However, the pI of viral proteins of algal (pI 7.08) and vertebrate (pI 7.09) hosts was in the basic range. The virtual 2D map of the viral proteome from different hosts exhibited host-dependent modalities. The virus proteome from algal hosts and archaea exhibited a bimodal distribution of molecular weight and pI, while the virus proteome of bacterial host exhibited a trimodal distribution, and the virus proteome of fungal, human, land plants, invertebrate, protozoa, and vertebrate hosts exhibited a unimodal distribution.A proteome-wide study of the virus kingdom based on 1.713 million protein sequences from 19,128 virus proteomes was conducted to construct an overall proteome map of the virus kingdom. Viral proteomes encode an average of 386.214 amino acids per protein with the variation in the number of protein-coding sequences being host-specific. The proteomes of viruses of fungi hosts (882.464) encoded the greatest number of amino acids, while the viral proteome of bacterial host (210.912) encoded the smallest number of amino acids. Viral proteomes were found to have a host-specific amino acid composition. Leu (8.556%) was the most abundant and Trp (1.274%) the least abundant amino acid in the collective proteome of viruses. Viruses were found to exhibit a host-dependent molecular weight and isoelectric point of encoded proteins. The isoelectric point ( They also found as virophage where a larger virus act as host for the smaller virus4. Viruses are sometimes referred to as a “piece of bad news” wrapped in a protein5 and lipid6. Viruses play a very important role in the ecology and evolution of the biosphere due to their potential ability to integrate into a host genome (except RNA virus and virophage) and utilize the transcription and translation machinery of their host8. Although most RNA viruses cannot, a small number of viruses (e.g. HIV-1) has the potential that can use reverse transcriptase to produce DNA that can integrate into the host genome9. Viruses are obligate parasites whose genomes have played an important role in the evolution of life10. Almost all viruses use single-stranded or double-stranded DNA or RNA for genome replication and expression12. The evolutionary origin of viruses, however, is a mystery due to their patchy molecular and functional attributes14. Although several theories and hypotheses15 have been postulated regarding the origin and evolution of viruses, none are backed by substantial evidence. Phylogenetic and taxonomic studies of viruses have gained enormous attention as the number of sequenced viral genomes has rapidly increased16. An understanding of the taxonomy and phylogeny of viruses will help to clarify the diversity and evolution of viruses in different host phyla16. Viruses have been extensively used in medicine and for genetic engineering due to their simple genetic architecture18. In addition, viral genomes have also been widely used to better understand the process of gene and DNA replication, transcription, RNA formation, translation, protein formation, and immunology19. Viruses are also used as vectors in genetic engineering applications and also as biological warfare agents. Millions of virus species have been projected to exist with approximately 6000 virus species thus far reported20.Viruses are the most abundant and ubiquitous obligate intracellular parasites22. The high level of sequence divergence and the smaller number of genes in viral genomes relative to prokaryotic and eukaryotic genomes, makes it difficult to identify genes that are conserved across and within families of viruses. The mutation rate of the viral genome varies from 1.5 × 10–3 mutation per nucleotide per genomic replication (RNA phage Qβ) to 1.8 × 10–8 mutations per nucleotide per replication (HSV-1)24. Notably, the mutation rate corresponds to the type of polymerase used in replication22. RNA viruses that use an RNA-dependent RNA polymerase were reported to mutate faster than viruses that use an RNA-dependent DNA polymerase (retrovirus) or reverse transcriptase22. Viruses that use an RNA-dependent RNA polymerase also mutate faster than viruses that use a DNA polymerase22. Drake et al. proposed a universal mutation rate in microorganisms of 3.4 × 10–3 mutations/genome/genomic replication25. The mutation rate of ssDNA (phage ϕX174), however, was reported to be quite higher than the level proposed by Drake et al.26. The high mutation rate makes it challenging to address problems associated with viral diseases. Although the genomic aspects of viruses are frequently studies, the proteome of viruses is poorly understood. Viral genomes, their role in immune modulation, and the use of viral genomes in vaccine development have gained enormous attention. Again, however, the viral proteome, its composition and function across hosts is poorly understood.The most challenging aspect of deciphering the origin and evolution of viruses is the presence of high sequence divergence due the high rate of mutation, genetic recombination, gene duplication/loss, and horizontal gene transfer that occurs in virusesProteomics is a very promising approach that can be used to better understand the molecular details of viruses. Increased knowledge of viral proteomes would enable a better understanding of the disease process and could be used to identify new biomarkers for the diagnosis and early detection of diseases and for drug development. Although proteomics has been used for biomarker development, its use in the development of broad-spectrum vaccines remains limited. The diversity of genomic and proteomic sequences in viruses may explain the basis of this problem. Although viral genomes, proteomes, and even the sequence of individual proteins are not conserved, a portion of a protein sequence can be used to develop a broad-spectrum vaccine against viral diseases. Therefore, characterizing the proteomic details of all known viruses was very important. Viruses are host specific and all viruses require a host to propagate. Therefore, the question arises, whether the genomic and proteomic content of a virus is also host specific? When viruses lack either conserved DNA or protein consensus sequences, it becomes essential to analyse viral genomes and proteins as they relate to host specificity. It is plausible that viruses may use similar molecular or biochemical mechanisms to propagate in specific hosts or host ranges. Understanding the molecular details of different groups of viruses in relation to their hosts may shed more light on the evolution and phylogeny of viruses. Therefore, we conducted a proteome-wide analysis of the proteome of the virus kingdom to provide details pertaining to the composition and structure of the proteome of the virus kingdom.Hyperionvirus sp. (GCA_003814165.1), with a genome size of 2.38 Mb, was found to encode the highest number of protein sequences (2493), followed by Pandoravirus inopinatum  with 1839 protein coding sequences. The average viral genome size was calculated to be 0.038 Mb and encodes an average of 48.662 protein sequences. Notably, a circular virus (GCA_004205475.1) was found to encode only one protein sequence within its 0.001043 Mb genome sequence.To understand the proteomic details of the virus kingdom, authors have downloaded 1.713 million predicted virus protein sequences from 19,128 viruses from National Center for Biotechnology Information database. Result showed, viruses exhibit great diversity in genome size, ranging from the largest at 49.31 Mb  to the smallest at 0.001043 Mb (GCA_004205475.1), the latter of which comprises a circular virus. A few other viruses with large genome sizes are 31.04 Mb (GCA_002003145.1), 29.61 Mb (GCA_002003185.1), 26.49 Mb (GCA_002013235.1), 25.31 Mb (GCA_002013255.1), 25.05 Mb (GCA_002003165.1), 21.19 Mb (GCA_002003205.1), 16.98 Mb (GCA_002003245.1), and 6.68 Mb (GCA_002003125.1) for uncultured marine viruses of invertebrate hosts. Due to the lack of properly annotated protein coding sequences, these viruses were excluded from the present study. Only viral genomes with complete protein annotation were utilized in the present analysis. Among these, Prochlorococcus phage P-SSM4 (accession AAX46881.1), ORF1ab  of the human SARS coronavirus (accession AAT98578.1), polyprotein  of the fungal Ceratobasidium endornavirus D (accession AOV81681.1), hypothetical protein  of the archaea Prokaryotic dsDNA virus sp. (accession QDP67171.1), and FhaB-like adhesion protein  of the protozoan Bodo saltans virus (accession ATZ80495.1).Viruses were found to encode a variable number of coding sequences, the number of which was based on their host. On average, the virus proteome was found to encode 386.214 amino acids per protein sequence. The average number of amino acids found in protein sequences of the virus proteome varied depending on their host species, as follows: algae (254.68), archaea (216.774), bacteria (210.912), fungi (882.464), humans (388.459), invertebrates (390.741), land plants (405.099), protozoans (329.364), and vertebrates (397.433). The protein sequences of bacterial viruses were found to be the smallest (210.912) and fungal viruses encoded he largest proteins (882.464). PSCNV polyprotein (AYD75846.1) of the Planarian secretory cell nidovirus was the largest and heaviest viral protein, comprising 13,556 amino acids and 1567.858 kDa. The largest viral proteins in other hosts were: fibronectin type III domain-containing protein  of algal Tetraselmis virus 1 (accession AUF82581.1), pp1ab protein  of the vertebrate Ball python nidovirus 1 (accession AIJ50565.1), hypothetical polyprotein  of the land plant Gentian Kobu-sho-associated virus (accession BAM78286.1), baseplate wedge initiator protein  of the bacterial Leishmania aethiopica RNA virus was found to encode eight amino acids, M-I-Q-C-S-T-V-A. Hypothetical protein (AAQ13720.1) in the archaea His 1 virus was found to encode 15 amino acids, M-Q-M-Q-E-K-G-W-K-I-I-I-E-E-Q, and hypothetical protein phiC2p81 (ABE99541.1) of the Clostridium virus was found to encode a small peptide of 15 amino acids, M-G-I-S-G-E-K-L-G-N-F-L-I-F-K. The fungal Sclerotinia sclerotiorum fusarivirus 1 was found to encode a small peptide of 28 amino acids .Viruses were also found to encode small peptides. Murine hepatitis virus (vertebrate host) was found to encode the smallest peptide (CAA28656.1) in the known virus kingdom with only 5 amino acids, M-S-S-T-T. A putative NTPase/helicase (AIW68525.1) in common midwife toad virus was found to encode seven amino acids, M-L-I-S-L-S-E. Hypothetical protein (AHZ10898.1) in the protozoan An analysis of 1.713 million viral protein sequences from 19,128 virus proteomes across many different hosts was conducted. The viruses were grouped according to their host. The host groups were Algae (99), Archaea (555), Bacteria,  Fungi (312), Human (540), Invertebrate (1956), Land plants (2546), Protozoa (87), and Vertebrates (2917). The amino acid composition in the different virus proteomes was analysed to characterize host-specific amino acid distribution (Supplementary Table Cys (0.983%), His 1.680%), and Pro (3.728%) amino acids were least abundant in Archaea viruses. Glu (5.310%), Ile (4.972%), Lys (4.795%), Asn (4.111%), and Tyr (3.525%) exhibited their lowest abundance in fungal viruses. The lowest percentage of Phe (3.755%) and Ser (6.123%) was found bacterial viruses, Met (2.202%), Arg (4.540%), Thr (5.425%), and Val (5.520%) exhibited their lowest abundance in Protozoan viruses  was the most abundant and Trp 1.274%) was the least abundant  between the amino acids , while fungal viruses encode the largest proteins (98.911 kDa) (Supplementary Table 2.942 kDaA correlation (Pearson’s) analysis was conducted to determine the relationship between the molecular weight of viral proteins and different hosts Fig. . ResultspI of the virus proteome was found to reside in the acidic pI range. The average pI of the virus proteome was 6.89. When viral proteomes were grouped by host, however, a host-related pI distribution was observed. The bacterial virus proteome exhibited the lowest average pI (6.3), while land plant virus proteome had the highest average pI (7.5) , human viruses (pI 6.85), invertebrate viruses (pI 6.96), and protozoan viruses (pI 6.85), all of which represent a basic pI.The pI of virus proteomes found in different hosts was land plants > vertebrates > algae > fungi > invertebrate > human > protozoa > archaea > bacteria (Supplementary Table pI protein in the virus kingdom was a pI 13.364 (BAH72951.1) found in the bacterial virus, Ralstonia phage phiRSL1, while the lowest pI in a virus proteome was pI 2.448 (ATZ81137.1), which was found in the protozoan virus Bodo saltans. The average acidic pI viral proteome was pI 5.507, while acidic pI range was 5.001–5.9  clustered together, while the basic pI viral proteomes of protozoa and vertebrate clustered in close proximity to each other   plant proteome was 424.34, while the nuclear-encoded (not from chloroplast) proteome of fungi was 459.31928. While Trp (1.28%) is the least abundant amino acid in the nuclear-encoded plant kingdom proteome, Cys (1.267%) is the least abundant amino acid in the nuclear-encoded fungal kingdom proteome28. Cysteine forms disulphide bonds in proteins and provides conformational stability. Disulphide bonds are typically present in extracellular proteins and rarely in intracellular proteins36. The low percentage of Cys in viral proteomes reflects their need to be targeted to intracellular compartments of the cell. We suggest that viruses encode a low percentage of Cys amino acids so that their encoded proteins can be readily targeted to the cytoplasm of the host cell. The most and least abundant amino acids in the virus proteome are more closely correlated with their amino acid abundance in the nuclear-encoded proteome of the plant proteome than the fungal proteome. The molecular weight of viral proteomes revealed that the proteomes of fungal viruses encode the heaviest average proteins with an average molecular weight of 98.911 kDa, while the proteomes of bacterial viruses encode proteins with an average weight of only 22.942 kDa. The molecular weight of a protein has a significant impact on translocation across cellular compartments and also represents an important aspect of the functional role of a protein. Viruses are able to manipulate host cells to transcribe and translate large proteins that are not needed by the host cells. Understanding the molecular components responsible for the translation of such large proteins in host cells can be crucial to eliminating virus-mediated diseases in host organisms. The largest protein encoded in the human genome contains more than 27,000 amino acids and possesses at least 34 functional domains, while the largest protein encoded by viruses and produced in human hosts is the ORF1ab polyprotein (AAT98578.1) of the human coronavirus, which contains 7182 amino acids  is the most abundant and Trp 1.274%) is the least abundant amino acid, respectively. Leu is also the most abundant amino acid in the nuclear-encoded plant (9.62%) and fungal (9.115%) proteome.274% is pI range (pI 6.89), which is similar to average pI of the nuclear-encoded plant and fungal proteomes28. The average algal virus proteome had a pI of 7.08 and the vertebrate viral proteome had a pI of 7.09, both of which are in the basic range. Although the collective virus proteome encodes a higher percentage of acidic than basic pI proteins, they only encode a few proteins with a neutral pI (7.0). The percentage of neutral pI viral proteins algal, archaea, bacterial, fungal, human, invertebrate, plant, protozoan, and vertebrate hosts was 0.159%, 0.079%, 0.115%, 0.285%, 0.231%, 0.241%, 0.215%, 0.16%, and 0.241%, respectively. The archaea virus proteomes had the lowest percentage of neutral pI proteins, while fungal virus proteomes had the highest percentage of neutral pI proteins.The average isoelectric point of the virus proteome is in the acidic 28. Schwartz et al. previously reported a trimodal distribution for the pI and molecular weight of all eukaryotic proteins38. The pH of cytoplasm of prokaryotic and eukaryotic cells is usually in the acidic pI range. Therefore, viruses have also evolved to produce proteins that have an acidic pI. However, the pH of chloroplasts is in the basic pI range, and as a result, proteins with a basic pI may be targeted to specific sub-cellular locations. Kirag et al. reported that the pI of a protein is based on their modality and taxonomic association39. Ecological niche and sub-cellular localization, however, have also been reported to play a critical role in determining the pI of a protein39. Schwartz et al. reported that the pI of a protein is correlated with its sub-cellular localization and the pH of the cytosol is below 738. Viruses, however, do not have any cellular or sub-cellular components and hence do not encode the pI of a protein based on these factors. The selection pressure on the pI of a viral protein is completely based on the cellular environment of its host. Modification in the host pI environment by external factors can assist in eliminating a virus from its host and decrease the virulence and pathogenicity of a virus. Although the estimated and experimentally-validated pI value of a protein can be different in vivo, they are typically in close agreement when evaluated on a 2-DE gel40. The variation in the modal distribution of virus proteomes may be attributed to host-dependent selection pressure. The observed variation in the modal distribution of different viral proteomes based on their hosts is intriguing. Protozoan and bacterial virus proteomes exhibited a trimodal distribution, while algal and archaea virus proteomes displayed a bimodal distribution. Although protozoa are eukaryotic organisms and bacteria are prokaryotes, they share a common, trimodal modality in molecular weight and isoelectric point of their proteome. Although, archaea are also prokaryotes, they exhibit a bimodal distribution in the molecular weight and pI of their proteomes. The proteomes of multi-cellular eukaryotic hosts, including fungi, humans, land plants, invertebrates, and vertebrates display a unimodal distribution. This perhaps explains why the proteomes of viruses that infect and reside in multicellular eukaryotic hosts also display a unimodal distribution.The virus proteome exhibited a host-dependent modal distribution of molecular weight and isoelectric point. Algal and archaea virus proteomes exhibited a bimodal distribution, while protozoan and bacterial virus proteomes exhibited a trimodal distribution. Fungal, human, land plant, invertebrate, and vertebrate viral proteomes exhibited a unimodal distribution. Nuclear-encoded proteomes of the plant kingdom have been reported to exhibit a trimodal distribution, while fungal proteomes exhibit a bimodal distributionViruses exhibit highly diverse and heterogeneous genome and replication mechanisms. They tend to undergo a high rate of mutation that contributes to a high degree of genetic and proteomic variation. Similarly, a single-stranded virus mutates more frequently than a double-stranded virus. The reverse transcribing DNA hepadnavirus can undergo a high-degree of genetic mutation that contributes to high genomic and proteomic diversity. In addition, they lack proofreading and lack a DNA repair mechanism. Although, these molecular mechanisms play important role in contributing to higher genetic and proteomic diversity, ecological and demographic factors are also responsible for the higher genetic diversity. Natural selection and random genetic drift generate great pressure to undergo evolution towards the genetic diversity in viruses. Although there is a great diversity between the virus population, the virus population structure for any particular host can be useful to develop broad range of antiviral agents/vaccines using artificial intelligence and a machine learning approach. The virus proteome study is just a starting point for the functional studies and discoveries uncovered through the proteomic study need extensive study to determine their functional significance. Virus proteome-based specific application tool need to be developed to understand valuable information on viral pathogenesis and their lifecycles as well as cellular functions. Amino acid composition, isoelectric point, and molecular weight of the virus protein can be very valuable towards development of such tools in future.41. All the analysis was conducted based on the host groups to understand the host-specific relationship and or differences. The calculated molecular weight and isoelectric point of the virus proteome were further processed using Microsoft excel 2016.All the protein sequences of the virus proteome were downloaded from the National Center of Biotechnology Information (NCBI). The virus proteome was downloaded based on its host. The viral host were algae, archaea, bacteria, fungi, humans, invertebrates, land plants, protozoa, and vertebrates. In total protein sequences of 19,128 virus proteome were downloaded that constituted 1.713 million predicted protein sequences. The downloaded protein sequences of the virus proteome were subjected to analysis of molecular weight and isoelectric point. The molecular weight of the virus proteins was calculated using the python-based command line “protein isoelectric point calculator” (IPC Python) in a Linux-based platform. The source code was used as mentioned by KozlowskipI proteins of the virus proteomes. Statistical software Unscrambler version 10.4 was used to conduct the principal component analysis. NIPLAS  model was used to conduct the PCA plot with 100 iterations. The role of calibrated to validated residual variance was 0.5 and the ratio of validated to calibrated residual variance was 0.75. Correlation and regression analysis was conducted using statistical software JAPS version 0.14.1.0. Pearson’s correlation (r) was used to run the correlation-regression plot with confidence interval 95% (p < 0.05) and prediction interval 95% (p < 0.05). The correlation heat-map plot was constructed using JAPS version 0.14.1.0 software using Pearson’s correlation (r) with confidence and prediction interval 95% (p < 0.05). The photograph of the virtual 2D proteome map of virus proteome was constructed using scatterplot online platform (https://scatterplot.online/).Statistical analysis was conducted to understand the similarity and difference between the virus proteome originated from a different host. Principal component analysis (PCA) is used to analyze exploratory data and for making predictive models by projecting each data points. It defined the direction and maximizes the variance of the projected data. Therefore, to understand the similarity and variances in the virus proteome data we conducted the PCA to understand the similarity and differences of the amino acid composition of the virus proteome. Further, PCA was also used to understand the similarity and variances in the acidic and basic Supplementary Information 1.Supplementary Information 2.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.


Hydroxycobolamine supplementation in hereditary cobolamine deficiency and serial biochemical follow‐up allow uncomplicated pregnancy outcome. Hydroxycobolamine supplementation in hereditary cobolamine deficiency and serial biochemical follow‐up allow uncomplicated pregnancy outcome. We report a pregnancy in a woman with known cobalamin C/D deficiency treated with hydroxocobalamin.Cobalamin vitamin B12) defects are rare, autosomal recessive inherited disorders in the general population causing defects in the remethylation pathway of homocysteine. Homocysteine is an amino acid derived from methionine. Under normal circumstances, homocysteine is subsequently converted into cysteine or it is remethylated, forming methionine again. Inherited cobalamin deficiency is a remethylation defect causing the accumulation of homocysteine and methylmalonic acid  and high urinaru methylmalonic acis  at the first visit. No other family members reported any symptoms, there were no other siblings in the family.Because of the recessive inheritance pattern of this disorder, her partner underwent genetic testing and he turned out not to be a carrier of cobalamin deficiency. This implicates that the baby has a minimal risk of having the same deficiency.For her cobalamin deficiency, she was treated with intramuscular injections of hydroxocobalamin 100 mg every 2 weeks, which she continued throughout the entire pregnancy. During pregnancy, she smoked two cigarettes a day. Ultrasound investigations at 16, 23, and 35 weeks gestation showed no structural abnormalities. Fetal growth was normal for the given gestational age. Metabolic follow‐up at 19 weeks gestation demonstrated undetectable homocysteine values in blood and urine.Right before delivery, a blood test given showed a hemoglobin value of 11 g/dL, a slightly elevated value of white blood cells (18 400/µL), normal ionogram value, and decreased creatinine value (0.40 mg/dL), all within the physiologic ranges for pregnancy. The value of vitamin B12 was >2000 ng/L, and methylmalonic acid was undetectable in urine. This implicated a control of the cobalamin C/D deficiency. The patient has always refused to follow any dietary restrictions.At 37 weeks and 6 days gestation, the patient went into spontaneous labor leading to a vaginal delivery. There were no complications, and on the same day, she delivered a term daughter of 2.230 kg (<10th percentile on the Flemish growth charts) with a length of 46 cm (50th percentile) and a head circumference of 33 cm (50th percentile). APGAR scores after 1, 5, and 10 minutes were 9, 10, and 10, respectively. A neonatal physical examination showed no abnormalities.Postpartum, the patient was observed for 5 days to ensure there were no problems concerning her cobalamin deficiency. During this period, there were no specific problems reported, and she was able to go home in good condition. Ammonia levels in the blood were elevated (27 µmol/L) the day after delivery, and they decreased to 21 µmol/L on the second day. After delivery, carnitine (carnitine syrup 1000 mg per day) was added to the treatment. At 3 months postpartum, her metabolic follow‐up was normal.3Different remethylation disorders are inherited in an autosomal recessive way, all of which result in deficient activity of methionine synthase Figure . These dDiagnosing a remethylation disorder in patients using the clinical picture might be difficult due to the highly heterogeneous presentation.In 1998, the European Journal of Pediatrics published guidelines for diagnosing and treating remethylation disorders. They stated that patients with a new cobalamin deficiency should first be treated with intravenous hydroxocobalamin and, after a few weeks, with intramuscular injections (1‐2 mg/dose). They also state that the addition of betaine (3‐6 g/day), folic acid (5 mg/day), and carnitine (50‐100 mg/kg/day) can be beneficial.The main treatment of cobalamin deficiency is hydroxocobalamin, which is used instead of cyanocobalamin because it seems more effective in patients with cblC deficiency.Our patient was treated with hydroxocobalamin and carnitine. The treatment with hydroxocobalamin was already initiated before conception, as she was receiving intramuscular injections of hydroxocobalamin 100 mg (1000 µg) of every 2 weeks. After delivery, she also received carnitine 1000 mg/day. This is a general measure we follow to counter the high metabolic demand. Her treatment did not include betaine.The main concern regarding metabolic disorders in pregnancy lies in the increase in amino acids. When they exceed the catalytic capacity of the enzymes needed for their metabolism, there is an accumulation of toxic metabolites, which could be harmful for the mother and the baby. Especially, the involution of the uterus postpartum leads to an increase in proteins and, thus, the risk of an accumulation of metabolites, such as ammonia.Few studies have been published concerning cobalamin deficiency in pregnancy; as far as we were able to determine, this case report is the fifth one published. Three previous case reports on pregnant women with cobalamin C (cblC) deficiency reported healthy neonatal outcomes and no maternal complications during pregnancy and postpartum. In two cases, the women started having treatment at 15 weeks gestation. One of the women was treated with a low protein diet, acetylsalicylic acid (80 mg/day), cobalamin, folic acid, and carnitine.In most cases, complementation studies to identify the genetic defect of the cobalamin deficiency are not accomplished. Therefore, these patients are categorized as having a cblC/D deficiency.Guidelines for the management of cobalamin deficiency in pregnancy do not exist. We emphasize serial monitoring and clinical and biochemical observation as the mainstays of management. Careful observation, anticipation, and management of maternal and fetal complications are key considerations.None declared.HK: involved in case description and reviewed the literature. YJ: involved in discussion and performed the data collection on the case.Published with written consent of the patient.


Insects are crucial for ecosystem functions and services and directly influence human well-being and health. Insects are among the most diverse classes of the animal kingdom, and therefore it is not surprising that they contain an immense degree of virus diversity, which remains largely unexplored. Until recently, investigations of insect virus diversity were focused on insect species of economic, veterinary, or medical importance. The deployment of high-throughput sequencing technologies has allowed for studies of virus diversity in a wider range of insect species and has led to an explosion in the discovery of new viral sequences in insect hosts, both from known virus groups and from entirely novel families. These studies have, in turn, inspired a re-assessment of the evolutionary relationships among, and taxonomic classification of, insect viruses. It is now clear that further studies of the biological effects of viruses on their insect hosts are required to understand the impact of diverse insect viromes on their host’s physiology, reproduction, and the potential of host insects to be viral vectors.Acheta domesticus) [Neohydatothrips variabilis) [Scathophaga furcata) [Aedes aegypti) from the Amazon basin [Apis mellifera) [Andrena spp.) [Varroa destructor mites, which are closely associated with both Western and Eastern honey bees  [Diabrotica undecimpunctata and D. virgifera) [Matsumuraeses phaseoli, an important legume pest, were also presented [The aim of this Special Issue was to invite research articles focusing on recent advances in virus discovery and the elucidation of virus diversity in insect hosts. The published papers illustrated the effectiveness of high-throughput sequencing approaches for the comprehensive analysis of virus communities, identification of novel virus variants, and discovery of novel viruses. This includes the discovery of previously unknown RNA viruses in house crickets (esticus) , soybeaniabilis) , termiteiabilis) , dung flfurcata) , mosquiton basin , hematopon basin , honey bllifera) ,8,9 and na spp.) , and evectively) . Reportsrgifera)  and a beresented .V. destructor and honey bees (A. mellifera) [Sequence data on the novel viruses identified in high-throughput sequencing studies could be a starting point for further analysis of virus–host interactions. Notably, some of the published papers already present such analyses, including the investigation of viral inter-species transmission between wild and managed honey bees  and betwllifera) , and anallifera) .


Zika viAmong live-born infants reported in USZPIR, 4.6%  had any Zika-associated birth defect. In the subgroup with positive Zika virus NAAT during pregnancy, 6.1%  infants had any Zika-associated birth defect. Among pregnancies with positive Zika virus NAAT results, and thus less likelihood of exposure misclassification, the frequency of any Zika-associated birth defect was higher among those with firstThe most frequent structural defects reported among live-born infants and children were microcephaly; corpus callosum abnormalities; intracranial calcification; abnormal cortical gyral patterns; ventriculomegaly; cerebral or cortical atrophy; chorioretinal atrophy, scarring, or pigmentary changes; and optic nerve abnormalities . A similAmong 325 pregnancies with laboratory evidence of confirmed or possible Zika virus infection that resulted in a pregnancy loss, 13 (4.0%) fetuses had any reported Zika-associated birth defect. Defects included microcephaly, cerebral or cortical atrophy, abnormal cortical gyral patterns, corpus callosum abnormalities, cerebellar abnormalities, hydranencephaly, ventriculomegaly or hydrocephaly, and brainstem abnormalities . ,During 2015–2017, large Zika virus outbreaks occurred throughout the United States (including U.S. territories and freely associated states). In the United States, infections during pregnancy were initially reported among U.S. travelers returning from affected countries.Certain individual brain and eye defects associated with Zika virus infection were frequently reported in USZPIR cohort. A similar subset of Zika-associated birth defects was found to have significantly higher prevalence ratios in areas of widespread local transmission compared with areas without local transmission in the Zika Birth Defects Surveillance System.The findings in this report are subject to at least five limitations. First, these data are based on information abstracted from medical records. Although CDC provided specific guidance for evaluation of all infants born from pregnancies with possible Zika virus exposure during pregnancy . Approximately 5% of infants in USZPIR had any Zika-associated brain or eye defect. Several individual brain and eye defects were more commonly reported. One third of infants with any Zika-associated birth defect had more than one defect reported.Certain brain and eye defects in infants might prompt suspicion of prenatal Zika virus infection and might provide a signal to the reemergence of Zika virus, particularly in geographic regions without ongoing comprehensive Zika virus surveillance.


Further, the electron microscopy of symptomatic plant samples explicated the presence of isometric particles of CMV and quasi spherical particles of tospoviruses. This is the first molecular evidence for the natural co-existence of chlorosis inducing CMV strain with CaCV and GBNV on hot pepper in India.Cucumo- and tospoviruses are the most destructive viruses infecting hot pepper (chilli). A diagnostic survey was conducted to assess the prevalence of cucumo and tospoviruses in chilli growing tracts of Tamil Nadu. Infected plants showing mosaic with chlorotic and necrotic rings, veinal necrosis, mosaic mottling, leaf filiformity and malformation were collected. Molecular indexing carried out through reverse transcription polymerase chain reaction (RT-PCR) with coat protein gene specific primer of Cucumber mosaic virus (CMV) and tospovirus degenerate primer corresponding to the L segment (RdRp). Ostensibly, amplifications were observed for both CMV and tospoviruses as sole as well for mixed infections. The sequence analysis indicated that the Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) to be involved with CMV in causing combined infections. The co-infection of CMV with CaCV was detected in 10.41% of the symptomatic plant samples and combined infection of CMV with GBNV was recorded in around 6.25% of the symptomatic plants surveyed. The amino acid substitution of Ser Capsicum annuum L.) is the most important spice crop with significant commercial value throughout the world due to its great phenotypic, horticultural, agricultural and biological diversity1. India is one of the world’s largest producer, consumer and exporter of chillies globally. Chilli is reported to be affected by more than 30 economically important viruses by causing 15–50% of yield losses in chilli cultivation3. It includes Cucumber mosaic virus4, Chilli leaf curl virus5, Tomato spotted wilt virus6, Groundnut bud necrosis virus7, Capsicum chlorosis virus8, Tobacco mosaic virus9, Potato virus Y6, Tomato leaf curl New Delhi virus10 and Chilli veinal mottle virus11. Among them the cucumo and tospoviruses are the most predominant viruses reported to infect almost all the vegetables crops and pose serious threat to chilli production12. CMV  consist of isometric particles having sense, ssRNA genome with tripartite segment designated as RNA1, RNA2 and RNA313. CMV causes severe mosaic symptoms like leaf distortion and fruit lesions which affect the significant marketable yield of chilli14. Tospoviruses  are also isometric, ambi-sense, ssRNA virus with tripartite genome of small (S), medium (M) and large (L) segment15. The symptoms appeared on young leaves as chlorotic lesions later turns to necrotic lesions or yellow spots coalesce to form mosaic pattern and develops concentric rings on mature leaves and fruits12. Basically, infection may be caused by a single virus or mixed infection by more than one virus together by forming disease complex7. In general, dynamics of mixed infection with two or more unrelated viruses are common in nature16. Indeed, mixed infection of CMV with Potato virus Y17, CMV with Pepper mottle virus18, CMV with Turnip yellow mosaic virus19, CMV with Watermelon mosaic virus20, CMV with Tomato leaf curl New Delhi virus21 and Cucurbit yellow stunting disorder virus with Zucchini yellow mosaic virus22 have been reported previously. The mixed infection allows diverse virus interactions and maintenance of viral genetic diversity. Symptoms induced by mixed viral infections were more severe than the single viral infection23. Besides, mixed infections tend to affect virulence, accumulation, symptomology and host range which ultimately results in unpredictable pathological consequences25. Moreover, virus interaction in host plant hews the evolutionary dynamic of viral populations26. Unraveling the viruses concerned with mixed infections is vital for better understanding of the ecology and evolution of viral diseases which are essential for viral disease prevention and formulating management strategies25. The prime objective of the present study is to explore the mixed infections of CMV with tospoviruses in chilli under natural field conditions of Tamil Nadu in India.The hot pepper commonly known as chilli . Initially, localized chlorotic lesions were observed on inoculated leaves of chilli plant at 2 dpi  were sap inoculated onto chilli and Inoculated plant samples were partially purified and observed under transmission electron microscope to determine the particle morphology of viruses associated with mixed infection. Electron micrograph of infected plants showed isometric virus particles of 24–52 nm in diameter and quasi spherical virus particles of 70–120 nm in the sample  substituted with serine (Ser129) at 129th position in coat protein of CMV  observed in coat protein comparatively with CMV isolate causing single infection.The nucleotide sequences of GBNV MT553997) showed 98.96% of identity with chilli isolate of Trichy (KU941833) followed by groundnut isolate of Coimbatore (MF491630). Similarly, CaCV (MT553996) had an identity of 97.5% at nucleotide level with chilli isolate of Coimbatore (KU941835) followed by groundnut isolate of China (KX078565). Likewise, CMV (MT647887 and MT647888) isolates showed 97.5–98% identity with chilli isolate of Karnataka (HM348786) followed by 97.2–97.5% of identity with coat protein gene of chilli isolate (KM272275). Genetic divergence of TN CaCV and GBNV isolates have been evaluated through phylogenetic analysis of TN isolates with other member isolates inferred using neighbor joining tree method with 1000 replicates. Thus, phylogenetic analysis depicts diversified cluster formation of CaCV and GBNV isolates based on RdRp gene segment with other member GBNV and CaCV isolates of India  infected chilli plants showed concentric rings on mature leaves and chlorotic spots on younger leaves12. Indeed, CMV infected chilli plants showed mosaic mottling, puckering, shoe string or filiformity and stunted growth27. Based on symptomology, symptomatic leaf samples of mosaic mottling along with concentric chlorotic ring spots and mosaic with veinal necrosis were suspected for mixed infection. Since, symptomology based detection would not be reliable and it has to be further confirmed and validated either by serological or by molecular method of detection12. RT-PCR based method will be the reliable to underpin the mixed infection and to index the associated viruses. It is more accurate to detect the viruses even at very low titer in the infected leaf samples than serology based assays28. From the analysis, it is clearly demonstrated that the tospoviruses associated with CMV were identified as Groundnut bud necrosis virus and Capsicum chlorosis virus. The symptomatological study depicts that GBNV and CaCV produces similar symptoms on chilli plant and it is very difficult to distinguish them based on symptomology. The sap inoculation of tospovirus (GBNV and CaCV) chilli isolate on Nicotiana and cowpea host plants produced localized chlorotic lesions followed by necrotic lesions at 4–5 dpi29. The CaCV certainly induces chlorotic spots initially and later developed necrosis around the central spot8. Intrinsically, mechanically inoculated plant samples after 12 dpi were positively amplified for both CMV and tospovirus in RT-PCR.Several epidemiological studies depicts that single host plant affected by more than one virus implies the occurrence of mixed virus infection. In general, mixed viral infections are more common30. Hence, electron microscopic examination of infected plant samples corroborated the co-existence of isometric cored particle of CMV with quasi spherical particle of tospovirus. The results have been substantiated with previous findings33.Electron microscopic visualization and evaluation helps to study the particle morphology for detection of mixed viral infections. Substantially, diagnosis of viral diseases by TEM utters rapid and accurate identification towards mixed infection34. Coat protein of CMV comprised of 657 nucleotides putatively translated into 219 amino acids. CMV can be categorized as chlorosis inducing strain (Ser129 or Leu129) and mosaic inducing strain (Pro129) based on the position of 129th amino acid of coat protein gene35. Certainly, amino acid 129 of coat protein is a genetic determinant responsible for symptom induction in host plant and in certain cases it act as virulence determinant36. Amino acid substitution of Ser129 over conserved Pro129 induces chlorosis in the host rather necrosis by disrupting the functions of chloroplast37. More likely, cholorosis induction in the infected plant determined by structural alteration in coat protein of CMV incited by conserved (pro129) amino acid substitution38. Comparative amino acid sequence alignment vindicated CMV isolates associated with mixed infection is a chlorosis inducing strain (substituted with Ser129). Whereas, CMV causing single infection are prevalently mosaic inducing strains (possess Pro129). Increased flexibility of βE-αEF loop (129–136 amino acids) of coat protein has direct correlation with pathogenesis of virus. Moreover, flexibility of βE-αEF loop is regulated by 129th amino acid properties39. In general, frequent reassortment and recombination occurs in segmented viruses over monopartite viruses40. Amicably, CMV undergoes prompt of genetic changes through reasssortment and recombination42. Thus, evolutionary mechanism is a characteristic attribution of multipartite viruses which plays significant role in emergence and interspecies transmission of viruses43. Any single variation in amino acid or nucleotides may effect on stability and infectivity of virus. Hence, thus apparent variation observed on multipartite TN CMV isolate suspected to be associated with co-adoption or co-existence of CMV with other viruses. Over all the accumulating evidences suggests that amino acid 129 (chlorosis induction) besides symptom determinant may also have correlation with co-existence of CMV with tospoviruses.The nucleotide sequences of coat protein of CMV indicates that CMV isolate belong to subgroup IB. Based on serology and nucleic acid hybridization, CMV isolates are classified into subgroup I and II and further CMV subgroup I divided into IA and IB based on coat protein gene sequence and phylogenetic analysisMixed virus infections will intensify the disease dynamic in host plants comparatively than single virus infection. In some cases, symptom induced by one virus can be masked by other virus in combined infections. Mixed infection in host plants will results in maintaining the genetic diversity of viruses. Thus may inflict to the emergence of novel genetic phenotypes or strains of virus. It is very crucial to study the significant role of mixed infection in altering genetic diversity of viral population. In the present study, we explicated the co-existence of Cucumber mosaic virus with Capsicum chlorosis virus and Groundnut bud necrosis virus in chilli under natural field conditions of Tamil Nadu in India. Indeed, CMV, GBNV and CaCV are the most notorious and economically important viruses reported to be causing mosaic and necrosis on most of vegetable crops. Hence, combined infection of these destructive viruses may menace to chilli cultivation. Moreover, frequency of occurrence of mixed infection of mosaic and necrosis disease in chlli becoming increasingly diverse and alarming currently in chilli growing areas of Tamil Nadu. To our knowledge, this is the first report for combined infection of cucumo- and tospoviruses in chilli. This preliminary study sought to help in further future studies on combined infections of CMV with other plant viruses. Although, the mechanism behind the interaction of viruses between different groups on a single host during mixed infections is yet to be studied in detail.Nicotiana benthamiana plants at 2–3 leaf stage by gentle aberration on carborundum dusted leaves43. The inoculated plants were maintained at 30 ± 2 °C in greenhouse of Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore (India) under insect proof cage for the symptom expression.A systemic field survey was conducted during 2018–2019 in Coimbatore, Thiruvannamalai, Dindigul, Tirunelveli, Namakkal, Salem, Krishnagiri and Theni districts of Tamil Nadu in India. Plants showing characteristic virus disease symptoms were identified and the symptomatic leaf samples with mosaic, veinal necrosis, chlorotic rings, necrotic ring spots, mosaic mottling, leaf malformation and stunted growth were collected. Infected chilli samples (observed for mixed infection) with 0.1 M phosphate buffer (pH 0.7) were mechanically inoculated on chilli and 44. 250 g of infected sample ground using liquid nitrogen and centrifuged at higher speed. The obtained pellet suspended in 200 µl of storage buffer containing 5 mM sodium borate and 0.5 mM EDTA. Briefly, 10 µl of 0.01% of BSA was loaded into 400 mesh copper grid and draw off from grid after few seconds. Subsequently, 2 µl of purified virus sample coated into grid kept for 3–5 min and then, washed thrice with sterile water. Consequently, virus particles have been stained with 2% phospho tungstic acid. The excess stain has been wiped off from edges of grid with filter paper and allowed for air dry. Eventually, grid was observed under TEM with EDAX  at 70,000–90,000 magnification and documented.The virus from infected plants was purified as per methodology45. The first stand complementary DNA (cDNA) was synthesized from the extracted RNA to detect the viruses associated with infections. For the cDNA synthesis of CMV, 20 µl reaction mixture  was used  and incubated at 42 °C for 60 min followed by 70 °C for 5 min46. For tospovirus, the reaction mixture contains total RNA, random primer and nuclease free water up to 12.5 µl were incubated at 65 °C for 5 min followed by 5× reaction buffer, 10 mM dNTP mix, RNase inhibitor and reverse transcriptase were added and incubated for 1 h at 42 °C47. The presence of CMV was confirmed through RT-PCR with CMV coat protein gene specific primer (RsCMV-F and RsCMV-R) with a cyclic condition of 94 °C for 2 min followed by 94 °C for 30 s of denaturation, 59 °C for 30 s of annealing, 72 °C for 60 s with final extension of 72 °C for 10 min48. The tospovirus was confirmed through RT-PCR using tospovirus universal primer (gL3637 and gL4435C) corresponding to L segment (RdRp) with the cycling condition of 94 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 56 °C for 1 min and 72 °C for 1 min with 72 °C for 10 min of final extension49.Total RNA was extracted from the collected symptomatic leaf samples using TRIzol reagenthttp://blast.ncbi.nlm.nih.gov) and comparative amino acid and nucleotide sequence analysis carried out using Clustal W (www.ebi.ac.uk). Sequences were aligned using Bio-Edit Sequence Editor program 7.2. The phylogenetic relationships among the isolates were analyzed using MEGA 7.0 software (www.megasoftware.net) by Neighbor joining tree method with 1000 bootstrap replication50.The positive amplicons of CMV (coat protein) and tospoviruses (replicase protein) were purified using GenJET PCR purification kit (Themo scientific Inc.) and cloned into pGEM-T easy vector (Promega). Two independent clones were sequenced at both orientations with M/s Barcode Biosciences, Bangalore. Data base searches performed with NCBI BLAST (Supplementary Informations.


The oral route of administration is by far the most convenient route, especially in the treatment of chronic conditions. However, many therapeutics present formulation difficulties which make them unsuitable for oral delivery. Recently, we synthesized a denatured whey protein isolate (dWPI) bead entrapped with insulin. Our present goal was to assess the suitability of this delivery system to the delivery of other potential molecules, both hydrophilic and hydrophobic. Beads of 1.2–1.5 mm in diameter were entrapped with four payloads representing a range of solubilities. The water-soluble payloads were sodium fluorescein (SF) and FITC dextran 4000 Da (FD4), while the hydrophobic ones were Fast Green and curcumin. Encapsulation efficiency (EE) was 73%, 84%, 70%, and 83% for SF, FD4, Fast Green, and curcumin-loaded beads, respectively. The corresponding loading capacity for each bead was 0.07%, 1.1%, 0.75%, and 1.1%, respectively. Each payload produced different release profiles in simulated gastric fluid (SGF) and simulated intestinal fluids (SIF). SF released steadily in both SGF and SIF. FD4 and curcumin release was not substantial in any buffers, while Fast Green release was low in SGF and high in SIF. The differences in release behaviour were likely due to the varying properties of the payloads. The effect of proteolysis on beads suggested that enzymatic degradation of the whey bead may promote payload release. The beads swelled rapidly in SGF compared to SIF, which likely contributed to the release from the beads, which was largely governed by solvent diffusion and polymer relaxation. Our results offer a systematic examination of the behaviour of hydrophilic and hydrophobic payloads in a dWPI delivery system. These beads may be further designed to orally deliver poorly permeable macromolecules and poorly soluble small molecules of pharmaceutical interest. Delivery of therapeutics via the oral route is preferred by patients, but it presents formulation difficulties for poorly permeable Biopharmaceutical Class System (BCS) Class III small molecules, peptides and proteins, as well as poorly soluble BCS Class II small molecules. The chief concerns are susceptibility to degradation, solubility, and/or poor permeability at the intestinal epithelium . The gaspDNA to rat colon specifically, and that it could be transfected effectively [Numerous strategies have been investigated in attempts to overcome obstacles to oral drug delivery, including the use of micro and nanocarriers, protease inhibitors, permeation enhancers (PE), chemical modifications, excipients to aid solubility, and use of ingestible microneedle and patch devices . While tectively . Anotherectively .Another interesting polymer is whey protein, a by-product of cheese manufacturing. Whey protein isolate (WPI) is used as an encapsulation matrix in micro and nanoparticles because of its capacity for gel formation. These gels can be formed by cold-set gelation, which does not subject the cargo to harsh temperatures or solvents, making it ideal for labile therapeutics . Cold-seWPI has also been shown to improve the storage stability of payloads and resistance to oxidation . The hyddWPI has also been used to encapsulate riboflavin (Log P: −1.46) , and vit®-200F was a gift from Evonik . Simulated intestinal fluid (SIF) powder was purchased from Biorelevant . All other reagents were purchased from Sigma Aldrich  unless stated otherwise.BiPro WPI (97%) was obtained from Davisco Foods International Inc. , Aerosilw/v) WPI was dissolved in HPLC grade water overnight. Following this, the WPI was denatured by stirring in a 78 °C water bath over 45 min. The solution was then rapidly cooled on ice for 1 h followed by overnight storage at 4 °C. The resulting dWPI was used over 2 days.Denaturation of WPI was carried out in advance of acid-induced gelation using our previous method . In thisDenaturation was confirmed by measuring the thiol content of native (nWPI) and denatured WPI as it has been reported that it increases after denaturation . The conw/v) dWPI and 0.01% (w/v) payload in the case of SF, or 0.1% (w/v) payload for FD4, Fast Green, and curcumin. Each payload was dissolved in a solvent prior to incorporation with dWPI: water for SF and FD4, ethanol (33% v/v) for Fast Green, and methanol (100% v/v) for curcumin). After ~15 min stirring, beads were produced by adding the mixture dropwise to a sodium acetate hardening solution (pH 4.6) using a 1 mL plastic syringe  with a 30G needle . This solution was prepared by combining equal volumes of 0.5 M acetic acid and 0.5 M sodium acetate, then adding Tween®-20 (0.5% v/v) to lower the surface tension. The beads were cured for 15 min before rinsing with deionized water several times with the aid of a nylon filter with 50 μm pores . Beads were then transferred to a Petri dish and air-dried for 1 h to remove surface water. Amorphous fumed silica  (4% wet weight) was then added to the Petri dish and the beads were stirred to allow the coating to take place and left overnight in a fume hood to air-dry. Aerosil®-200F was used to adsorb water and prevent the beads from sticking to one another. Once completely dry, the Aerosil®-200F was sieved off using a wire mesh sieve (pore size 300 μm). The sieved Aerosil®-200F was collected and weighed to monitor the amount of silica that attached to the beads. Unloaded beads were made in the same manner using water in place of a payload. The theoretical loading for each payload was calculated according to Equation (1).The dWPI was combined under gentle stirring with four separate payloads with each chosen according to MW or solubility. The water-soluble payloads were sodium fluorescein  , and flu® software. High-quality images of beads were also taken using a stereomicroscope .Standard images of loaded and unloaded dWPI beads for sizing were taken with a Google Pixel smartphone, and the beads were sized using Image J0 samples were immediately removed, the SGF/SIF was discarded, and the beads were placed on a paper towel before being weighed. This was repeated for all time points up to 2 h. The degree of swelling was calculated according to Equation (2).Dry unloaded beads were weighed and placed into a series of Eppendorf tubes. Then, 1 mL simulated gastric fluid  was added to one set of tubes and 1 mL SIF (pH 6.8) was added to a second set. Both simulated fluids were prepared according to United States Pharmacopoeia (USP) specifications . Both seprovided .® Gemini, San Jose, CA, USA) at an excitation of 490 nm and an emission of 525 nm, while curcumin was analysed at an excitation of 420 nm and an emission of 535 nm. Fast Green was analysed using a spectrophotometer  at 628 nm. Concentrations were calculated from calibration curves for each payload. Encapsulation efficiency (EE), loading, and final loading were calculated according to Equations (3)–(5) below.Loaded beads were ground using a mortar and pestle and the resulting powder was added to 25 mL SIF. For curcumin, the powder was added to 25 mL methanol due to its low solubility in SIF. These solutions were left being stirred vigorously for 4–6 h until the powders had completely dissolved. Then, 200 μL samples were added to a 96-well plate  along with a buffer control and analysed. SF and FD4 were analysed using a spectrofluorimeter , and then at 0, 30, 60, 90, 120, and 180 min (for SIF). For FD4 and curcumin samples, 50 μL NaOH (0.1 M) was added to all SGF samples to improve the fluorescent signal. Samples were analysed at the wavelengths specified above and concentrations were calculated using the calibration curves. Cumulative release as a percentage of total bead loading was then calculated. Release data were fitted to mathematical drug release models including zero-order, first order, Higuchi, and Korsmeyer–Peppas using the MicrosoftDDSolver . The equw/v) pancreatin (4 × USP) was aliquoted and centrifuged for 30 min at 14,000 rpm at 4 °C. The supernatants were combined into a working stock. The release study was then carried out using a water bath on a hot plate to maintain 37 °C. A beaker containing 25 mL FaSSIF with 3% of the pancreatin working solution was placed in the bath and stirred at 200 rpm using a paddle stirrer. This method was modified from Presas et al. [0 sample was immediately withdrawn and replaced with FaSSIF containing 3% of the pancreatin working solution. Samples were taken at 0, 15, 30, 60, 120, 180, and 240 min and pipetted into a black 96-well plate. As with the SGF samples, NaOH was added to FaSSGF samples and they were analysed at a wavelength of 420 ex, 565 em (nm). FaSSIF samples were read at a wavelength of 420 ex, 535 em (nm). A protein assay was carried out on the FaSSIF + P solution to monitor the pancreatin concentration. This was performed using a Quant-iT™ protein assay kit according to the manufacturer’s instructions .Curcumin release was further evaluated in FaSSGF and FaSSIF. Since low aqueous solubility is an issue for curcumin, these beads were chosen to test in FaSSGF/FaSSIF because bile salts in such buffers can improve the solubility of highly hydrophobic compounds such as curcumin . A seconp < 0.05 compared to control. Statistical analysis was carried out using GraphPad® Prism Version 5.0 .One-way ANOVA with a Bonferroni post-test was used to compare EE and loading for each of the payloads. The swelling study and release data for curcumin were analysed by 2-way ANOVA with Bonferroni’s post-test. Data were presented as mean ± the standard deviation (SD). Results were considered significant if The swelling study showed a significant difference in weight between unloaded beads incubated in SGF (pH 1.2) and SIF (pH 6.8) A, and al® on the surface. Ridges can also be seen on the surface of the beads at the higher magnification, along with indents  and moderate loading values (0.07–1.1%)  describes the release mechanism. For spherical particles when 0.43 < n < 0.85 the release is non-Fickian anomalous transport. This is when diffusion and polymer relaxation occur to the same extent [n > 0.85 for spherical particles, release followed non-Fickian Super Case II transport. Fast Green and curcumin release fell into this category. In this type of release, the diffusion rate is higher than polymer relaxation and the solvent penetrates the particles at an accelerated rate [n values of the Korsmeyer–Peppas model also indicate the release kinetics are haracterized by diffusion and polymer relaxation [SF beads quickly released 50% of payloads in SGF over 1 h, followed by a slow continuous release of the remainder over 3 h in SIF A. The ree extent . This wated rate . FD4 rellaxation . Though Curcumin beads were further examined in FaSSGF and FaSSIF to see if the presence of bile salts would aid solubility and increase release A. No staEncapsulation of peptides and other unstable molecules in delivery vehicles is a common strategy used in oral drug delivery to overcome low solubility, proteolysis, poor intestinal permeability, and to improve storage stability. The milk-derived protein, dWPI, has multiple functional properties that make it an ideal encapsulation matrix for oral delivery of unstable cargos. Oral peptide formulations, in particular, can benefit from the dWPI-based oral delivery system as it may offer mucoadhesion, enzyme inhibition, and permeation enhancement without the need for additional excipients. dWPI beads encapsulate a range of molecules with different properties, and this is demonstrated in our study. All of the payloads used here showed high encapsulation efficiency and good loading irrespective of molecular weight and aqueous solubility. Of the characteristics examined, release from the beads was the characteristic most affected by the payload properties. This is particularly interesting as therapeutics with similar physiochemical properties could behave in a comparable way to the payloads used in this study. This work could provide a starting point in the development of dWPI-based formulations by giving an insight into the characteristics that could be observed for similar payloads. The development of effective oral formulations for unstable bioactive molecules is a long and risky process and our study could provide valuable information which may prevent costly failures.The Korsmeyer–Peppas model of drug release suggested that solvent diffusion and polymer relaxation were important steps in the bead-release mechanism. These events are also necessary for swelling to occur . This meNone of the other three payloads demonstrated gastric release to any great extent. This could suggest that there were other factors involved in the release, but an examination of the swelling behaviour of these beads could help elucidate the release pattern. FD4  release from the beads was much slower than sodium fluorescein, which could be due to the molecular weight or perhaps an interaction between the payload and dWPI. It has been reported that dextran and dWPI can form covalent bonds ,31, thouThe release characteristics presented here are an example of how hydrophilic and hydrophobic therapeutics may behave when released from dWPI beads. However, another benefit of dWPI-based beads is that they are highly modifiable. Release patterns in the small intestine can be altered by changing the composition and thickness of coatings . The dryThis dWPI-based delivery system is a low-cost and versatile option for the oral delivery of a range of molecules. The delivery system is applicable to both hydrophilic and hydrophobic compounds, and the payloads examined here were encapsulated with high encapsulation efficiency and reasonable loading regardless of their physiochemical properties. Examination of bead release and release kinetics revealed that the payload properties affected the release behaviour of the beads more than they affected any other bead characteristic. This release was also likely facilitated by bead swelling. There was also some evidence to suggest that enzymatic degradation of the beads in the intestine may promote further payload release. This delivery system has the potential to be useful in the oral delivery of hydrophilic and hydrophobic therapeutics.


Secale cereale is an important crop in the Triticeae tribe of the Poaceae family, and it has unique agronomic characteristics and genome properties. It possesses resistance to many diseases and serves as an important resource for the breeding of other Triticeae crops. We performed a genome-wide study on S. cereale to identify the largest group of plant disease resistance genes (R genes), the nucleotide-binding site-leucine-rich repeat receptor (NBS-LRR) genes. In its genome, 582 NBS-LRR genes were identified, including one from the RNL subclass and 581 from the CNL subclass. The NBS-LRR gene number in the S. cereale genome is greater than that in barley and the diploid wheat genomes. S. cereale chromosome 4 contains the largest number of NBS-LRR genes among the seven chromosomes, which is different from the pattern in barley and the genomes B and D of wheat but similar to that in the genome A of wheat. Further synteny analysis suggests that more NBS-LRR genes on chromosome 4 have been inherited from a common ancestor by S. cereale and the wheat genome A than the wheat genomes B and D. Phylogenetic analysis revealed that at least 740 NBS-LRR lineages are present in the common ancestor of S. cereale, Hordeum vulgare and Triticum urartu. However, most of them have only been inherited by one or two species, with only 65 of them preserved in all three species. The S. cereale genome inherited 382 of these ancestral NBS-LRR lineages, but 120 of them have been lost in both H. vulgare and T. urartu. This study provides the full NBS-LRR profile of the S. cereale genome, which is a resource for S. cereale breeding and indicates that S. cereale can be an important material for the molecular breeding of other Triticeae crops. To overty (PTI) . These ety (ETI) . Among oe family . The proe family . The seqe family . In conte family .In angiosperms, NBS-LRR genes are divided into three subclasses based on their phylogenetic relationship . CharactArabidopsis thaliana  and barley (Hordeum vulgare) are important crops in the Triticeae tribe of the Poaceae family. S. cereale has unique characteristics in both agronomic performance and genome properties . Genes without a conserved NBS domain were removed from the datasets. All of the non-redundant candidate sequences were compared with the NCBI Conserved Domains Database (CDD) to further verify the CC, TIR (Pfam accession number: PF01582), RPW8 (Pfam accession number: PF05659), LRR and other integrated domains.NBS-LRR gene identification was performed on the annotated proteins of eviously . BrieflyMEME analysis  was perfS. cereale genome was determined by extracting and parsing genomic locations of the NBS-LRR genes from the GFF3 annotation file. A sliding window analysis was performed with a window size of 250 kb to identify the number of genes that appeared in a cluster on a chromosome as described by The distribution of NBS-LRR genes on the chromosomes of the S. cereale, T. urartu and H. vulgare were aligned using ClustalW with default options and then manually corrected in MEGA 7.0  and slightly more than that in the diploid wheat T. urartu genome (537 genes)  . This sueviously . The res genomes , mirrorimonocots .S. cereale NBS-LRR proteins revealed that not all of them have the characteristic domains at the N-terminal and the LRR domain at the C-terminal. In contrast, the NBS-LRR proteins show high domain composition and structure diversity  were found at the N-terminal and/or the C-terminal of 49 of 581 CNL genes , formingS. cereale NBS-LRR proteins were detected by the Multiple Em for Motif Elicitation (MEME) suite  suite . The resE) suite . In contproteins . Analyziproteins  revealedS. cereale, the physical locations of NBS-LRR genes were retrieved from the GFF3 annotation file. The result showed that S. cereale NBS-LRR genes are unevenly distributed on the seven chromosomes , and this is nearly two times the number of NBS-LRR genes on chromosome 2 (54). Chromosomes 1, 3, 5, 6 and 7 have 77, 74, 71, 104 and 61 NBS-LRR genes, respectively. We also compared the chromosome distribution pattern of NBS-LRR genes in the S. cereale genome with that in the barley and wheat genomes  of wheat  were generated from segmental duplications , while only 152 ancestral NBS-LRR lineages were shared by the two species . Among them, only 60 ancestral NBS-LRR lineages were inherited by all three species. Most of them conservatively evolved in each species without large duplication . In contrast, more than 90% (680) of ancestral NBS-LRR lineages were only inherited by one or two species after speciation , includireserved . However species . The resR gene Hm1 resistant to the fungus Cochliobolus carbonum race 1 was cloned from maize nearly 30 years ago, over 300 functional R genes have been identified. Most of these are from plant species that are important crops including rice, wheat and tomato  has recently been cloned by analysis of the wheat pan-genome (R gene enrichment sequencing in wild diploid wheat (Thinopyrum ponticum), Sr26 and Sr61, against stem rust have been cloned from wheat by mutational genomics and targeted exome capture methods (The Triticeae is a tribe of the Poaceae family that contains many important grain crops, including wheat, barley, ae crops , only a se crops . The rec species . For exan-genome . Four stid wheat . Two CNL methods . All of S. cereale have been used to transfer genetic materials to wheat to improve its quality. The disease-resistance genes carried by the 1RS chromosome arm of S. cereale have been transferred to the wheat genome to confer resistance against powdery mildew and stripe rust diseases (R gene profile on the transferred chromosomal segment is unclear. In this study, genome-wide analysis identified 582 NBS-LRR genes in S. cereale. The number of NBS-LRR genes in S. cereale is larger than that in barley and T. urartu (S. cereale would be an important resource for mining and transfer of functional R genes to barley and wheat. The distribution of NBS-LRR genes on different chromosomes of S. cereale was determined and serves as fundamental molecular information for the introduction of chromosomal segments from S. cereale to other Triticeae crops. An interesting finding is that both the chromosome 4 of S. cereale and the A genome of wheat retained a larger number of NBS-LRR genes than barley and genomes B and D of wheat. This result highlights chromosome 4 of S. cereale as an important resource for disease resistance breeding and also implies that chromosome 4 of wheat genome A has experienced a different evolutionary history compared to genomes B and D, at least for the NBS-LRR genes.Besides the wild wheat resources, both barley and diseases . However. urartu . The higS. cereale and their distinct genetic origin with NBS-LRR genes in barley and wheat suggest that S. cereale could be an important resource for obtaining functional NBS-LRR genes for molecular breeding. This also provides a molecular basis for developing S. cereale as a donor material for Triticeae breeding.Although the number of NBS-LRR genes in the three Triticeae does not differ dramatically, a large proportion of the NBS-LRR genes from the three species are inherited from different ancestral NBS-LRR lineages that diverged in the common ancestor of the three species. The result suggested that the NBS-LRR diversity would be significantly increased if the three Triticeae species are considered as a ‘Triticeae NBS-LRR gene pool’ in resistance-gene mining. The high abundance of NBS-LRR genes in S. cereale and compared the chromosomal distribution and evolutionary history of this gene family in three Triticeae species. This information provides a fundamental resource for mining functional R genes from S. cereale. Since cross-species transformation of genomic segments has been frequently used for the molecular breeding of Triticeae species, the NBS-LRR profile of S. cereale expands the gene pool for Triticeae molecular breeding.In conclusion, we uncovered the NBS-LRR profile in


Legumes, one of the most important sources of human food and animal feed, are known to be susceptible to a plethora of plant viruses. Many of these viruses cause diseases which severely impact legume production worldwide. The causal agents of some important virus-like diseases remain unknown. In recent years, high-throughput sequencing technologies have enabled us to identify many new viruses in various crops, including legumes. This review aims to present an updated list of legume-infecting viruses. Until 2020, a total of 168 plant viruses belonging to 39 genera and 16 families, officially recognized by the International Committee on Taxonomy of Viruses (ICTV), were reported to naturally infect common bean, cowpea, chickpea, faba-bean, groundnut, lentil, peas, alfalfa, clovers, and/or annual medics. Several novel legume viruses are still pending approval by ICTV. The epidemiology of many of the legume viruses are of specific interest due to their seed-transmission and their dynamic spread by insect-vectors. In this review, major aspects of legume virus epidemiology and integrated control approaches are also summarized. Legume is a term used for the plant or the fruit/seed of plants belonging to the Fabaceae (Leguminosae) family. Pulses or grain legumes include species the seeds of which are used as a protein source for humans and animals, and in oil production, while forage legumes are sown in pastures and are used for silage and forage. Furthermore, legumes are known to improve the fertility potential of the soil, therefore they may constitute major components for the sustainable management of farming systems .Cultivated legumes are known to be susceptible to natural infection by a plethora of viruses. Several comprehensive publications have reviewed the viruses affecting specific legume crops or production areas i.e., ,4,5,6,7),5,6,74,5Cicer arietinum L.), faba or broad bean (Vicia faba L.), lentil (Lens culinaris Medik.), field pea (Pisum sativum L.), and the warm season ones, common bean (Phaseolus vulgaris L.), cowpea (Vigna unguiculata (L.) Walp.), and groundnut or peanut (Arachis hypogaea L.). The most important forage legumes considered were the perennial alfalfa or lucerne (Medicago sativa L.), clovers (Trifolium spp.), and annual medics (Medicago spp.).This review aims to present an updated list, though non-exhaustive, of the viruses found to naturally infect legumes. Species taken into consideration were the most important annual cool season legumes, including chickpea or garbanzo bean  to mottle or light green vein banding and rings, accompanied by leaf deformation and plant stunting. ALCV causes plant stunting, leaf curling, crumpling, and shriveling. CMV infections cause light-green mosaic or mottle on slightly malformed leaves. LTSV causes transient yellow streaks or flecks along the leaf veins, while BLRV induces mild, transient yellowing of older leaves. The LALV infections of alfalfa remain latent. Alfalfa also exhibits a number of viral symptoms of still unknown etiology .Nucleorhabdovirus), alfalfa ringspot-associated virus , cnidium vein yellowing virus , lychnis mottle virus , medicago sativa marafivirus 1 , and Zhuye pepper nepovirus  [The list of alfalfa viruses is continuously increasing, and thanks to the use of HTS when exploiting some alfalfa disease syndromes, the identification of numerous novel viruses, such as ALCV, alfalfa dwarf virus, alfalfa enamovirus 1, alfalfa virus S, and alfalfa virus F, was made possible, and alfalfa was also confirmed as a new host for known viruses, such as cactus virus X, chickpea chlorosis Australia virus (CpCAV), strawberry latent ringspot virus (SLRSV), and cycas necrotic stunt virus ,22,23,24povirus) ,22,23,25Trifolium spp.) (Nepovirus), red clover-associated varicosavirus (Rhabdoviridae family), and phasey bean mild yellows virus  are suggested as new clover pathogens still pending official classification [Thirty-two viruses  have been reported to infect clovers (um spp.)  [14,26,2fication ,32,33.Medicago spp) have been reported to be infected by 10 viruses, from seven genera and six families  the lethal “black root” disease which is characterized by red-brown expanding spots on leaves that become necrotic. Infections by begomoviruses are mostly associated with yellow mosaic diseases. BYMV-infected beans show crinkling, yellow mottling, and necrotic areas along the veins of infected leaves. AMV infections are associated with mosaic or mottle, while CMV strains can induce mild mosaic to severe plant malformation. CCMV is the causal agent of “bean yellow stipple disease” ,39,41,66Badnavirus, family Caulimoviridae), which is still pending official classification [Recently, sequences of the Ethiopian tobacco bushy top virus were retrieved from common bean using HTS . Common fication .Cowpea is susceptible to at least 33 legume viruses from 16 genera and 11 families  5,68,69,71,72,73The potyvirids CABMV and BCMV-blackeye cowpea mosaic strain produce on susceptible cowpeas an indistinguishable veinal and interveinal chlorosis, or dark-green vein banding. CPCMV, CPGMV CPMV, CSMV, and CMV cause different mosaic symptoms. SBMV may induce from symptomless infections to severe mottle or mosaic with leaf deformations ,7,73,74.Recently-discovered cowpea-infecting viruses that are pending official classification are cowpea polerovirus 1 (CPPV1), cowpea polerovirus 2 (CPPV2), and possibly two tombusviruses ,76.Chickpeas are reported to be naturally infected with at least 40 viruses, from 19 genera and 11 families worldwide  3,5,7,3,82,83,84The main viral disease affecting chickpea is the “stunt disease” with symptoms of stunting, leaf rolling, yellowing (or reddening), premature necrosis and, if the plants survive, poor pod-set. Poor pod-set was initially attributed only to BLRV and FBNYV, but recently it has been associated with infections by: the poleroviruses BLRV, BWYV, CpCSV, CLRDV, and CABYV; the nanoviruses FBNYV, FBYLV, FBNSV, MDV, PNYDV, and SCSV; the luteovirus SbDV; or the mastreviruses CpCDV, CpYDV, CpCV, CpRLV, and CpYV. AMV, BYMV, CMV, and PSbMV are associated with mosaic and mottle symptoms ,6,7,82.Hostuviroid, family Pospiviroidae) [Deep sequencing approaches identified chickpea as a new host of tomato mottle mosaic virus and of hop stunt viroid  . Chickpeiroidae) , that isWorldwide, faba bean is naturally infected by at least 40 viruses from 20 genera and 11 families  3,4,5,1,89,90,91FBNYV and related viruses cause necrotic yellow symptoms in faba bean plants, stunting, and poorly developed new shoots, leaves, and flowers. Leaves are small, rolled, and present interveinal chlorosis, which develops to necrosis. Infections might result in the death of the plants. Infection with BLRV (or related viruses) causes a leaf roll disease characterized by interveinal chlorosis, yellowing, stunting, leaf rolling, reddening and thickening of the leaves, suppression of flowering, and pod setting; infections before blooming may result in compete yield loss. BYMV is associated with mosaic and necrosis and BBSV with mottling, marbling, diffuse mosaic, and often with leaf malformation and plant stunting ,7,84,89.Pospiviroid, family: Pospiviroidae) [Except viruses, citrus exocortis viroid   and the iroidae)  have beeWorldwide, around 27 viruses from 11 genera and 10 families have been reported to naturally infect groundnut. Groundnut bud necrosis orthotospovirus (GBNV), tomato spotted wilt orthotospovirus (TSWV), Indian peanut clump virus (IPCV), peanut clump virus (PCV), peanut mottle virus (PeMoV), and BCMV (synonym peanut stripe virus), tobacco streak virus (TSV), CMV, and the symbiotic association of groundnut rosette virus (GRV) with groundnut rosette assistor virus (GRAV) are considered the most economically important ones. Groundnut rosette  disease is caused by the complex of GRAV, GRV, and a satellite RNA. Often IPCV and PCV infections can be confused with green rosette. TSWV induces spotted wilt and GBNV bud necrosis disease, which are similar in appearance. TSV-infected groundnut plants show stem necrosis. PeMoV and PStV infections are associated with mottle and stripe, and CMV infections with yellow mosaic ,94,95,96Worldwide, peas can be naturally infected with at least 42 viruses from 18 genera and 11 families  3,4,5,3,101,102.AMV-infected pea plants exhibit curling, chlorosis, and sometimes necrotic lesions on leaves, streaks on the stem, malformations, and general stunting. BLRV symptoms include chlorosis, upward curling of leaves (top yellows), and stunting. BYMV and PSbMV infections are mostly associated with mosaic and veinal chlorosis symptoms. PSbMV-infected plants may also show severe stunting, especially in seed-borne infections. PEMV-infected peas exhibit mosaic, leaves show chlorotic and translucent spots, vein clearing, downward curling (at the early stages of infection), growth deformations , and enations (hyperplastic outgrowths) associated with the veins underneath the leaf lamina, while pods may be distorted with wards (at later stage of infection). PSV causes brown necrotic streaks on stems and petioles, necrotic lesions on leaves, and wilting of the plant. BWYV and TuYV cause yellowing, rolling, thickening of leaves, and stunting of plants. PNYDV-infected peas show severe dwarfing of plants, yellowing, and leaf-rolling. PEBV causes purplish-brown necrotic discoloration and plant necrosis. The RCVMV-infected pea plants exhibit severe stunting, rosetting of leaves, and plant necrosis in early stages of infection ,103,104.Fimoviridae family) and PBMYV (genus Polerovirus) [Recent efforts to explore the pea virome identified pea-associated emaravirus (PaEV) (rovirus) ,102 whicAbout 24 viruses from 16 genera and 9 families have been reported to infect lentil in nature . The aphPlant viruses are obligate pathogens, and their survival depends on their dispersal mechanisms. They can often cause destructive epidemics, especially those transmitted by insects ,106. TheThe insect-transmitted viruses are characterized by specific interactions with their vectors during transmission. Depending on the time the vector remains viruliferous, they are classified as non-persistent (with short retention time), semi-persistent (with intermediate retention time) or persistent (with extended retention time). Depending on the site at which the virus is retained or the route of movement of the virus within its vector, viruses are termed as non-circulative (associated with the mouthparts or foregut) or circulative (cross multiple membrane barriers and are transported within the vector’s haemolymph). The non-circulative viruses are either non-persistent or semi-persistent, while among the circulative viruses some replicate in the vector’s tissues (circulative propagative viruses), while others do not (circulative non-propagative viruses) ,112,113.Alfamovirus, Cucumovirus, Fabavirus, Macluravirus, and Potyvirus. Those in the Carlavirus genus are also non-persistently transmitted by their aphid or whitefly vectors . Due to the short time needed for their transmission, they are transmitted with low vector specificity, by several aphid species, mostly the ones that visit, while seeking a feeding site, but do not colonize the crop (transient species). These viruses are non-circulative, only being associated with the insect’s mouthparts . As a result, they can be transmitted immediately after acquisition (no latent period), but they are lost after aphid moulting. Due to the external root of transmission, the vector remains viruliferous for only short periods of time (minutes), thus they can be only spread over short distances. Therefore, their sources of infection are within the crop or in close proximity. The non-persistent legume aphid-transmitted viruses belong to the genera  vectors  [11,12,1Caulimovirus , and the whitefly-transmitted genus Crinivirus. The beetle-transmitted Bromovirus, and Comovirus are also reported to be transmitted in a semi-persistent manner  for acquisition and transmission. These viruses do not enter the insect’s body, but they are associated with its foregut  and their retention period extends from hours to days. However, similar to the non-persistent viruses, they do not require a latent period, and they are lost when the vector moults. Due to the extended feeding time needed for transmission, they have a narrower range of vector species that colonize the crop. Semi-persistent transmission may be carried out by aphids, whiteflies, and leafhoppers. The semi-persistent legume viruses belong to the aphid-transmitted genera of t manner  [11,12,1Capulavirus, Enamovirus, Luteovirus, Nanovirus, Polerovirus, and possibly to the viruses not assigned to a specific genus of the Luteoviridae family. Indirectly, the circulative transmission applies to viruses in the genus Umbravirus that use an assistor luteovirus for their transmission. In addition, the whitefly-transmitted viruses of the Begomovirus genus or the leafhopper-transmitted viruses of the genera Becurtovirus, Curtovirus, and Mastrevirus are also transmitted in a persistent, circulative manner. On the other hand, the circulative propagative legume viruses belong to the thrips-transmitted Orthotospovirus, the aphid-transmitted Cytorhabdovirus, and the leafhopper-transmitted Marafivirus genera  to be efficiently acquired and transmitted by their insect vectors. As a result, the persistent viruses can only be transmitted by species which colonize the crop. They circulate inside the vector’s body, traversing insect tissue membranes to invade multiple organs and they pass through moult. Therefore, a latent period is needed after acquisition for these viruses to finally reach and invade the salivary glands to be successfully transmitted. Persistent viruses exhibit a high degree of vector specificity and vectors may remain viruliferous for some weeks (the circulative viruses) or for their lifetime (the circulative propagative viruses). As a result, they can be spread over long distances and individual insects can infect many plants. Some propagative viruses may be also transmitted to the vector’s offspring. Among the legume-infecting persistent viruses, circulative transmission can be found in the aphid-transmitted genera of s genera  [11,12,1Alexivirus genus are transmitted in a semi-persistent manner. The same applies to the nematode transmitted viruses in the genera of Cheravirus, Nepovirus, Tobravirus, and possibly the non-assigned to a specific genus SLRSV (Secoviridae family). The chythrid-transmitted Alphanecrovirus, Dianthovirus, Tombusvirus genera, and pea stem necrosis virus (Tombusviridae family), and the plasmophorid-transmitted genera Pecluvirus and Pomovirus, show characteristics of persistent transmission. In the field, legume viruses in the Potexvirus, Sobemovirus, Soymovirus, Tombusvirus, and Tobamovirus are readily transmitted by contact, and may be sometimes mediated by insects. Viruses in the Dianthovirus and Tombusvirus genera may be also readily transmitted via soil without a vector. Viruses in the Bromovirus, Comovirus, and Tymovirus genera are reported to be semi-persistently transmitted by beetles, but their transmission is sometimes considered by contact; this possibly applies also to the unassigned bean mild mosaic virus (Tombusviridae family). Viruses in the Ilarvirus genus are transmitted by pollen and their transmission may be facilitated by thrips. Finally, the vector(s) and transmission type of the legume viruses belonging to the genus Soymovirus, as well as of alfalfa betanucleorhabdovirus, remain unknown , alternative hosts, and/or viruliferous vectors (for the persistent viruses) may serve as their sources ,114,115.An estimated 50% of the legume viruses are seed-borne; and most of them (the non-persistently transmitted ones) are found in the cotyledon/embryo (referred to as true seed transmission), but some  could be carried on the seed coat, as a surface contaminant . The seeAlfamovirus, Cucumovirus, Fabavirus Macluravirus and Potyvirus; (ii) persistently-transmitted by aphids of the genus Enamovirus; (iii) transmitted by aphids or whiteflies in Carlavirus genus; (iv) vectored by nematodes from the Tobravirus, and Nepovirus genus including SLRSV; (v) transmitted by pollen of the Ilarvirus genus; and (vi) transmitted by plasmodiophorids and belong to the Pecluvirus and Pomovirus genera. Viruses in the Potexvirus and Tobamovirus genera (transmitted by contact) and possibly in the Comovirus and Sobemovirus (beetle- and contact-transmitted) are carried by seeds as contaminants [A non-exhaustive list of the seed transmitted legume viruses include the following: (i) non-persistently transmitted by aphids, belonging to the genera aminants ,13.For the few legume viruses with a broad host range, e.g., CMV that infects at least 1060 species in 100 families , multiplThe persistently transmitted viruses can be also perpetuated in the absence of susceptible hosts in their viruliferous vectors via transovarial  transmission from infected females to their progeny. Surviving, over winter or summer, virouliferous vectors on stems of perennial legumes may serve also as source of the virus .Epidemics of insect-transmitted viruses start with virus introduction from external virus sources (primary spread), which is followed by further spread among field plants (secondary spread). The vector species involved and the abundance and proximity of virus sources are major components in virus epidemiology. The necessary feeding time for virus acquisition and transmission shapes the vector specificity (crop colonizing or non-colonizing) and the plethora of possible vectors. Likewise, virus retention time in the insect vector determines the vicinity of the virus sources for further virus spread. For the non-persistently transmitted viruses, which are mostly transmitted by transient/migratory aphid species, the most important factor affecting the spread is the proximity to substantial virus reservoirs (due to the short retention time of the virus in the vector). On the other hand, semi- and persistently transmitted viruses might originate from distant virus sources due to the increased retention time in the insect-vectors. These viruses are introduced mostly in random distribution, by only colonizing species, and their spread is mainly affected by the vector population built up and activity in the crop ,114,119.Plant viruses cause systemic infections and are capable of destructive epidemics, especially those that are transmitted by insects. In the absence of antiviral substances, only integrated management approaches which combine various measures may efficiently control the virus spread. Such approaches aim to prevent virus introduction into and further spread within the crop by minimizing virus and vector reservoir(s), and reduction of the size of vector population and its movement onto the crop. Notwithstanding, in order to obtain the optimal combination of such measures, knowledge of the identity of the viruses that infect legume crops in a specific area and the epidemiological aspects of their spread is of major importance ,120,121.The use of virus-free or virus-tested seed is the main measure employed for the control of legume viruses. The use of virus tested, i.e., seeds with infection rates below accepted threshold values, is of primary importance especially for local landraces conserved in situ by farmers. Removal of volunteering legume plants or other virus and vector sources, such as weeds within or in close proximity of the crop, should be performed before sowing, while infected crop legumes should be removed (roguing) early in the season before the onset of the activity of the insect vectors. Crop rotation with cereals may also reduce virus and vector sources originating from volunteer legumes.Other potentially infected crops  and especially perennial pastures  that may host legume viruses and aphid vectors should be avoided through spatial isolation. The effective radius for this isolation highly depends on the virus transmission mode; for the non-persistently transmitted viruses, the sources are in close proximity to the crop, while semi- and persistently transmitted viruses may also originate from distant sources, sometimes difficult to avoid.The use of virus-resistant or -tolerant genotypes to avoid or alleviate crop losses is considered the most economical, practical, and environmentally-friendly approach. However, lack of resistant cultivars is one of the major limitations of the approach and screening of legume germplasm for resistance and its integration in elite varieties is sometimes a challenge.Avoidance of infection is a strategy that employs several cultural practices in order to prevent incoming vectors to feed on the legume crop and they are based on the knowledge of the insect’s behavior. Some insects tend to alight preferentially or to plants surrounded by bare soil. Therefore, when establishing the crop, planting in large compact blocks of uniform age decrease the proportion of plants in the vulnerable peripheral areas. Sowing a non-host of the virus around the crop (border planting), may also delay the entry of migrating aphid into the crop and decrease the load of non-persistent viruses due to their short retention time and the probing activity performed on the non-hosts. The spread of persistent viruses may be also decreased in case the viruliferous insects colonize the non-host border and, therefore, only their non-viruliferous progenies migrate to the crop if the colonies get overcrowded.Likewise, increasing plant density  and early canopy development resulting in early bare soil cover is reducing the landing rate of incoming viruliferous vectors on young plants. Close spacing proved to decrease the proportion of infected plants within a stand. Shaded, virus-infected plants are also less vigorous and, therefore, they are not available for secondary virus spread. Groundcover or mulches may have the same effect in decreasing vector landing and exposure of young plants. Early planting is another measure that may promote early growth and the most vulnerable young plants will not be exposed to peak vector populations.Insecticide applications that aim to reduce vector populations may sometimes play only a limited role in plant virus control. Their efficacy highly depends on their mode of action, the mechanisms of transmission of the targeted viruses, and the type of virus spread in the field (primary or secondary) . InsectiLegumes have gained an increasing importance in human health and sustainable agriculture in recent times. Plant viruses are considered a major constraint for legume production in many areas of the world. In the absence of efficient chemicals to treat virus-infected plants, the identification of the causal virus is of major importance in order to employ the most efficient control measures. Cutting-edge technologies explore the virome of legume species in order to identify the causal agents of major diseases, thus resulting in the discovery of new viruses. This changes our knowledge and perception of the virus status and challenges virus systematics, which should be continuously updated. However, it is the first step toward understanding the epidemiology of plant virus diseases and limiting their spread in order to ensure our future food and feed security.