. As a result, alginate-calcium was decoupling and the gel dissolved in the phosphate buffer. Oppositely, in SIF, BSO-loaded alginate beads rapidly expanded . All forThese BSO-loaded alginate beads can effectively shield the encapsulated therapeutic ingredients from the harsh-lytic condition of the stomach throughout its travel through the gastrointestinal tract. Therefore, these formulations can favourably deliver the drug to the specific targeted site, such as the intestine or colon, by the influence of the pH environment. The drug release from alginate beads relies on the dissolution medium penetration into alginate beads, swelling and dissolution of the alginate hydrogel, and the dissolution of the encapsulated drug subsequent to leakage through the swollen hydrogel. The drug release pattern of BSO-loaded wet and dried beads in SGF (pH 1.2) and SIF (pH 6.8) at 37 °C is shown in The amount of drug released from BSO-loaded beads was less than 5% in SGF and more than 90% in SIF at 2 h. This finding suggests that BSO-loaded beads can be used as a useful drug carrier for specific-site targeted drug delivery, such as intestine/colon-targeted drug delivery, without premature drug release during its transition period in the stomach.p < 0.05). The low percentage of oil release in this media could be due to the absence of any potential swelling rate in the simulated gastric fluid. In the meantime, the study observed a positive correlation between the erosion rate and the release of oil. According to Corstens et al. (2017), the physical characteristics of the alginate beads are largely influenced by the pH and ionic concentrations . In thisMore oil was released from the beads as the erosion rate in the intestinal phase increased . It was 2) and release exponent (n) values (2) value of BSO-loaded beads (F1–F9) in SGF were found to fit first order kinetic model (r2 = 0.818–0.981) over the predetermined release time of 2 h. However, r2 data for all the formulations (F1–F9) in SIF followed zero order (r2 = 0.926–0.980) and Korsemeyer–Peppas (r2 = 0.900–0.997) kinetic model at 2 h. The diffusional or release exponent (n) values of the formulations  ranged from 0.49 to 0.61, indicating non-Fickian diffusion or anomalous transport in acidic media (pH 1.2). This is regarded as two different mechanisms such as couple diffusion or followed by polymer relaxation (erosion), whereas the rest of formulations followed the super case II transport model. On the other hand, the release exponent value for all the formulations (F1–F9) in SIF were ˃0.85 (0.86–1.66), indicating that the BSO release from alginate beads followed super case II transport mechanism. This release mechanism occurred through the polymer dissolution controlled and polymeric chain expansion or polymer relaxation/swelling. This is in line with the swelling study which showed the same pattern in release behaviour mechanism.To better comprehend the mechanism of BSO release from electrohydrodynamic-assisted alginate beads, the cumulative percentage of drug release data was fitted into different equation models to obtain the correlation coefficient (r) values . The corThe ex vivo wash-off time revealed that the beads could adhere to the mucosal surface of the intestine up to the experimental period. In this part, data were acquired according to predetermined time interval from 1 to 6 h with two different wash-off media. Firstly, in SIF the beads showed a good mucoadhesive strength whereby the percentage of remaining beads was recovered from the mucosal surface of the employed intestine as 98.26 ± 1.15% at 1 h and 38.83 ± 2.51% at 6 h. This practically supports its potential mucoadhesive properties in intestinal media (pH 6.8). Secondly, the mucoadhesive properties in SGI showed very weak mucoadhesive strength (65.42%) . The preFollowing oral administration of fixed number of beads, more than 90 ± 4.62% beads were recovered from the stomach after 1 h, but at the same time, less than 10% (7.0 ± 0.75%) were in the duodenum followed by 3% in the jejunum. However, at a certain time interval (3 h), the maximum number of beads were transferred or moved from the stomach to the duodenum (14 ± 1.21%) and the lower part of intestine, including the major sections of jejunum-1 (23 ± 2.33%), jejunum-2 (21 ± 1.98%), jejunum-3 (23 ± 2%), ileum (11 ± 1%) and colon (6 ± 0.54%). Furthermore, after 6 h of administration, significantly, a large quantity of beads (6%) was counted from different parts of the jejunum  and ileum. After 1 h of oral administration, almost all beads were retained in the stomach, while beads were not observed in the rest of the intestine sections except the duodenum. After 3 h of beads administration, the maximum number of beads in the stomach moved to the duodenum and jejunum regions of the lower intestine which was exposed to UV fluorescence. Moreover, various sections of the intestine at 6 h of administration were not noticed. Furthermore, mucoadhesive and swelling characteristics of the beads in intestinal fluid facilitated the erosion of the polymer matrix and the drug was released in the specific site of the intestine in the next 2 h. Due to erosion, beads were not seen in the lower sections of the intestine at 6 h of administration. In the distribution study, qualitative image analysis showed the uniform distribution of beads at 3 h of oral administration in rats.Novel BSO-loaded alginate beads were prepared as a pH-sensitive carrier for intestinal site-specific drug delivery. It showed pH-dependent swelling and drug release behaviour. The swelling of BSO-loaded beads was less in SGF compared to SIF, which can be advantageous as an intestinal-specific drug carrier. The release of BSO from the BSO-loaded beads was dependent on pH. The rate of BSO released from the beads was higher in SIF due to the extended swelling of the beads. These results indicate that BSO-loaded beads could be an oral drug delivery system to deliver drugs more specifically to the intestine.


Aim of the present study is the evaluation of ultrasound as a physical method for virus inactivation in human plasma products prior to transfusion. Our study is focused on achieving a high level of virus inactivation simultaneously leaving blood products unaltered, measured by the level of degradation of coagulation factors, especially in third world countries where virus contamination of blood products poses a major problem. Virus inactivation plays an important role, especially in the light of newly discovered or unknown viruses, which cannot be safely excluded via prior testing.Taking into account the necessary protection of the relevant coagulation activity for plasma, the basis for a sterile virus inactivation under shielding gas insufflation was developed for future practical use. Influence of frequency and power density in the range of soft and hard cavitation on the inactivation of transfusion-relevant model viruses for Hepatitis-(BVDV = bovine diarrhea virus), for Herpes- and Parvovirus B19 (PPV = porcine parvovirus) were examined. Coagulation activity was examined via standard time parameters to minimize reduction of functionality of coagulation proteins. A fragmentation of coagulation proteins via ultrasound was ruled out via gel electrophoresis. The resulting virus titer was examined using end point titration.2 shielding gas insufflation—to avoid radical emergence effects—the coagulation activity was less affected and the time window for virus inactivation substantially widened. In case of the non-lipidated model virus , the complete destruction of the virus capsid through hard cavitation was proven via scanning electron microscopy (SEM). This can be traced back to microjets and shockwaves occurring in hard cavitation. The degree of inactivation seems to depend on size and compactness of the type of viruses. Using our pre-tested and subsequently chosen process parameters with the exception of the small PPV, all model viruses were successfully inactivated and reduced by up to log 3 factor. For a broad clinical usage, protection of the coagulation activities may require further optimization.Through COBuilding upon the information gained, an optimum inactivation can be reached via raising of power density up to 1200 W and simultaneous lowering of frequency down to 27 kHz. In addition, the combination of the two physical methods UV treatment and ultrasound may yield optimum results without the need of substance removal after the procedure. Since the introduction of blood transfusion security levels of the blood transfer process between individual humans for medical purposes have been a key issue, periodically aggravated by current virus circulations in society. Initially the topic had become a major concern after the discovery of HIV. The current security concept includes careful donor recruiting under elimination of high-risk groups, testing for presence or absence of virus in donor blood and utilization of newly developed measures for virus inactivation.In Germany, all blood and plasma products are regarded as pharmaceuticals and thus are subject to pharmaceutical legislation. In 1998 in Germany, a new law on blood transfusion was passed and amended in 2007 and 2017 [Blood and blood component transfusions can cause transfer of parasites, bacteria and viruses resulting in serious infections . Since 1Various methods for virus depletion and inactivation are available  as outliThe solvent detergent method poses the best known and most common, feasible method for lipidated viruses primarily for use on plasma pools. Methylene blue inactivation is not yet authorized in Germany, but is employed in other EU states. It exerts its inactivation via interaction of methylene blue with the genome of the virus and subsequent radiation with sodium light. This generally works for all viruses, but cannot inactivate some non-lipidated viruses such as, e.g., PPV. Inactivation methods using psoralenes (furocumarine), gene toxic substances, are currently in development , while tMost of the above described methods require separation of the plasma from the employed chemicals, posing an additional effort and the need for testing of rest substance. Thus, ultrasound as a purely physical method poses a novel, effective and easily usable alternative.Ultrasound used for sonication describes sound waves at a frequency above approximately 20 kHz, non-audible to humans, generated by electrically triggered quartz resonators and radiated in potent bundle format. The transmittable energy finds a high range of use in industry and military as well as in medical setups, e.g., to detach bacteria in clinical microbiological diagnostics or to detect implant-associated infections . To inacThe goal of the underlying study is the detailed examination of ultrasound treatment for inactivation of transfusion-associated viruses including proof of virus inactivation by ultrasound, detection of optimal physical parameters such as time, frequency and power of ultrasound treatment and evaluation of ultrasound’s effects on the coagulation activity of plasma proteins. Ultrasound triggers chemical  and mechanical (cavitation) reactions. In presence of oxygen, the radical reactions can destroy proteins, including coagulation factors . InitialInitially, reduction of global tests such as Quick, elongation of aPTT and reduction of factors fibrinogen, FIX and FVIII after 60-min ultrasound treatment in varying frequency were tested and compared. Each test was repeated five times. Ultrasound baths of various sizes and sources for various frequencies from 27 kHz up to 250 kHz were employed, yielding a variation of power density, as described in Table ® bloodbags, the connecting tube shortened to 1 cm and the interface connected via a 3-way-connector. The bag was then stabilized by a holding frame inside the ultrasound container. For CO2 fumigation the blood bags were inserted into a custom-made pressure chamber.Virus inactivation was conducted in standardized 150 ml Compoflex2. Two blood bags were prepared, one as a control group, the other as the treatment test. Times of sampling (5 ml per sampling time) can be seen in Table Ultrasound treatment of standardized and documented virus suspensions in PBS and plasma as outlined in Table 2 control group, the other was used for ultrasound sampling.For sequential testing of plasma, virus suspension and PBS, three bags were employed as shown in Fig. The positive controls were kept for an equal time period in the water container under the exact same conditions. Acoustic exposure was then conducted for 5 h and samples taken in pre-defined time intervals. High power density tests were sampled every 30 min, low power every 60 min as described in Table Fresh frozen plasma for the conduction of all tests was obtained from blood donation service Baden-Württemberg. Inactivation testing of virus loaded plasma was conducted at the Paul-Ehrlich-Institute as outlined below. Plasma was thawed at 37 °C, portioned in 50-ml units and processed immediately.Inactivation of the model viruses was conducted in PBS. Sample viruses employed are listed in Table 2 saturation of plasma , partial pressures of CO2, O2, and pH values were tracked using the blood gas measurement system Synthesis 10 .To measure maximum COAdV-luc is a replication-deficient Adenovirus of human serotype 5 sized at roughly 90 nm with a double-stranded DNA. The so-called firefly luciferase gene is expressed under the control of the CMV promotor.Biologic activity of coagulation proteins was conducted via measurement of the global thromboplastin time using the Quick test (PT), as well as the activated partial thromboplastin time (aPTT).Virus count was conducted after staining with trypan blue to differentiate viability of cells. Cell count was conducted via microscope in a Neubauer counting chamber. Cytotoxicity was evaluated using the WST-1 assay.2, −US and +CO2, +US) at the beginning (0 h) and end of sonication (5 h) were applied, followed by samples of plasma after 1, 2, 3, 4 and 5 h, separated in 8–16% Tris-Glycine-Gradient Gel at a voltage of 125 V and output of 57 mA. The recombined SDS-Page-Protein-Marker was used as a mass standard in the range of 10–150 kDa. Prior to electrophoretic analysis of the plasma samples, the albumin fraction was reduced, using an albumin removal kit by diluting 60 µl of plasma with 540 µl of binding buffer.Samples sonicated at 27 kHz under shielding gas protection were used for electrophoretic examination. Initially, the positive control samples  are shown in Table Virus inactivation in blood plasma can only be regarded successful if simultaneously proteins for coagulation remain more or less intact by choosing an optimum time window and energy level. Influence of sonication treatments at 27 and 42 kHz for 60 min on coagulation parameters n =  are show2 insufflation prior to the ultrasound treatment. Displacement results of O2 by CO2 as a shielding gas by inserting carbon dioxide at 0.5 bar pressure can be seen in Fig. Preliminary literature and testing showed the protective function of CO2 of 48 mmHg and pO2 of 151 mmHg. Introduction of CO2 led to a steep rise in pCO2 an equivalent linear decrease in pH from 7.3 to 6.3. In contrast, pO2 decreased relatively slowly.The used plasma samples showed an initial partial pressure of CO2 gas insufflation showed a marginal influence on overall clotting tests such as thromboplastin time and aPTT. On the other hand, activity of FVIII decreased by 10% and FIX by 18% on average.Testing the activity of plasma coagulation factors after COExamination of the influence of ultrasound treatment on Quick, on aPTT, or coagulation factors such as fibrinogen, Factor IX and Factor VIII in summary showed the following: Extensive length of acoustic exposure yielded a reduction of coagulation activity in plasma, strongest influence displayed at low frequencies with high power density. Strongest influence was shown at 250 kHz on factor VIII activity, yielding a reduction of 68% (180 min) and 75% (300 min). Lowest influence was proven for a frequency of 70 kHz as shown in Table Pre-testing for side effects has thus proven the frequency of 250 kHz to be unsuitable. For subsequent testing purposes both lipidated and non-lipidated viruses were examined. The observed viruses differ in size, structure of particles as well as structure of nucleic acid and cover the entire range of transfusion-associated viruses regarding characteristics as RNA-virus, DNA-virus, lipidated and non-lipidated viruses.2 shielding gas protection are displayed in Tables Results of influence on coagulation and virus count reduction under COSee Table See Tables Rising quantity of blood transfusions as well as a variety of infectious sources requires modern affordable and reliable methods for inactivation of viruses in blood while simultaneously protecting blood coagulation factors from negative influences. Success and failure of virus inactivation is measured in terms of multiple logarithmic reduction of virus titers. Influence of ultrasound on virus inactivation has been tested and described in literature yielding varying rates of success  partiallPrevious authors have tested similar methodologies, including pasteurization with regard to its effectiveness on virus inactivation and similar to our research proven the necessity of additional protective measures . Others The method examined in our test series has proven to be effective in virus inactivation yet protective for blood’s hemostatic properties. The examined mechanical method yields satisfying results with regard to virus inactivation through hard cavitation as described earlier . The effOther authors describe interaction of free radical species development during sonolysis . Hard veLimitations of this study include the small selection of comparable virus stems compared. Yet these virus stems were chosen diligently to allow for a high degree of comparability. The examined method is both time and labor intensive as well as only functional for small amounts of treated blood cells. Consequently, the technology is hardly scalable for industry in developed regions. However, the described method allows a significant increase in safety of blood transfusions in technically less developed regions.It was clearly shown that ultrasound treatment yields positive results with regard to virus inactivation with minor side effects on coagulation proteins and no signs for cytotoxicity. In addition, hard and soft cavitation effects were compared which clearly showed that influences on virus inactivation and on coagulation factors occurred in different energy ranges. This allows for optimization of mechanical effects. In addition, the new method could potentially be combined with existing physical and chemical methods such as methylene blue or UV radiation.Further studies may examine the effectiveness on a larger scale, industrialized model. In addition, cost and time consumption could be evaluated to understand the economic effects caused by this new technology.


This paper presents dielectric measurements of talc, glass beads, and their mixtures under different moisture and salinity levels. The measurements were conducted using a prototype seven-rod probe  connected to a single port of vector network analyzer. The samples were moistened with distilled water and KCl solutions in order to obtain six different moisture content levels. The complex dielectric permittivity was determined from vector network analyzer reflection-coefficient measurements based on the open-water-liquid calibration procedure. Next, the fitting of volumetric water content–real part of dielectric permittivity calibration curves was performed for each material at selected frequencies, and the obtained relations were compared with well-known calibration equations. Additionally, a salinity index for the tested materials was calculated. It was concluded that pure talc is not an optimal material for the calibration and verification of dielectric methods. The calibration curves obtained for glass beads and the mixtures of glass beads with talc gave results close to well-known reference calibration functions. Additionally, the addition of talc caused the data points to be less scattered. Moreover, the values of the salinity index for the tested materials were in a good agreement with literature data for sand. The obtained results indicated that glass beads with the addition of talc can be used as a reference material for the calibration and verification of dielectric methods and devices for soil moisture measurement. The monitoring of soil water content provides essential information that is necessary to mitigate the impact of natural disasters like floods or droughts, and it is also a valuable tool for a precise irrigation, which, in turn, can bring reductions in water consumption ,2. ThereElectromagnetic techniques rely on the measurements of dielectric properties of materials . RelativIt is recognised that SIn the literature, there are a number of theoretical and empirical models describing the relation among the loped in , where SThe application of dielectric techniques in order to estimate soil moisture content has resulted in the development of an increasing number of sensors working at various frequencies from kHz up to several GHz . TDR senFrom a user point of view, a dielectric sensor should be able to operate in different types of soil and should be capable of adapting to changing environmental conditions. The manufacturers usually offer commercial sensors with the calibration equation that relates mination . Howevermination . The firmpirical  or semi-The electromagnetic sensors are usually evaluated using the determination of permittivity in the soil over a range of water contents . These sElectromagnetic simulations of the seven-rod probe with different dimensions using the Ansys HFSS software .−1) using the short seven-rod probe connected to a PNA-L N5230C VNA  in the operational frequency range of the probe.Measurements of the Comparison between Estimation of repeatability of the materials under test.Evaluation of the salinity index model.The aim of the study was to measure the complex dielectric permittivity of talc, glass beads, and their mixtures under different moisture and salinity levels. This paper presents the following:3. The probe was manufactured at the Laboratory of Dielectric Spectroscopy of the Institute of Agrophysics PAS, Lublin, Poland  and with three potassium chloride (KCl) solutions of electrical conductivities of  was calculated.The glass beads, 90–106 µm , and mixtures of glass beads with 5% and 10% of talc (Sigma Aldrich) were moistened with distilled water θ=0.0HydraProbe (HP) calibration for mineral and loam soils at 50 MHz :(7)θ=0.1ML3 ThetaProbe (TP) calibration for mineral soils at 100 MHz :(8)θ=0.1Calibration function for an FDR sensor obtained in the frequency range of 390–480 MHz :(9)θ=0.1Topp’s equation obtained for TDR :(10)θ=−5Calibration equation for 10 soils at 20, 50, and 100 MHz :(11)θ=0.R2 and RMSE values obtained for each material under different moisture and conductivity conditions for selected frequencies, are presented in The application of pure talc as a reference material was under consideration. However, as can be seen in R2, which increased with an increase of frequency. The highest value of R2 was observed in the glass beads with 10% of talc. It also occurred that the maximum value of the RMSE was obtained at 20 MHz. For each material, the RMSE decreased with an increase in frequency. The smallest differences between the maximum and minimum values of the RMSE among the tested materials was observed for glass beads with 10% of talc.Generally, the materials under test gave high values of The SI values for measurement materials were calculated using Equation (3). Following [R2 > 0.95. The highest R2 was obtained for glass beads with the addition of talc.As expected, the addition of salt increased the value of the ollowing , εa was SI and R2 (l is the slope of a relation between SI and the l = 0.0124 ± 0.000194, which matched the l = 0.0128 value that was calculated using TDR for sand [ high R2 , where lfor sand . Similarfor sand .ɛ’ between repetitions did not exceed 1%. This showed that the glass beads with talc met repeatability requirements, which is the one of important parameter for a reference dielectric material.Next, the repeatability of the dielectric properties of the most promising reference material was examined. In this case, the mixture of glass beads with 5% of talc moistened with distilled water was measured in three independent repetitions. It was calculated that the differences in the value of l of SI versus the This paper presented the dielectric properties of glass beads and mixtures of glass beads with talc. It was concluded that the addition of talc to the glass beads provided more repeatability and stability of the material, which was observed through the diminished scattering of the data points in comparison to the results from the glass beads alone. The fitting equations obtained for the mixture of glass beads with talc, especially for the 5% addition of talc, gave results close to well-known reference calibration functions. It was also observed that pure talc stood out from the rest of the tested materials and the calibration curves, which makes pure talc a less optimal calibration material than the aforementioned mixtures. Additionally, the addition of 5% of talc also provided the highest linear correlations between


A native of Russia, Tatyana Golovkina graduated in 1985 with a MS in biology from Moscow State University .At UIC in Ross’ lab, Golovkina studied genetic elements of the mouse mammary tumor virus (MMTV) that direct expression of the genes in tissue specific fashions, specifically in the mammary gland. However, she soon realized that she was most interested in studying host–pathogen interactions. Ross encouraged her to start series of experiments with MMTV, which led to Golovkina’s first significant publication . In thatIn 1994, Golovkina moved with Ross to the Cancer Center and Department of Microbiology at the University of Pennsylvania. It was there that Golovkina realized that the traditional discipline of virology alone was not enough to answer the many questions she had begun to ponder related to host–pathogen interactions. She started learning more about immunology and genetics to understand how the immune system responds to pathogens, and how the genetic composition of a given host makes it resistant or susceptible to particular viruses. These questions led her to take a new position as an independent investigator at The Jackson Laboratory, where she took advantage of their vast mouse genetic resource and worked for the next 8 years studying susceptibility and resistance to viral infections in various strains of mice. During this time she published another foundational paper , in whicIn 2005, Golovkina moved to the University of Chicago along with her husband, joining the tenured faculty in the Department of Microbiology, where her lab has continued to focus on host–pathogen interactions. Specifically, they are studying how the innate immune system detects retroviral infection and initiates virus-neutralizing adaptive immune responses, and the mechanisms evolved by retroviruses to overcome host protective responses. To investigate these important questions, her group employs virus-resistant mice capable of controlling retroviruses from distinct genera. Using mice from retrovirus-resistant strains and mouse retroviruses, in 2011, Golovkina’s team found that endosomal Toll-like receptor 7 (TLR7) is an innate immune receptor that detects mouse retroviruses and signals to stimulate the production of virus-neutralizing antibodies .Most viruses enter the host through surfaces exposed to commensal bacteria that protect the host from incoming pathogens. In 2011, Golovkina’s team performed pioneering work showing that a mucosally transmitted mouse retrovirus, mouse mammary tumor virus (MMTV), exploits gut commensal bacteria for sufficient transmission and spread. Specifically, they demonstrated that the MMTV virion-associated Gram-negative bacterial wall component lipopolysaccharide (LPS) activates the Toll-like receptor 4 (TLR4), stimulating production of the immunosuppressive cytokine IL-10, aiding virus evasion of the host immune response .In a completely independent study conducted by Julie Pfeiffer’s group at University of Texas Southwestern, it was shown that human picornavirus and reovirus transmission also depends on the gut microbiota . Since tGolovkina continued her work studying how some hosts are resistant to persistent viral infections while others remain susceptible. A long journey into forward genetics using positional cloning and other methods culminated in 2017 when her group discovered that the specific allele of MHC Class II gene H2-Ob in I/Ln J mice supports the production of virus-neutralizing antibodies . H2-Ob (That 2017 study was the result of nearly 20 years of work, a matter of patience and persistence Golovkina credits with keeping her lab running through the early days as a young researcher when she struggled to secure funding. Experiments with animal models can be time-consuming and expensive, often taking years of painstaking research to yield results. From the teachings of Kryukova, Ross, and other mentors, along with Chervonsky’s constant partnership, Golovkina learned that she needed to maintain multiple projects in parallel, any one of which might lead to interesting scientific conclusions. She begins most of her research projects at the bench herself to determine whether or not a particular project is worth pursuing. If so, she can then hand it off to her students and postdocs, and then repeat the cycle with another project. This continuous supplement of new ideas is crucial when it may take up to 20 years to find the right answer. In addition, Golovkina really likes to be at the bench, which she still truly enjoys.Golovkina thinks that the definitions of scientific disciplines like microbiology, immunology, genetics, or neurobiology are becoming obsolete as new tools and technologies allow scientists to cross these traditional boundaries. If there is an interesting scientific question, researchers now have the ability to delve into other fields and connect with experts to help them answer it.During her career, Golovkina has advised over 20 undergraduate and graduate students, two postdoctoral scholars, and has taught many courses in virology and microbiology. She credits her teaching style to one of her former teachers, Vadim Agol, an eminent Russian virologist who lectured from simple written note cards and a blackboard. She thinks that in an age when students are constantly immersed in their laptops, smartphones, and digital media, this simple strategy helps engage students in a more direct way.Golovkina has served on numerous committees at UChicago and is a member of the American Society for Microbiology. In 2018, she was named as a Fellow of the American Academy of Microbiology, and in 2019 as a Fellow of the American Association for the Advancement of Science.


Aedes sollicitans mosquitoes collected in a saltwater marsh in East Texas, USA. Full genome sequencing and phylogenetic analyses indicate that PTBV is distinct but genetically related to Drosophila X virus and mosquito X virus, which are assigned to species in the genus Entomobirnavirus, family Birnaviridae. PTBV produced cytopathic effect (CPE) in cultures of mosquito (C6/36) cells, but not in Vero cell cultures. Ultrastructural studies of PTBV in infected C6/36 cells demonstrated unenveloped virus particles about 55 nm in diameter.This report describes and characterizes a novel entomobirnavirus, designated Port Bolivar virus (PTBV), that was isolated from a pool of  Aquabirnavirus, Avibunyavirus and Blosnavirus) infect vertebrates, whereas members of four genera  infect invertebrates.The family Birnaviridae consists of viruses with two segmented dsRNA genomes, that form icosahedral, non-enveloped single-shelled particles with a diameter of approximately 65 nm . The famEntomobirnavirus, only two viruses have been classified taxonomically to date: Drosophila X virus  was detected in laboratory-reared fruit flies (Drosophila melanogaster) in France . Two. Two4]. viridae) . Drosophila X virus and Mosquito X virus) have been assigned in the genus Entomobirnavirus by the International Committee on Taxonomy of Viruses (ICTV), and no formal species demarcation criteria appear to have been published. Phylogenetically, PTBV clearly falls within this genus and, based upon amino acid sequence divergence in VP1 RdRp and the VP2-VP4-VP3 polyprotein, it is clearly distinct from both DXV  and MXV   and are sufficiently divergent in amino acid sequence  to be assigned to distinct new entomobirnavirus species.Currently, only two species (ctively) . AlthougTo date, the strongest association of entomobirnaviruses has been with mosquitoes; but the effects of this group of viruses on their mosquito hosts are still unknown. With the current interest in mosquito microbiomes, this should be an area for future study. Also, given their wide geographic distribution and the diversity of mosquito hosts, it seems likely that many other entomobirnaviruses exist in nature in mosquitoes and possibly other insects and arthropods.