Viruses have been infecting their host cells since the dawn of life, and this extremely long-term coevolution gave rise to some surprising consequences for the entire tree of life. It is hypothesised that viruses might have contributed to the formation of the first cellular life form, or that even the eukaryotic cell nucleus originates from an infection by a coated virus. The continuous struggle between viruses and their hosts to maintain at least a constant fitness level led to the development of an unceasing arms race, where weapons are often shuttled between the participants. In this literature review we try to give a short insight into some general consequences or traits of virus–host coevolution, and after this we zoom in to the viral clades of adenoviruses, herpesviruses, nucleo-cytoplasmic large DNA viruses, polyomaviruses and, finally, circoviruses. Viruses are obligatory cellular parasites, developing with their hosts since the dawn of life. Coevolution, when the virus and the host reciprocally affect each other’s evolution, is often detected. According to the Red Queen Hypothesis, both the parasite and the host are perpetually struggling to maintain a constant fitness level  the primordial virus world or the virus first hypothesis claims that the ancestors of viruses existed already in the pre-cellular world; (ii) the escaped genes theory describes viruses as mobile genetic elements, which became independent of their host cells; (iii) whereas according to the cellular regression theory, viruses are regressed intracellular parasites Holmes .According to the primordial virus world hypothesis, in the primordial soup multiple pre-cellular and pre-viral Ur-organisms competed with each other. The last universal common ancestor—the first cellular life form—emerged from these primordial replicators, and at least a fraction of current viruses might originate from the remaining ones  are viral genes or sometimes complete genomes inserted into host genomes. As genome integration is a compulsory step in retroviral replication, most of these elements are of retroviral origin, but further virus families were detected as well: hepadnaviruses, adeno-associated viruses, herpesviruses and others  or organ  against these genetic parasites to protect themselves and their genomic integrity.Most strikingly, antiviral defence mechanisms often have viral origins too  A, F and G  fibre seems to be of atadenoviral origin , while the family Alloherpesviridae contains the HVs of amphibians and fish (Anamnia), and the family Malacoherpesviridae contains the HVs of molluscs. The three families were clustered under the novel order Herpesvirales  implying the common origin of HVs and tailed bacteriophages  are large (150–200 nm) icosahedral, enveloped viruses having a so-called tegument layer between the capsid and the envelope. HVs possess a linear, non-segmented, double-stranded DNA genome (135–295 kbp)  of Herpesviridae emerged probably before the mammalian radiation, 60 to 80 million years ago, while the diversification time point of the three subfamilies had been around 200 million years ago  existed around 400 million years ago based on similar phylogenetic branching patterns of HVs and host lineages  has been isolated from cattle worldwide and this species was thought to be the original host of the virus. However, BoHV-4 has been reported from wild buffalo and other ruminants as well   (Aswad and Katzourakis Branchiostoma floridae (Florida lancelet or amphioxus) contains several genes showing homology to herpesviral genes, implying the existence of a HV associated with this invertebrate chordate. The terminase and polymerase gene sequences from the putative amphioxus HV show higher similarity to those of the mollusc HVs than to any vertebrate HVs ] to collect the above-mentioned virus families  are a group of DNA viruses including the families Mimiviridae and pandoraviruses. Branch #2 gathered the families Asco-, Irido-, Marseille- and Pithoviridae. This branch contained the widest variation of genome sizes (100 kb to 1500 kb). Branch #3 included only non-giants in two distinct clades: Asfarviridae (African swine fever virus and its protist-infecting relatives) and Poxviridae. According to the present knowledge, a major host switch took place two times along this branch. Once during the early history of poxviruses, which infect both arthropods and vertebrates, two animal phyla that radiated from the common ancestor more than 500 million years ago  viruses are the most prominent gene gainers, whereas NCLDVs infecting animal hosts have undergone considerable gene losses, a sort of ‘genome contraction’ during their evolution from ancestral protist viruses. It was hypothesised that in animals, the selective pressure for virus genome size is stronger than in protists, yet its mechanism needs to be analysed  according to the ICTV. The taxonomical classification of PyVs is based on the genetic distance of the large tumour antigen. Still, some PyVs cannot be categorised into any accepted genus, so these require additional genera in the future  are non-enveloped icosahedral DNA tumour viruses with a double-stranded, cca. 5000-bp-long circular DNA genome. Approximately 80 accepted PyV species form the family The organisation of the circular PyV genome is highly conserved: the 5–7 genes are located on both strands, and distributed into early and late regions. There is a non-coding control region as well. The early coding region contains regulatory proteins: the small and the large tumour antigen. The late coding region contains the genes of the structural proteins, like the major (VP1) and two minor capsid proteins. The approximately 500-bp-long non-coding control region contains regulatory elements and transcription promoters, the origin of DNA replication  is oncogenic like several other mammalian PyVs.PyVs are known to infect mammals and birds, but using viral metagenomics, PyVs were identified and characterised also in fish and arthropods  infects birds of diverse families  are vertebrate-infecting, single-stranded DNA (ssDNA) viruses with one of the smallest (~2 kb) virus genomes (Parrish rep and cap) encoding the replication-associated (Rep) and the capsid (Cap) proteins, respectively  and broadened the perspective for the coevolutionary studies. (3) Concerning NCLDVs, we have discussed that many earlier unrelated virus families with large/giant capsid and genome sizes and even more divergent host range (from protists to vertebrates) can be phylogenetically linked based on a subset of core genes. This phylogenetic reconstruction demonstrated a ‘turbulent evolution’, which was dominated by gene gain and gene loss via horizontal gene transfer both from other viruses and from host genomes. (4) In connection with polyomaviruses (PyV) the recombination among PyV lineages generating novel virus species was also mentioned, and the importance of the intra-host molecular evolution was discussed in more detail. It was demonstrated that point mutations and rearrangements in non-coding control regions both can critically alter the pathogenic potential, immunogenicity and target organs of related PyVs of a single host species. (5) In the case of circoviruses (or more broadly CRESS DNA viruses), higher mutation rates and rolling circle type replication of their ssDNA, alongside with recombination events and integration into host genomes as well as presumed cross-species transmissions of distant hosts were simultaneously forming the coevolutionary processes.As seen from the example of this latest mentioned dispute concerning the appearance of circoviruses, there are a number of unsettled questions in connection with the coevolutionary processes observed in DNA viruses and their hosts. In the paragraphs above, we have listed these major processes. (1) Apparent speciation and coevolution of adenovirus lineages (genera) with the different vertebrate families. Here, host switches to new taxa were marked both with shift in the genome content  and more severe pathology in the non-coevolved hosts. (2) In connection with herpesviruses (HVs) a longer coevolutionary past was supposed, largely synchronous with the more divergent (vertebrate and invertebrate) host lineages. Integrated HV genomes in host chromosomes were demonstrated to provide new data about the genome content of ancient HVs and a new HVs lineage. Moreover, the discovery of a HV core gene homologue in tailed bacteriophages established the phylogenetic relatedness between two seemingly unrelated virus orders  hosts (Welch et al. Viruses have always been around. Their parasitic lifestyle had an enormous driving effect on the evolution of all organisms, and thus life would not be the same without them. Future research will hopefully shed light on the exact origin and coevolutionary history of all viruses.


Given the global trend of aging societies, medical expenditure has hit record highs in many countries. Because medical advice-seeking behaviors can affect the health of whole societies, how members of a society make medical-related decisions with limited available resources is worthy of investigation. Although transaction cost theory has been extensively applied in commercial research, it is yet to be applied in studies on medical advice-seeking behaviors.This study conducted in-depth interviews with 15 participants and verified that transaction cost theory is applicable for analyzing people’s medical advice-seeking behaviors.This study verified that transaction cost theory influenced the participants’ choices of physicians and treatment methods, which implies that improved transparency of medical information could considerably reduce transaction costs in relation to medical behaviors and enhance people’s well-being. Population aging is an international phenomenon revealing a steadily increasing life expectancy among geriatric people. With the most rapidly aging society worldwide, Taiwan is poised to become an aged society. According to statistics from the Executive Yuan, the aged population in Taiwan will reach 4.75 million by 2025, denoting that one in every five citizens will be over 65 years of age. The status of Taiwan’s society as aging is aggravated by the increasing occurrence of chronic diseases among the geriatric population. Shortages of medical resources have prevented large numbers of older adults with chronic diseases from receiving proper medical attention and treatment, thereby leading to numerous acute complications and consequential medical consumption behaviors, which have caused not only considerable consumption of scarce medical resources but also a decline in medical care quality that has induced numerous problems [The idea and importance of transaction costs was initially described by Ronald Coase in 1937. In 1975, Oliver E. Williamson developed a comprehensive framework for transaction cost theory based on Coase and related literature. Transaction cost theory is a principal theory for management and organization studies .Medical advice-seeking behaviors can influence the prognosis of a disease, medical expenses, and equity in access to medical resources. “Transactions” involving medical services constitute critical decisions many people must make in their lives. Decisions or “transactions” involving choices of food, clothing, education, and jobs broadly and deeply influence human lives.Although most related studies have concentrated on medical advice-seeking behaviors in relation to various diseases or variations in medical advice-seeking behaviors between different societal groups, the present study, used the transaction cost theory to deconstruct medical advice-seeking behaviors from the standpoint of the users Tables , 2.TableThe Nature of the Firm published in 1937. Coase questioned the validity of zero transaction cost as described by conventional economic theories and asserted that in the process of a transaction, uncertainty in the environment and the limited rationality of human beings might incur additional costs for the parties involved. Coase termed these costs “transaction costs.” Coase believed that transaction costs referred to the following three types of cost: (1) search cost: the cost of search for required goods; (2) bargaining cost: the cost of negotiating and reaching an agreement; and (3) enforcement cost: the cost of executing a contract and supervising its execution after the transaction has been validated [Transaction cost theory was introduced by Ronald Coase, a professor at the University of Chicago, in In 1975, Williamson developed and expanded Coase’s views to promote better awareness of transaction cost theory among economists. He proposed that failure to complete a transaction usually stems from market failure caused by human and environmental factors, which cause difficulties in market transactions and increase transaction costs. The human factors highlighted by Williamson included the bounded rationality and opportunism of human beings, whereas the environmental factors included uncertainty, complexity, and small transaction volumes. In addition to these factors, information asymmetry and the atmosphere were recognized as factors that could increase transaction costs. In 1985, Williamson further proposed that the suppositions of asset specificity and the frequency and uncertainty of transactions could cause organizational failures and increased transaction costs. Therefore, transaction costs were defined as the costs incurred from the transaction process in addition to that agreed by the parties involved. Such costs include those incurred from information searching, negotiating, drafting, maladaption, haggling, setup and running, and bonding .In the present study, interviews were conducted in October and November 2016 with 15 Taipei-based students all from different regions in Taiwan and various social and economic backgrounds.In-depth interviews were conducted to ensure data completeness and accuracy. Before each interview, the participant was asked to provide consent for notes and an audio recording of the interview to be taken. Upon completion of each interview, the audio recording was transcribed verbatim for further analysis. After each interview had been transcribed in full, the interviewers compiled a memorandum detailing their observations regarding the body language, mood, and speech of the participant as a reference for follow-up research. In addition, to improve precision and for greater reliability and validity, the research team followed the principle of triangulation tests by using multiple sources for evidence, asking the interviewer to examine the transcript, and establishing construct validity . Based on the interviewed participant and location revealed in the transcript; or Based on the research objectives and constructs determined through the literature review.The research team composed interview guidelines based on the research objectives and conferred with other researchers about the content corresponding to the research questions, which were subsequently used as bases for the interview questions. The qualitative interview relied on the data analysis helix in Creswell et al.  to proce#1:41-50:S2 “So how much time would you spend to make sure the information is correct?”“About half an hour.”“You’d spend half an hour checking it and then you would go to the clinic, right?”“Right.”#11:1-3:S3“What, in your opinion, are the costs of medical advice-seeking behaviors?”“Looking up a clinic, registration fees, and waiting times. Before going to see a doctor, I usually prepare myself by making a mental list of my symptoms so that I can readily consult with the doctor and see how I can be cured quickly.”Fan  emphasiz#9:1-1:B2“What, in your opinion, are the costs of medical advice-seeking behaviors?”“Registration fees and time.”#6:1-1:B3“What, in your opinion, are the costs of medical advice-seeking behaviors?”“Registration fees, transportation time, carfare, and waiting time.”#8:3-5:B4“Speaking of transportation, if you go to see a doctor by riding a scooter, the cost is the fuel cost, and if you go by mass transportation, then it’s the carfare. Oh, yes, and the registration fee, too.”#12:2-6:B5“Suppose I have to make an appointment with a doctor. I may have to sacrifice part of my working time or school time. If it’s working time, then it will affect my income. When it comes to money, the costs also include transportation fees and fees for treatment like registration fees.”Liao  proposed#3:7-11:D1 Decision-making costs: Trust “So why did you choose that clinic?”“Because it was close to my place.”“Locational advantage?”“Yes.”“Was there any reason other than locational advantage? Like, maybe you find that doctor trustworthy?”“That doctor has always been very gentle to patients, which puts you at ease, and he has always been very patient, too.”“So, simply put, you have grown to trust that doctor.”“Right, I trust him.”#5:18-25:D2 Decision-making costs: Habits“So you are from Tainan? Do you habitually go to the same clinic or doctor when you are in Tainan?”“Yes, there is one, and I always go there unless it is not open that day.”“Is there a reason that you choose this particular clinic or this particular doctor?”“Because I have been going there since I was little. Also, I find the treatment and the medicine the doctor gives me very effective. Usually, I only have to make one appointment and then I’m cured, unless I’ve got a really bad cold. In that case, I’d usually be cured after the second visit. In addition, I trust that doctor, and there has never been any trouble with him regarding the doctor–patient relationship.”“So, you are used to that clinic and you trust them?”“Yes, and I have a very high opinion of them.”#14:10-11:D3 Decision-making costs: Consultation (expertise)“What if you are seriously ill and the doctor at the clinic is unable to cure you?”“My doctor will refer me to another specialist. He will write a referral letter for me.”Mutual trust can lower the information searching costs incurred by both parties on acquiring the transaction opportunity. Negotiation costs can be flexibly reduced when both parties are willing to make concessions based on expectations of future gains from each other .#3:7-11:#2:70-73:M1“So, would you feel worried… I mean, after seeing this doctor, would you still feel worried and seek a second opinion?”“Yes.”“Does that mean you still fear the doctor may make an inaccurate diagnosis?”“Yes. If I am cured with no trouble, then I won’t be worried, but if the sickness persists, I will surely have doubts.”#10:68-74:M2“Would you, for example, search for information online first if you feel very sick and have symptoms that you have never experienced before?”“Yes, I would search for information on the Internet.”“What if you find your situation to be kind of different from the doctor’s diagnosis, or even in conflict with it? Which side will you take when that happens?”“I will show the information to the doctor. I mean, I will show it to him and tell him that it seems to contradict what he said, and then ask him to explain it to me. If his explanation makes sense to me, then of course I will follow his instructions; otherwise, I will go to another clinic for a second opinion.”#15:33-40:M3“Since you just mentioned that you habitually go to the same clinic whether you are in Taipei or in Miaoli, do you also look for medical advice from other sources?”“Do you mean the Internet, or any source?”“Newspaper supplements for example; some people who want to lose weight may follow a diet regime they found in a supplement. Some people with gynecological problems will try traditional Chinese medicine to ‘strengthen the body’ in addition to going to a gynecologist. If you don’t have full confidence in this doctor, you may want to get a second opinion from another doctor and compare what they have to say. Do you do such things?”“I do. I will check what is good for my situation.”“So you have no problem with traditional Chinese medicine or things like food therapy?”“That’s right.”Two types of uncertainty are inherent in transactions, both of which can increase policing costs. The first consists of predicable or unpredictable chance events induced by limited rationality and the second is uncertainty in insufficient information rendered by withholding, falsification, or distortion .#2:70-7#1:61-69:E1“Would you still feel worried after seeing the doctor? Like, maybe sometimes you will want to see another doctor or conduct an online search of the medicine the doctor prescribed.”“I’d only worry about the side effects.”“You mean harm to the body from side effects or failed therapy?”“Yes.”“So, in the face of uncertainty, would you look up information on the medicine prescribed or maybe even stop taking it altogether?”“I think I would visit another doctor.”“Do you mean you would counter the uncertainty by changing to another doctor?”#7:81-87:E2“What would you do if it turned out that the mole removal had failed?”“I think I would be very angry.”“What else?”“If they dare ask me to give them a rating, I’d give them the worst one and demand compensation.”“Would you go back to them for remedial measures?”“No. I’d change to another clinic.”“You’d switch clinic?”“Of course, it’s for my face after all.”“Suppose the original clinic offers method A for mole removal and the clinic you intend to switch to offers method B. Would you still want to switch to the new method?”“I suppose I would. The old method has failed anyway, so why not give another method a try? Yes, I would try it. I would be open to anything that could solve the problem.”#3:39-44:E3“Would you be worried if the doctor asked you whether you are allergic to anything or feel uncomfortable about taking certain medicines? Would you perhaps worry that the doctor may overlook certain small things at your expense; for example, giving you unsuitable medicine without making sure you aren’t allergic to it?”“Not at all.”“Would you change therapy if you felt uncomfortable after taking the medicine? Would you switch treatments or even switch to another clinic?”“Yes, if that happened I would.”After a contract has been agreed upon, both parties must verify whether the other has fulfilled the terms of the contract, and in the event of noncompliance, enforce the terms. The costs that ensure the execution of a contract are contract execution costs. Upon completion of a contract, both parties may choose to renew their transaction or one may choose to relinquish their part of the agreement to another partner, in which case, the costs incurred are switching costs .#1:61-6The interview transcripts indicate that there is a tight relationship between transaction cost theory and medical-seeking behavior. Although, the public has only a weak conception of transaction costs. The transcripts, traces of understanding of transaction costs can be detected in relation to medical advice-seeking behaviors. Most of the discernable elements corroborate the findings of Dahlman , includiIn addition to transaction costs, another factor tentatively referred to as transaction assets has been mentioned repeatedly. This factor may be accumulated at the expense of multiple costs and may facilitate the reduction of future costs. The possession of transaction assets can effectively reduce transaction costs and such assets may be the result of trust, habits, and connections. Experienced people tend to rely on such assets to effectively reduce transaction costs, or in other words, obtain access to equal or preferential medical treatment; for example, some participants stated that they need not spend much time searching for clinics when at home because they are already acquainted with local physicians. However, after leaving home for further education or work, people must spend more time or use connections to build up trust. Therefore, transaction assets can be understood as reducing search and information costs, negotiation costs, policing costs, and contract execution costs.Generally, when faced with familiar symptoms, people tend to seek medical advice from medical institutions that incur lower transaction costs such as those that are closer to home, charge less, or do not have long waiting times. By contrast, when faced with unfamiliar symptoms, people feel inclined to incur higher search and information and negotiation costs to prevent the illness from developing beyond their control, thereby ultimately incurring higher policing and contract execution costs.The present study employed transaction cost theory to analyze considerations in medical advice-seeking behaviors from the perspective of patients. The following conclusions were drawn from the in-depth interviews: transaction costs play a critical role in the selection and decision-making processes inherent in medical advice-seeking behaviors. Although search and information costs may initially appear substantial, the acquisition of accurate information can considerably reduce follow-up transportation times, transportation fees, policing costs, and contract execution costs for failed treatment.After discovering the relationship between medical-seeking behavior and transaction cost theory, we can understand the concern and the logic of patient when seeking medical treatment. This understanding can serve as the foundation for the healthcare provider and government when caring patient and the public. For example, the government can implicate family medicine policy, and by having a trustworthy physician close to patient's house, mean to help the patient to acquire a transaction asset that reduces future transaction cost.In addition to verifying the role of transaction costs in medical advice-seeking behaviors, this study detected a number of behaviors extended from transaction cost considerations, the most notable of which being (1) the inclination to accumulate transaction assets such as trust to reduce future transaction costs; and (2) reliance on personal experience to determine the severity of an illness and transaction costs to be invested.Except for the aforementioned findings, the limited sample size prevented this study from addressing other questions such as how various societal groups recognize transaction costs or the methods adopted by groups to reduce transaction costs. Such questions require further study.


Potyviridae. This slippage results in either an insertion or a substitution, depending on whether the RNA duplex realigns following the insertion. In this study we investigated whether this phenomenon is a conserved feature of superfamily I viral RdRps, by inserting a range of potyvirus-derived slip-prone sequences into a picornavirus, Theiler’s murine encephalomyelitis virus (TMEV). Deep-sequencing analysis of viral transcripts indicates that the TMEV polymerase ‘slips’ at the sequences U6–7 and A6–7 to insert additional nucleotides. Such sequences are under-represented within picornaviral genomes, suggesting that slip-prone sequences create a fitness cost. Nonetheless, the TMEV insertional and substitutional spectrum differed from that previously determined for the potyvirus polymerase.The substitution rates of viral polymerases have been studied extensively. However less is known about the tendency of these enzymes to ‘slip’ during RNA synthesis to produce progeny RNAs with nucleotide insertions or deletions. We recently described the functional utilization of programmed polymerase slippage in the family  Paramyxoviridae, Rhabdoviridae and Orthomyxoviridae, the RNA-dependent RNA polymerase (RdRp) ‘stutters’ on short poly(U) tracts to polyadenylate the mRNAs [Ebolavirus and Paramyxoviridae have long been known to use polymerase slippage for gene expression [Potyviridae, the largest family of positive-sense RNA viruses of plants [6) sequence, giving rise to transcripts with an additional adenylate. Translation of these transcripts allows expression of an essential ‘transframe’-encoded protein, P3N-PIPO. In a subgroup of potyviruses, slippage also occurs at a second GA6 site, enabling expression of the ‘transframe’-encoded protein, P1N-PISPO [In members of the negative-sense RNA virus families he mRNAs . Polymerhe mRNAs , 3. Polypression , 5. Moref plants , 7. In t1N-PISPO .6 slip site. When the reverse complement of the slippage site is present, substitutions are observed instead at the −1 position. The position of these substitutions allows determination of whether ‘to–fro’ slippage occurs during positive- or negative-sense synthesis. For the potyvirus polymerase, ‘to–fro’ slippage occurs mainly during synthesis of poly(A) rather than poly(U) regardless of the orientation of the GA6 sequence [In potyviruses, the single nucleotide insertion rate varies between 0.8 and 2% (P3N-PIPO expression) –9 and 5 sequence .Picornaviridae and Potyviridae RdRps [n and Un (n≥6) homopolymeric sequences when the functionally utilized potyviral slippage sites were excluded [n and Un sequences might also occur at the translational level as a result of ribosomal slippage, the picornavirus analysis was performed in all three reading frames. Selection against An and Un sequences may reflect a propensity of the RdRp to slip at such sites, leading to a reduction in virus productivity from packaging of the defective transcripts. Slippage events may potentially also lead to more serious in trans antiviral effects, such as dominant negative interference by truncated versions of viral proteins and potential preferential MHC class I antigen presentation of slippage products [Given the evolutionary relatedness of the ae RdRps , it is rand Un n≥ homopolyproducts –15.Cardiovirus. As with other picornaviruses, TMEV has a polyadenylated positive-sense RNA genome of ~8 kB that encodes a polyprotein which is processed to produce the structural and non-structural viral proteins (6 tract (in the region encoding 3C), one U6 tract (in the 5′ UTR) and no A7 or U7 tracts.In this study, we wished to investigate to what extent potyviral slippage sites lead to polymerase slippage in Theiler’s murine encephalomyelitis virus (TMEV), a model picornavirus in the genus proteins . The 5′ in cis for replication (at least for the related poliovirus) [We inserted candidate polymerase slippage sites into the coding region, rather than the UTRs, so that insertions or deletions would lead to defective genomes that could not amplify without a helper virus. Indeed, as translation beyond the 2A region is required iovirus) , such geet al. [6 slip sites , slip sites with an extra adenylate inserted (GA7) (TuMV+A) and their reverse complements . A PISPO+A mutant could not be rescued and was not used. Recombinant viruses were obtained from BHK21 cells transfected with T7 polymerase in vitro transcripts of the mutant plasmids. Following infection of naïve cells (MOI of 10), virus was harvested at multiple time points and titrated by plaque assay as previously described [Slippage mutant viruses were generated by the ligation of dsDNA linkers into a digested pTMEV-2SG backbone. Clones were designed based upon two potyviral slip-prone sequences . Followiet al. , we use escribed . The mutin vitro-transcribed RNA for each mutant viral genome was transfected into BHK21 cells in duplicate. Four hours post-transfection, cells were washed thoroughly and one replicate was frozen . Supernatant was harvested from the remaining replicate at 24 h post-transfection (‘Virus 1’). The supernatant was used to infect naïve cells at an MOI of 0.1, which were subsequently harvested at 24 h post-infection  at which point an additional supernatant sample was harvested (‘Virus 2’)  . RNA was6 slip site, T7 slippage was previously measured at ~2.8% [The T7 polymerase is known to slip on poly(A) and poly(U) homopolymeric sequences . At the in vitro T7 transcripts. To validate this assumption, the experiment was repeated (using independently transcribed in vitro transcripts) where a sample of T7 RNA was sequenced prior to transfection, alongside the remaining three samples for each mutant virus. This second dataset exhibited markedly similar results to the first, thus supporting the previous results , the ‘Virus 1’, ‘Cell I’ and ‘Virus 2’ samples had a 2.2- to 3.1-fold increase in single-nucleotide insertions compared to the input, reaching levels of 0.71±0.16 and 0.76±0.16% for the TuMV RC and PISPO RC mutants, and 4.2±0.5 and 3.7±0.5% for the TuMV+A RC and PISPO+A RC mutants, respectively , the ‘Virus 1’, ‘Cell I’ and ‘Virus 2’ samples had decreased levels of insertions compared to the input, indicating that the viral RdRp has a lower tendency to slip on these sequences than the T7 polymerase. Since slippage may also occur during library preparation, these values – which ranged through 0.50±0.12% (TuMV WT), 1.1±0.16% (PISPO WT) and 1.6±0.24% (TuMV+A) – should be considered as upper bounds on the viral RdRp slippage rates.For the four reverse complement mutants  by T7 polymerase have been noted previously and are an essential component of efficient transcription termination [6 or U6C slip-site viruses.We also quantified deletional slippage  of the Uet al. focussed on sites with potyvirus RdRp substitution levels ≥0.5% [6), G-to-A substitutions at position +7 were 36-fold lower than previously observed with the potyvirus RdRp (0.015±0.003 versus 0.54±0.07%); and for the TuMV RC slip site (U6C), C-to-U substitutions at position −1 were 33-fold lower than with the potyvirus RdRp (0.065±0.014 versus 2.2±0.5%) [Due to the background of spurious mutations, Olspert ls ≥0.5% . For the.2±0.5%) .6–7 and U6–7 sequences, with higher levels of slippage occurring when poly(U) is present in the positive strand. The highest levels of ‘to–fro’ slippage occur at position +7 for the TuMV+A RC slip site, suggesting that slippage on U6–7 sequences may occur predominantly during positive-sense synthesis. Picornaviral RdRps are thought to use RdRp stuttering to maintain genomic poly(A) tail length during replication [To summarize, our data indicate that the TMEV RdRp permits insertional slippage at levels of 0.46–4.3% on Alication , where tlication . It is tCardiovirus genus) has a long poly(C) tract in its 5′ UTR that is associated with heightened virulence [6-7 tracts – they are less prone to spurious slippage events than poly(A) or poly(U).It remains possible that picornavirus polymerases may have evolved an increased propensity for slippage in specific genera or species, or that polymerase slippage may be utilized on specific sequences that differ from the potyvirus-derived sequences tested herein. For example, encephalomyocarditis virus (also in the irulence . However6 regardless of sense. Whereas potyviruses utilize polymerase slippage to access novel ORFs, there are no known cases of this occurring in picornaviruses. As mentioned above, non-programmed polymerase slippage within coding sequences results in defective transcripts which may lead to various negative effects [6–7 and A6–7 sequences within picornaviral genomes. This research contributes to our understanding of the mechanisms that shape RNA virus genomic diversity and highlights differences between related viral polymerases, where the potyvirus RdRp may have co-evolved with the expression of the essential P3N-PIPO protein to be specifically tuned to facilitate slippage on the GA6 slip sites that potyviruses functionally use for gene expression.Although both potyviruses and cardioviruses possess superfamily I RdRps , the ten effects –15, and


Zika virus has been associated with a wide range of neurological complications. Neurologists in areas without current active transmission of the virus may be confronted with Zika-associated neurological disease, as a large number of returning travellers with Zika virus infection have been reported and the virus continues to spread to previously unaffected regions. This review provides an overview of Zika virus-associated neurological disease and aims to support neurologists who may encounter patients returning from endemic areas. The exponential increase in travelling has major consequences for the epidemiology of infectious and postinfectious disease, constituting a global public health challenge.Flavivirus genus in the Flaviviridae family. Other flaviviruses include dengue, yellow fever, West Nile virus and Japanese encephalitis virus, many of which are associated with neurological disease.Aedes aegypti mosquito, which transmits the virus between humans and is widespread in tropical regions globally encephalitis.Myelitis.Acute disseminated encephalomyelitis (ADEM).Encephalopathy.*There have also been some cases of combined central and peripheral disease.17ZIKV is known to be neurotropic: in neural progenitor cells infection it halts proliferation and may induce cell death, which is the likely disease mechanism of ZIKV-related microcephaly cases.17As such, any traveller returning from an area with ZIKV transmission, or with a sexual partner who has returned from such an area, who develops an acute neurological illness should be screened for ZIKV infection. Other cocirculating arboviruses should also be considered, such as chikungunya and dengue virus, which have been linked to neurological complications.6 17Box 3 A 60-year-old woman who had returned to the Netherlands from Suriname 10 days before presented with diarrhoea, low-grade fever, nausea and vomiting, and an unsteady gait. Over 4 days, she developed bilateral facial palsy, symmetrical paresis of all limbs and distal hypoesthesia with low to absent tendon reflexes. Cerebrospinal fluid (CSF) had a normal cell count and increased protein concentration; electrophysiology showed signs of a demyelinating polyneuropathy. Her urine and blood were positive for Zika virus (ZIKV) RNA, and serology for other recent infections associated with Guillain-Barré syndrome (GBS) was negative. She received a 5-day course of intravenous immunoglobulin (0.4 g/L daily). At 1-year follow-up she was limited in her daily activities by fatigue and minor weakness in the legs but could walk without aid and had returned to part-time work.Since the start of the global ZIKV epidemic, 23 countries have reported an increased incidence in GBS in parallel with the rising incidence of ZIKV infection. An association between GBS and ZIKV was first established in a case–control study in French Polynesia and many case reports and series have since substantially linked ZIKV and GBS.Campylobacter jejuni, cytomegalovirus, hepatitis E virus, Mycoplasma pneumoniae and Epstein-Barr virus. ZIKV is the latest pathogen to be added to this list. The current paradigm is that in GBS the preceding infection triggers an immune response that causes nerve injury. This has been best described in cases with preceding C. jejuni infection in which there is carbohydrate mimicry between lipo-oligosaccharides on C. jejuni and gangliosides on human peripheral nerves and cross-reactive antibodies to these structures.23GBS is an acute polyradiculoneuropathy and its classic form is characterised by a rapidly progressive symmetrical weakness of muscles in legs and arms with sensory symptoms and reduced tendon reflexes.ZIKV does not seem to be associated with a specific GBS phenotype, although most cases have a sensorimotor variant with facial nerve palsy, ventilatory insufficiency and a demyelinating pattern on clinical neurophysiology. There have also been more reported cases with paraparesis and intact reflexes than expected based on previous studies.There have been sporadic case reports linking other peripheral nerve and neuromuscular diseases to ZIKV infection, including two cases with acute onset of chronic inflammatory demyelinating polyneuropathy (CIDP), three cases of transient polyneuritis and two cases of myasthenia gravis.It is not yet clear if these reported cases are mere coincidence or if ZIKV is indeed a causal factor but, in patients with flaccid paralysis following ZIKV infection, clinicians should consider these differential diagnoses. Furthermore, patients diagnosed with GBS after ZIKV need careful follow-up as it may transpire that they have acute-onset CIDP.26Inflammation of the meninges (meningitis) or brain parenchyma  may result from viral infection of the brain. There has been no robust study associating ZIKV with CNS disease. However, meningoencephalitis in the context of recent ZIKV infection following foreign travel was first reported in early 2016 when, 10 days after returning from a cruise around New Caledonia, a patient who developed focal neurological symptoms and a concurrent rash was found to have ZIKV reverse transcription (RT)-PCR positive CSF.27ZIKV-associated encephalitis is associated with a range of clinical outcomes, from full recovery to death.30Myelitis is a spinal cord inflammation that often occurs following infection and may appear in isolation, as part of a spectrum of inflammatory nervous system pathology or in multisystem disease. Patients typically present with rapid-onset motor and sensory changes that are usually bilateral and associated with a defined sensory level, as with transverse lesions. Autonomic dysfunction is common and CSF or MRI may give evidence of an inflammatory myelopathy.31In contrast, some neurotropic viruses, particularly enteroviruses such as polio and flaviviruses such as Japanese encephalitis virus, directly attack the anterior horn cells of the spinal cord. This causes a longitudinal anterior myelitis on imaging, and a poliomyelitis-like flaccid paralysis clinically.31 32ZIKV-associated cases of myelitis appear to have elements of both transverse and anterior patterns, including evidence of motor, sensory and autonomic signs, 1–2 weeks after systemic symptom onset. Spinal cord lesions on MRI may be anterior and longitudinally extensive. Clinical improvement may follow standard treatment with corticosteroids or plasma exchange.17 33 34Acute disseminated encephalomyelitis (ADEM) is a parainfective or postinfective immunological disease associated with multifocal neurological symptoms and encephalopathy, often occurring in young adults and children as is seemingly the case with ZIKV-associated ADEM. The relationship between viral infection and disease onset is heterogeneous among reported cases and there are no known ZIKV-specific clinical or MRI features .35 3635 3637Reliable detection of viral RNA is possible for around 14 days in serum and urine. However, as there is a variable window of time between viral infection and neurological symptoms, PCR may well be performed when viral RNA is no longer identifiable.9ZIKV-specific IgM antibody of serum or CSF can be detected from around 4 days to at least 12 weeks after exposure, although tests have poor specificity due to cross-reactivity with antibodies to other structurally similar viruses such as dengue virus.There is no specific antiviral treatment available for ZIKV infection. Management of systemic symptoms should be supportive and standard practice should be followed for cases of ZIKV-related neurological disease.There are several vaccines in development and two have entered phase 1 clinical trials, although their progress is complicated by multiple factors.The rapid emergence of ZIKV as a potential cause of severe neurological disease has significant implications in endemic areas and beyond. Unsuspecting travellers and an abundance of potential vectors have facilitated its prolific spread, as illustrated by the number of imported cases to date and the extent of at-risk areas for future outbreaks. We advise neurologists working in areas without current active transmission of the virus to consider a preceding ZIKV infection in patients with acute inflammation of the central or peripheral nervous system returning from areas with ongoing active viral transmission, or with sexual partners who have returned from such areas. There are currently no antiviral drugs available for ZIKV and recommended treatment does not differ from standard practice.We advise testing for Zika virus in patients with suspected inflammatory neuropathy, unexplained myelitis or meningoencephalitis who have been in an area with local transmission or who have had sexual contact with a confirmed Zika virus case, with or without preceding viral symptoms.The recommended treatment in Zika virus-associated neurological disease does not differ from standard practice and there are currently no effective antiviral drugs.There are several vaccine candidates against Zika virus in development.


Lactobacillus plantarum) during heat processing to incorporate in mango juice. The solutions of sodium alginate-soy protein isolate (SA-SPI) with probiotic cells were dropped into the gelation bath containing calcium chloride (3% w/v) solution to develop various types of hydrogel beads. The level of survival of probiotics in encapsulated beads under acidic conditions  and bile salt (0.5 and 1.0% w/v) was evaluated. The survival of the encapsulated probiotics to thermal processing was evaluated by treating the beads in saline solution (0.9% w/v) at 30, 50, 63, and 72°C. The encapsulated probiotic bacteria were found alive even after treatment at 72°C for 90 s. Most of the free cells did not survive at the temperature higher than 50°C and very low pH (pH 2 and 3). The survival of probiotic cells was found higher with the hybrid hydrogel beads containing alginate and soy protein isolate (1:8 w/w). Furthermore, mango juice fortified with encapsulated L. plantarum in hydrogel beads was subjected to thermal pasteurization at 72°C for 90 s.Hybrid alginate-soy protein isolate-based hydrogel beads were prepared and evaluated to enhance the survival of the encapsulated probiotics ( Lactobacillus and Bifidobacterium are the most important probiotic microorganisms commonly associated with gastrointestinal tract [The functional beverage business has recently launched the new ready-to-drink with enhanced bioactive compounds. The functionality of food and beverage is enhanced via biological conversions such as by exploiting the activity of probiotics such as lactic acid bacteria (LAB) with a long history of safe use in the food industry. Probiotics have commonly been added to a wide range of food and beverage products such as yoghurt, sour milk, fermented vegetables, and fruit juices –3. Probial tract . The proal tract . Howeveral tract . Bifidobacterium bifidum RO71 in polysaccharide-protein gel beads decreased by about 2 log CFU mL−1 after treatment with simulated gastric fluid (pH 2.5) for 2 h. Most of the free cells did not survive at the same condition. Microencapsulated Lactobacillus casei NCDC 298 in alginate beads showed a higher survival rate than that of the free probiotics at low pH (1.5) and high bile salt concentration and under heat treatment  [ L. acidophilus readily decreased in harsh environment conditions, such as gastric (low pH in stomach) and thermal conditions (higher temperatures during pasteurization), when compared with hydrogel beads comprising WPI and alginate [ Lactobacillus reuteri encapsulated in chitosan-alginate beads was found to have better tolerance toward stress conditions encountered in food processing as well as to preserve its functional properties [Encapsulation of probiotics improves the stabilizing viability during processing and gastrointestinal transit (GIT). Alginate is one of the most frequently used biopolymers for encapsulating the cells and bioactive compounds because of its simplicity, nontoxicity, biodegradability, biocompatibility, low cost, and especially its pH-sensitive profile , 8. The nd 65°C) . Viable alginate . Lactobaoperties . L. plantarum probiotics under acidic conditions, and improve the survival of probiotic bacteria in fruit juices during pasteurization.None of the studies has yet been reported about alginate-soy protein isolate (SPI) based hydrogel beads incorporating probiotic cells. Soy protein isolate (SPI) possesses nonpolar and polar functionalities with both acidic- and basic-charged amino acids that are suitable for encapsulating various active substances. This study aims to determine the synergetic effects from the combination of alginate and SPI as encapsulation matrices, using the widely employed extrusion gelation technique, on the survival ofAlginic acid  extracted from brown algae (molecular weight 200 kD) with a glucuronic acid (60%) and mannuronic acid (40%) were obtained from Rama Production Co. Ltd. . Soy protein isolate (SPI) was obtained from Shandong Sinoglory Health Food Co., Ltd. . The viscosity of the solutions with different ratios of biopolymers  was measured by Brookfield viscometer (The DV-II+ PRO) with a spindle LV-3 at 25°C. The test was performed three times for each sample. L. plantarum (TISTR 050) was acquired from Thailand Institute of Scientific and Technological Research (TISTR), Thailand. Mahachanok mango puree was purchased from SWIFT Co. Ltd. . Culture media and all other analytical grade chemicals were of purchased from Merck .Pure culture of L. plantarum TISTR 050 was cultured in MRS medium at 37°C. The cells in early stationary phase were harvested by centrifugation at 5000 rpm  for 10 min at 4°C. The cell pellets were further washed twice with 10 mL of saline solution (0.9% w/v) followed by centrifugation at 5000 rpm  for 10 min at 4°C.The L. plantarum TISTR 050 probiotics (1011 CFU mL−1). The encapsulated beads were produced by extrusion technique using encapsulator as shown in −1 using a peristaltic pump . After 30 min of incubation for gelation, the hydrogel beads were washed twice with saline solution (0.9% w/v) and further stored at 4°C for further use.Various types of hydrogel beads were prepared and named after their multivalent components: alginate, soy protein isolate. Alginate-soy protein isolate-based hydrogel beads were produced by the gelation method as described by Bhopatkar et al.  with sliThe particle size of the 100 hydrogel beads was measured with a micrometer . The average values are presented.The encapsulation efficiency of the probiotic cells in alginate-soy protein isolate hydrogel beads was determined by digestion method as described by Anal and Stevens  with sli L. plantarum were observed under scanning electron microscopy . The hydrogel beads were mounted on copper stubs followed by sputter-coated in a metallizer (Agar Sputter Coater) with gold-palladium to reach a thickness of coating of 100 A° and then observed in high vacuum mode in different magnifications.The shape and surface morphology of hydrogel beads loaded with L. plantarum probiotics encapsulated in the hydrogel beads under acidic conditions was performed at pH 2.0, 3.0, and 6.5. Free cells were used as control. L. plantarum-encapsulated in various hydrogel beads (1010 CFU mL−1) were added to test tubes containing 9 mL of NGYC medium (comprising nonfat skim milk (12% w/v), glucose (2% w/v), yeast extract (1% w/v), and cysteine (0.05% w/v) and adjusted to the desired pH with 5 M HCl or 1 M NaOH). After incubation at 37°C for 3 h, the samples were centrifuged (5000 rpm for 10 min at 4°C) and the hydrogel beads were further allowed to disintegrate in 9 mL phosphate buffer saline solution  to release the cells from the beads. The survival of the free and encapsulated L. plantarum probiotics was evaluated by standard plate count (SPC) method.The survival of the L. plantarum were evaluated for their survival in 0, 0.5, and 1.0% (w/v) porcine bile salt solutions following the method of Hansen et al. [ L. plantarum-encapsulated beads (1010 CFU mL−1) were added to different test tubes containing 9 mL of milk–yeast extract medium at pH 6.9  containing 0.5, 1.0% (w/v) porcine bile salt. The samples were further incubated at 37°C for 3 and 6 h. The viable cell counts were enumerated as described in earlier section.Free cells and encapsulatedn et al.  with sli L. plantarum-encapsulated hydrogel beads dispersed in normal saline solution (0.9% w/v) was exposed to heat treatment following the method descried by Fang et al. [ L. plantarum-encapsulated beads (1010 CFU mL−1) were placed in test tubes containing 9 mL of normal saline solution (0.9% w/v). The test tubes were further incubated in a water bath at various temperatures  for 2 min. Aliquots were collected at different intervals of time after incubation. The samples were cooled down to room temperature (~25°C). The viability of the free and encapsulated L. plantarum probiotics was obtained as described above for heat treatment using SPC.The viability ofg et al.  with sli L. plantarum were placed in test tubes containing 9 mL of preheated mango juice. The test tubes were incubated in a water bath at 72°C for 90 s followed by storing at 4°C. The samples were further taken every 7 days to analyze the probiotic cells in juice and pH. All experiments were conducted in triplicate.The mango juice (50% w/v) was prepared by dissolving the mango concentrate in sterilized deionized water. Free cell andThe pH of mango juice was measured using pH meter . Titratable acidity (TA) was determined using titration method with standardized alkali solution (0.1 N NaOH). TA expressed as the percentage of citric acid per 100 g of juice.p < 0.05).All experiments were repeated at least three times. Results are reported as mean ± standard deviation using IBM SPSS statistics program . The statistical significance among various samples of free cell and encapsulated beads was evaluated with one-way ANOVA, with significant level  seems to exert a synergetic effect on the survival of encapsulated probiotics. The results additionally indicated that, at pH 2 and 3, increasing the content of SPI at a given SA content  influenced the survival of probiotic cells. The probiotic in bead D formulation achieved the highest survival rate (p < 0.05) with 6.23 ± 0.04 and 7.62 ± 0.10 log CFU mL−1 when submerged in NGYC medium pH 2 and 3 for 3 h, respectively. The variations in the probiotics survival may be due to differing complexion between SPI and SA when the relative content of SPI exceeds 8% w/v (at 1% SA).−1, respectively, and 1.0% w/v bile salt of 6 h incubation was 8.05 ± 0.20 and 8.51 ± 0.10 log CFU mL−1, respectively. However, the survival of the probiotic in the SA/SPI beads remained nearly the same as that of the starting content (9 log CFU mL−1) after 6 h owing to protection from the electrostatically charged SA and SPI complexion in the hybrid beads. Hence, bile salt concentrations of 0.5 and 1.0% w/v (high bile) had no diminishing effects on the survival level of the probiotics encapsulated in the SA/SPI beads after 6 h of incubation.Survival of free and encapsulated probiotics in SA and SA/SPI hydrogel beads in different bile salt concentrations  at 37°C was also evaluated. Viability of free and encapsulated probiotics in SA hydrogel bead remained unchanged after 6 h of incubation in milk–yeast extract medium without bile salt. In contrast, the viability of the encapsulated probiotics in the SA/SPI beads increased gradually from 3 to 6 h of incubation with increasing SA/SPI ratios to 1:8. This is due to the growth of the probiotics encapsulated in the SA/SPI beads; a milk–yeast extract medium, used as nutrient, penetrated the interface between the electrostatically charged SA and SPI complexion particles and the SA wavy matrix. Increasing the bile salt concentrations from 0.5 to 1.0 % (w/v), the survival of the free cells and the probiotic cells encapsulated in SA beads only decreased with increasing incubation periods from 3 to 6 h. The viability of free cells and encapsulated probiotic in SA beads after 6 h of incubation in 0.5% w/v bile salt was 8.17 ± 0.04 and 8.62 ± 0.08 log CFU mL−1) after heat treatment at 72°C for 1 min. There was no significant difference (p < 0.05) between the survival of probiotic in beads D and E formulations. However, the survival of probiotic in SA beads was not found after treat with heat at 72°C for 2 min. Importantly, adequate active amount of probiotic (6-8 log CFU mL−1) in the bead B to E formulations survived for 1 min under heat treatment at 63 and 72°C. The results confirmed the presence of probiotic bacteria at minimum levels of 6-7 log CFU mL−1 which is recommended in functional foods [ L. acidophilus more than free and alginate encapsulated cells.In order for probiotic cells to be effective and remain viable in food and beverage products, they must withstand the recommended pasteurization temperatures and/or other industrial processing parameters. Survival of probiotic cells in SA/SPI beads was therefore evaluated under the heat treatment at 50, 63, and 72°C and illustrated in al foods –19. Fang−1 (from 9.10 ± 0.04 log CFU mL−1 to 8.11 ± 0.13 log CFU mL−1) was found decreased. Therefore, it was proven that the encapsulated probiotic bacteria with SA/SPI can protect probiotics from thermal pasteurization.The hydrogel beads (formulation D) were chosen to fortify with mango juice.  L. plantarum bead was observed during the storage. The survival of L. plantarum TISTR 050 beads in mango juice was decreased from 8.56 ± 0.02 log CFU mL−1 to 5.83 ± 0.03 log CFU mL−1 during 28 days of storage. The number of cell during storages is decreased with large amount when compared to another study [ L. plantarum in encapsulated bead of our study passing the pasteurization process and this is the main cause of making the cell injury and decreasing to almost 2.8 log CFU mL−1 during storage. However, there was not any microorganism on pasteurized mango juice with free cell all the period of storage found. Therefore, encapsulated probiotics in SA/SPI bead were able to maintain viability in acidic fruit juice up to 25 days after pasteurization in which the probiotic remained at least 6 log CFU mL−1.er study , due to  L. plantarum TISTR 050) was 4.7. The pH of pasteurized mango juice with free cell remained constant during storage (28 days) because all L. plantarum was killed during pasteurization as illustrated in  L. plantarum bead remained constant at 4.7 during storage. In case of acidity of both pasteurized mango juice with free cell and pasteurized mango juice with encapsulated L. plantarum TISTR 050, the hydrogel beads were constant during 35 days of storage (1.25%). There was no significant difference (ρ < 0.05) between the pH and the acidity at day 0 and day 28 for two types of stored juice. Therefore, it was found that encapsulation of probiotic was able to maintain the pH and acidity of fruit juice during storage. As illustrated in  L. plantarum TISTR 050 free cells and pasteurized mango juice with encapsulated L. plantarum TISTR 050, the hydrogel beads remained constant during 35 days of storage (1.25%). There was no significant difference (p < 0.05) of acidity at day 35 between two types of storage juices.The initial pH of all mango juice samples at day 0 (before the addition of L. plantarum (TISTR 050) probiotics were successfully obtained by extrusion gelation technique of various ratios of pure SA and SA/SPI into CaCl2 solution. The encapsulated beads with SA and SA/SPI can protect L. plantarum from gastric condition; however, SA/SPI beads provided better protection. The bile salt concentrations of 0.5 and 1.0% (w/v) had no diminishing effects on the survival level of the probiotics encapsulated in the SA/SPI beads after 6 h of incubation. Furthermore, the probiotics encapsulated in the SA/SPI beads survived longer relative to those encapsulated in the SA beads and the nonencapsulated probiotics subjected to heat treatment. The application of encapsulated bead of L. plantarum in mango juice under pasteurization process showed successful resistance to thermal conditions (72°C for 90 s). The similar result is reported in case of encapsulated probiotics in alginate—whey protein isolate-based hydrogel beads [Encapsulated beads of


In this study, the innovative and multifunctional nanoparticles–hydrogel nanocomposites made with chitosan hydrogel beads and solid lipid–polymer hybrid nanoparticles (SLPN) were prepared through conjugation between SLPN and chitosan beads. The SLPNs were first fabricated via coating the bovine serum albumin (BSA)-emulsified solid lipid nanoparticles with oxidized dextran. The aldehyde groups of the oxidized dextran on the surface of the SLPN enabled an in situ conjugation with the chitosan beads through the Schiff base linkage. The obtained nano-on-beads composite exhibited a spherical shape with a homogeneous size distribution. The successful conjugation of SLPN on the chitosan beads was confirmed by a Fourier transform infrared spectroscopy and a scanning electron microscope. The effects of the beads dosage  and the incubation duration  on the conjugation efficiency of SLPN onto the beads were comprehensively optimized. The optimal formulations were found to be a 200 bead dosage, with 30–90 min incubation duration groups. The optimal formulations were then used to encapsulate thymol, an antibacterial agent, which was studied as a model compound. After encapsulation, the thymol exhibited sustained release profiles in the phosphate buffer saline. The as-prepared nanoparticles–hydrogel nanocomposites reported in this proof-of-concept study hold promising features as a controlled-release antibacterial approach for improving food safety. Hydrogels are highly hydrated three-dimensional network structures that are able to absorb large quantities of water and can swell without being dissolved, as a result of chemical or physical cross-linking . BecauseCurrently, the functionalization of chitosan hydrogel has become more and more popular, as it could confer additional functions to native chitosan hydrogel. In particular, nanoparticles–chitosan hydrogel composites have been developed and studied over decades, because of their enhanced properties compared with their individual components . For insIn our previous study, we successfully prepared SLPN from oxidized dextran (OD), bovine serum albumin (BSA), and solid lipid . OD is a4). The structure of OD was confirmed by nuclear magnetic resonance (NMR) spectroscopy and Fourier-transform infrared spectroscopy (FTIR). The prepared OD was then used in the formulation of SLPN by cross-linking it with BSA in order to confer the aldehyde groups on the surface of the nanoparticles, resulting in the formation of surface-active SLPNs capable of reacting with other amino-containing polymers, such as chitosan. Therefore, in the present study, we first prepared the chitosan beads using an antisolvent coagulation method, using a concentrated sodium hydroxide (NaOH) solution, and then these beads were subsequently incubated with surface-active SLPN for functionalization via the Schiff base linkage  and incubation duration  on the conjugation efficiency of SLPN onto the chitosan beads, the protein concentration in the SLPN dispersion was measured at different time points during the functionalization process. The protein, BSA, was used as a natural emulsifier during the SLPN preparation, and it formed the intermediate layer between the OD coating and the solid lipid core. While there may be free OD in the SLPN dispersion, the BSA molecules, as the intermediate and amphiphilic layer in the SLPN structure, shall be adsorbed on the solid lipid core. Thus, the change in the BSA concentration in the SLPN dispersion during the conjugation process was correlated to the change of the SLPN concentration. By subtracting the measured residual BSA concentration in the dispersion from the total BSA concentration used to prepare the SLPN, one can deduce the SLPN conjugated to the chitosan beads and thus calculate the conjugation rate and efficiency. Moreover, the protein content was normalized based on the number of beads, and the calculated protein concentration per bead was higher at lower bead dosage group B, sugges−1 were detected and assigned to the amide I and amide II stretching vibrations, respectively  and δ(C–H) vibrational modes [–1 was observed in the OD spectrum (−1 for the C=O stretching (amide I), N–H bending (amine II), and C–N stretching (amide III), respectively [−1, respectively  were successfully fabricated and were denoted as T-SLPN1, T-SLPN2, and T-SLPN3, respectively. The particulate characteristics and encapsulation efficiency are shown in SLPNs with thymol loading at 0.05, 0.075, and 0.1 mg/mg (2). The Higuchi model explained that thymol was first to be dissolved in a solid lipid matrix and was diffused to the surface of the matrix, then partitioned to amphiphilic protein layer, followed by the hydrophilic OD layer. Finally, the thymol was released to the surrounding medium. Among all of the groups, the 200B-30m group presented the highest R2 in the zero-order model , which means that the release of thymol was a better control in the 200B-30m group than in the other groups. As we had demonstrated that SLPNs maintained their integrity, and that the detachment of BSA from the composite beads was not detected for over 24 h of incubation in the same PBS medium, one could conclude that the sustained release of thymol from the composite beads was governed by diffusion.The cumulative release of thymol from the composite beads in the phosphate buffered saline (PBS) buffer is presented in L. monocytogenes, S. typhimurium, E. coli O157:H7, and B. thermosphacta [L. monocytogenes and S. typhimurium ranged from 0.25 to 0.5 μL/mL (0.24 mg to 0.48 mg/mL), while E. coli O157:H7 was less inhibited by thymol, with MIC and MBC ranging from 0.5 to 1 μL/mL (0.48 mg to 0.96 mg/mL). In our study, the thymol amount per bead (T-SLPN2) was 0.0013, 0.0017, and 0.0021 mg for 200B-30m, 200B-60m, and 200B-90m, respectively. The nanoencapsulation of thymol in SLPN is expected to enhance its antimicrobial activity by providing a large surface area, and it thus has better contact with the bacteria. By developing the nano-on-beads composite, thymol-loaded nanoparticles are immobilized on the chitosan beads, and when an appropriate number  of composite beads is used, it will ensure that the concentration of thymol reaches its MIC and MBC values against different bacteria, enabling the antimicrobial potency against common pathogenic pathogens.Thymol has been proven to have potent antimicrobial activities against a wide spectrum of pathogenic pathogens found in food products, including osphacta ,34,35,36® ATO 5 was a kind gift from Gattefossé. Dextran (40 kDa), thymol, sodium periodate (NaIO4), bovine serum albumin (BSA), and low molecular chitosan (75–85% deacetylated) were purchased from Sigma-Aldrich . Hydrochloric acid (HCl), sodium hydroxide (NaOH), and ethanol were obtained from Fisher Scientific Co. . Unless noted otherwise, all of the chemicals were of analytical grade and were used without further purification.Precirolw/v) was prepared by dissolving chitosan in 1% (v/v) acetic acid at room temperature overnight, with gentle stirring. In order to eliminate the gas bubbles, the chitosan solution was sonicated for 3 min, and then gently stirred overnight for complete hydration. To prepare the chitosan beads, 5 mL of the chitosan solution was extruded dropwise into a beaker containing 25 mL of 1 M sodium hydroxide, through tubing connected to a 200 µL pipette tip, and the extrusion was powered by a Fisherbrand™ FH30 peristaltic pump . The obtained chitosan beads were solidified in sodium hydroxide for 2 h while stirring at 300 rpm. Then, the alkaline solution was slowly decanted and the chitosan beads were collected using filtration. The beads were successively washed with ultrapure water, until the eluted water was neutral.The chitosan solution  aqueous phase containing 1 mg/mL BSA and 1 mg/mL OD solution in pure water was added into the above melted lipid under homogenization at 25,000 rpm for 30 s, to obtain a coarse emulsion, followed by 3 min sonication using a probe-type sonicator . The obtained sample was incubated under 70 °C for 30 min to initiate the in situ conjugation between the BSA and OD. Subsequently, the mixture was rapidly cooled down in an ice bath to solidify the solid lipid core.Empty SLPNs were prepared using a homogenization and sonication method, as previously reported . BrieflyWith the attempt to prepare antimicrobial hydrogel nanocomposite, thymol, an essential oil with a potent antimicrobial activity, was loaded into SLPN. Briefly, thymol was dissolved in ethanol at a concentration of 10 mg/mL as a stock solution. Then, different amounts of thymol stock solution  were mixed with the melted solid lipid to form new lipid phases, and were incubated together at 70 °C for 1 min. The subsequent procedures were the same as described above for the preparation of the empty SLPNs.® Ultra centrifugation device with a molecular weight cutoff of 10 kDa. After centrifugation under 10,000× g for 15 min, the solution in the receiving reservoir was collected and diluted five times with hexane, so as to extract the free thymol. The concentration of the extracted thymol was measured using a UV-VIS spectrophotometer at 275 nm, with a pre-established thymol standard curve (0.01–0.1 mg/mL). The EE was calculated using the following equation:The concentration of the thymol encapsulated in the SLPNs was measured using UV-VIS spectroscopy. The encapsulation efficiency (EE) was determined by measuring the concentration of the un-encapsulated thymol, using an AmiconA certain dosage of chitosan hydrogel beads  were directly added into a solution of freshly prepared SLPN solution (with or without thymol loading). The mixture was incubated under 70 °C for 30, 60, 90, 120, and 150 min so as to initiate the functionalization via a Schiff base reaction between the amino groups of the chitosan and aldehyde groups of the OD. After incubation, the mixture was cooled down in an ice bath. To determine the adsorption efficiency of SLPN onto the chitosan beads, the concentration of protein in the supernatant was determined at designated time points  using a protein assay kit, according to the manufacturer′s protocol. To determine the particulate characteristics of the SLPNs after functionalization, the particle size, PDI, and zeta potential of the SLPNs were measured using a Zetasizer Nano ZS . The particle size was determined by dynamic light scattering (DLS) at a 173° scattering angle. The PDI, which is a parameter to evaluate the homogeneity of the nanoparticles, was recorded together with particle size measurement. The zeta potential was calculated from the electrophoretic mobility of the sample. The SLPN functionalized chitosan beads are hereafter denoted based on their beads amount and incubation time. For example, the 50 chitosan beads with 30 min incubation group was labelled as 50B-30m.−1. The results were analyzed using OMNIC software, version 8.0.Selected samples  were placed in aluminum foil plate and dried in the oven for 2 h at 40 °C. The preliminary data indicated that drying at 40 °C for 2 h was sufficient to completely remove any water from the beads by measuring the weight change as a function of the drying time. After drying, the Fourier-transform infrared spectroscopy (FTIR) spectra of dried beads were recorded using a NicoletTM iSTM5 FT-IR spectrophotometer . in the range of 500–4000 cmThe oven-dried bead samples were directly placed on double-sided carbon tape. All of the samples were coated with gold using a sputter coater, before being observed under a scanning electron microscope .To evaluate the detachment of the SLPN from the chitosan beads, samples were incubated in water under mild stirring, and the incubation medium was collected at designated time points  for the measurement of the protein concentration using a Lowry assay, according to the manufacturer′s protocol.The release rate of the thymol from the nano-on-beads composite was conducted in phosphate buffered saline . The selected thymol loaded nano-on-beads composite (200 beads) were directly placed in a PBS release medium (50 mL). The release system was carefully closed so as to prevent evaporation during the release test. During the experiment, 2 mL of the release medium was withdrawn at predetermined time intervals, with the replacement of the same volume of fresh medium in order to keep the volume constant. The collected release medium was then lyophilized for 24 h, and then the thymol from the dried powder of the release medium was extracted with 5 mL of hexane. The concentration of thymol was determined using a UV-VIS spectrophotometer.p) was set as 0.05.All of the results were presented as the mean ± standard deviation (SD) of at least triplicate determinations. The data were analyzed using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test to compare the significance among the samples. The significant level  with unaffected physicochemical characteristics of SLPN after conjugation. After the optimization, the thymol was pre-loaded into the SLPN to prepare the antimicrobial nano-on-beads composites that exhibited a sustained release of thymol and thus held great potential for food safety-related applications.


Carica papaya L.) in Ecuador, named papaya virus E, was sequenced and characterized. The negative-sense single-stranded RNA genome consists of 13,469 nucleotides with six canonical open reading frames (ORFs) and two accessory short ORFs predicted between ORFs corresponding to P3 (movement protein) and M (matrix protein). Phylogenetic analyses using amino acid sequences from the nucleocapsid, glycoprotein and polymerase, grouped the virus with members of the genus Cytorhabdovirus, with rice stripe mosaic virus, yerba mate chlorosis-associated virus and Colocasia bobone disease-associated virus as closest relatives. The 3’ leader and 5’ trailer sequences were 144 and 167 nt long, respectively, containing partially complementary motifs. The motif 3’-AUUCUUUUUG-5’, conserved across rhabdoviruses, was identified in all but one intergenic regions; whereas the motif 3’-ACAAAAACACA-5’ was found in three intergenic junctions. This is the first complete genome sequence of a cytorhabdovirus infecting papaya. The virus was prevalent in commercial plantings of Los Ríos, the most important papaya producing province of Ecuador. Recently, the genome sequence of bean-associated cytorhabdovirus was reported. The genome is 97% identical to that of papaya virus E, indicating that both should be considered strains of the same virus.The complete genome of a new rhabdovirus infecting papaya ( Rhabdoviridae, a negative-sense RNA virus family, contains viruses that infect a wide range of hosts including vertebrates, invertebrates and plants [Rhabdoviridae [The d plants . Virionsd plants . Rhabdovoviridae ,4.Based on host type, genomic organization and other biological features, rhabdoviruses currently are organized in 18 genera . High-thl) and 5’ trailer (t) [The genome organization of rhabdoviruses has a canonical arrangement of five genes: 3’-N-P-M-G-L-5’ that encode the nucleocapsid, phosphoprotein, matrix protein, glycoprotein and the large polymerase, respectively. Terminal regions have non-coding regulatory sequences denoted, respectively, as 3’-leader (iler (t) ,10. Addiiler (t) .Cytorhabdovirus or Nucleorhabdovirus, based on their cytoplasmic or nuclear, site of replication in the cell, respectively [Dichorhavirus and Varicosavirus, have been created to classify bi-partite plant-infecting rhabdoviruses [Monopartite plant-infecting rhabdoviruses have long been classified into the genera ectively ,12. Thisoviruses ,4.Although more than 80 monopartite plant rhabdoviruses have been reported, based on cytopathology studies, complete genomes are only available for a few members of each genus. A genomic feature common to both cyto- and nucleorhabdoviruses is the presence of an additional open reading frame (ORF) between the P and M genes. The product of this ORF is considered the movement protein (MP) as it has been demonstrated to have cell-to-cell movement function ,14. AddiPlant infecting rhabdoviruses have been found in a wide range of hosts including monocots such as rice, maize, wheat and barley, and dicots such as potato, lettuce, carrot and strawberry, among others .Carica papaya L.), the occurrence of putative nucleorhabdoviruses in commercial plantings in Venezuela, Florida and Mexico was documented as early as 1980 [In papaya , previously used as part of a study on the epidemiology of papaya virus Q (PpVQ) and its relationship with papaya ringspot virus (PRSV) ,20, was National Center for Biotechnology Information (NCBI).Total RNA was extracted from the selected plant using the protocol described by Quito-Avila, et al. ; followeHTS data was verified by Sanger sequencing of RT-PCR amplified overlapping fragments, which were generated by specific primers. Terminal sequences were confirmed by RACE using total RNA as a template for cDNA, and specific primers near the ends as recommended by the manufacturer . Sequence comparisons, alignments and prediction of open reading frames (ORFs) were done using Geneious R 11 , the NCBI conserved domain database  and the Leaf tissue was dissected into one mm pieces using a biopsy punch, fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, 0.2% Tween-20, 0.5M Na cacodylate and processed in a Pelco BioWave microwave as previously described . TEM griBemisia tabaci) genome (acc. numbers: KJ994255-KJ994264), were identified and included in the analysis. Amino acid sequences were aligned using structural information with Expresso [Protein sequences corresponding to the nucleocapsid (N), glycoprotein (G) and polymerase (L) were downloaded from all the nucleo- and cytorhabdoviruses available in GenBank. In addition, cytorhabdovirus-like sequences annotated as part of the whitefly (Expresso . The conExpresso . Two chaExpresso .5’- CGCAAAACTCGATTGTTCCG-3’ and (R) 5’-CCTGCTGATGATCCTATCTCC-3’, which amplify a 779 nt fragment spanning a portion of the 3’ leader and the nucleocapsid gene.A total of 180 papaya plants from commercial fields in five Ecuadorean provinces  were tested for the virus as described . PCR was5’-GAGAAGTGGAACCTCAATTTCC-3’ and (R) 5’-CTGAAGAGAGAAGGGTCGGT-3’. Amplicons (860 nt long) were cloned and sequenced. Sequence comparisons were performed using ClustalW [In addition, up to five positive samples from each location were used to amplify a fragment of the virus polymerase using primers: (F) ClustalW  to deterThe annealing temperature for both primer pairs used in PCR was set at 55C.A total of 32,375,528 paired-end 100 nt reads were obtained from the HTS cDNA library. These reads were assembled into 61,033 contigs. Twenty-three contigs were identified as associated with plant viruses. One contig >13 kb revealed similarities to cytorhabdoviruses. The remaining contigs showed homology to PRSV. In subsequent analysis using Geneious R11  these contigs were assembled into a ~10 kb PRSV genome. Only about 75 thousand reads (0.23%) mapped to the cytorhabdovirus, while 3.8 million (11.68%) identified with the PRSV contig.The entire genome of the new virus, provisionally named papaya virus E (PpVE) (GenBank accession no. MH282832) has 13,469 nt. The antigenomic strand contains six ORFs organized in the classical canonical order of monopartite plant rhabdoviruses, plus two short accessory ORFs (ORFs 4 and 5) .AUAUG), encode two small hypothetical proteins  of unknown function. ORFs 6, 7 and 8 encode, respectively, the putative matrix protein (M), glycoprotein (G) and the polymerase (L) , phosphoprotein (P) and movement protein (MP) P3. ORFs 4 and 5, arranged in a contiguous fashion with overlapping termination/initiation codons (e.g. rase (L) .Phaseolus vulgaris L.) in Brazil [PpVE has an overall nucleotide sequence identity of 97% with the genome of bean associated cytorhabdovirus (BaCV), which was found recently from common bean , yerba mate chlorosis-associated virus (YmCaV) and Colocasia bobone disease-associated virus (CBDaV) as closest relatives [BLASTx searches performed on each ORF of PpVE and BaCV revealed homology, with low sequence identities, to rhabdovirus proteins from different members of the genus elatives ,32,33 , northern cereal mosaic virus  and strawberry crinkle virus . Interestingly, the two most closely related cytorhabdoviruses, RSMV and YmCaV, have shorter 3’ leaders. The 5’ trailer of PpVE is 167 nt long, similar in size to its counterpart from YmCaV .3’-GAUAAAA-5’ and 3’-CUAUUUU-5’ located at nt positions 12–18 and 13,430–13,436, respectively, complement each other, as reported for terminal regions of rhabdoviruses [UUCUUUUAA-5’3’- was identified at both the leader (nt 62–70) and trailer  regions.Several motifs were identified in both the leader and trailer regions of PpVE, which are fully or partially complementary to each other. For instance, motifs oviruses . In addiThe multiple sequence alignments for N, G and L proteins were 831, 936, and 3,071 amino acids (aa) long, respectively. After eliminating ambiguous fragments, the resulting alignment lengths were 284, 269, and 1,692 aa for each protein, respectively. The best evolution model for N and L was LG+G  was the The topology of the multi-locus tree was identical to the polymerase one and congruent with those inferred by analyses of the nucleocapsid and glycoprotein. Cyto- and nucleorhabdoviruses are monophyletic and each genus contains clades with well supported nodes corresponding with their vectors . For maiCytorhabdovirus genus, in a clade that includes the dicot-infecting YmCaV and CBDaV, and the monocot-infecting NCMV, MYSV, RSMV and barley yellow striate mosaic virus (BYSV), which are known (except for YmCaV) to be transmitted by Delphacidae/Cicadellidae vectors. Interestingly, virus-like sequences annotated as part of the B. tabaci genome grouped closely with PpVE and BaCV  of samples (cv. Sunrise) collected from one-year old plants; whereas 13% (n = 30) were positive in an adjacent four-month-old field. In Guayas province, the virus was only detected in one out of 30 plants tested from a two-year-old field. In Manabí, 20% of plants tested positive from a three-year-old field (n = 30). The virus was not detected in selected fields of Sucumbíos, a forest province where ‘Criolla’ papaya was grown, or Santa Elena, where the Hawaiian cultivar Sunset was sampled.All the plants that tested positive for the papaya cytorhabdovirus were also positive for PRSV. However, no differences in leaf symptoms were observed between PRSV-singly infected plants and plants co-infected with both viruses.Genome diversity was inferred by comparing an 860 nt fragment of the virus polymerase. Five isolates from Los Ríos, five from Manabí, and only one from Guayas, were selected for RT-PCR. Sequence alignments of the amplified fragment showed a 99% identity across isolates from different provinces in Ecuador.Rhabdoviridae is one of the most diverse virus families as it contains viruses that infect arthropods, vertebrates and plants [The d plants . Here, wCytorhabdovirus genus, with rice stripe mosaic virus, yerba mate chlorosis-associated virus and Colocasia bobone disease-associated virus as closest relatives.The new virus, provisionally named papaya virus E (PpVE), grouped with members of the In the concatenated alignment, 75.4% of the total length corresponded to the polymerase. The resulting tree was identical to the topology of the polymerase alone, supporting other studies that indicate using the polymerase is an accurate representation of the phylogeny for rhabdoviruses .Delphacidae or Cicadellidae. However, formal transmission experiments are needed to confirm this hypothesis. An interesting finding in this study was the genetic closeness (80% nucleotide identity) observed between PpVE and sequences from a whitefly genome annotated by Kumar and Upadhyay . We hypothesize that whiteflies used for the genomic analysis were either infected by a cythorhabdovirus or were carrying  a cytorhabdovirus acquired from a plant host.Furthermore, this study confirmed the strong correlation between phylogenetically-related species and their vectors . Accordil-N-P-M-G-L-t-5’3’-.The genome organization of rhabdoviruses includes five genes flanked by leader and trailer sequences at the 3’ and 5’ ends, respectively, resulting in the canonical arrangement: l-N-P-P3(MP)-M-G-L-t-5’3’- arrangement [In plants, cyto- and nucleorhabdoviruses have an additional ORF between the P and M genes, whose product has cell-to-cell movement activity, resulting in the typical angement ,14. Howeangement ,12,39.termination upstream ribosome binding site), which includes a pentanucleotide motif that is complementary to the loop region of helix 26 of 18S rRNA [In PpVE, two short ORFs (namely ORFs 4 and 5) were predicted with overlapping termination/initiation codons. This feature is commonly associated with a translation-reinitiation mechanism and has been documented, among others, for some animal rhabdoviruses . The rei18S rRNA . TURBS-l3’-AUUCUUUUUG-5’ not only in intergenic regions (except in the P3(MP)-P4 junction), but also in the 5’ (t) region, supporting its involvement in transcription termination of the corresponding preceding gene.One of the genomic hallmarks of rhabdoviruses is the presence of transcription regulatory signals, such as conserved intergenic motifs and self-complementary sequences located at terminal regions . The genIn addition, a second conserved motif was detected in gene junctions P3(MP)-P4, P5-M and G-L of PpVE . AlthougThis is the first report of a cytorhabdovirus in papaya and the first sequence deposited for any rhabdovirus of this host. Both its phylogenetic relatedness to cytorhabdoviruses and the TEM observations showing virus accumulation in the cytoplasm support the notion that this virus is not related to the papaya nucleorhabdoviruses reported in the early 1980s ,17,18. SLastly, in January 2019, the genome of BaCV was documented from Brazil . BaCV shS1 FigYellow arrows denote P4 from BaCV or YmCaV; light-blue arrow indicates the concatenated P4-P5 in PpVE. Conserved residues are black-shaded. Percentage identities between PpVE and BaCV or YmCaV are indicated on the right.(PNG)Click here for additional data file.S2 FigArrow points viruses whose evolutionary history is not congruent among proteins. Numbers above the nodes represent posterior probabilities. Papaya virus E and bean associated cytorhabdovirus, which are 97% identical throughout the genome, are shown in red.(PNG)Click here for additional data file.


A Ugandan child with an unexplained encephalitis was investigated using viral metagenomics. Several sequences from all segments of a novel orthobunyavirus were found. The S-segment, used for typing, showed 41% amino acid diversity to its closest relative. The virus was named Ntwetwe virus, after the hometown of the patient. In sub-Saharan Africa, children often present to hospitals with fever and acute alterations of consciousness, yet causes remain unidentified in up to 75% of such cases . Recent Cerebrospinal fluid (CSF) and ethylenediaminetetraacetic acid plasma samples were collected from the patient 2 weeks after onset of symptoms at the Mulago National Referral Hospital in Kampala, Uganda, and stored at −80°C. Viral metagenomic sequencing was performed on the CSF and plasma samples. Viral sequences were extended by genome walking, and the resulting contigs were used to infer an amino acid phylogeny and identity matrix. A reverse transcription quantitative polymerase chain reaction was developed, and the virus was quantified in CSF and plasma. Inflammatory biomarkers were measured in CSF and plasma using a Luminex panel. Details of the assays are provided in the In February 2016, a previously-healthy 3-year-old Ugandan girl fell ill in her hometown, Ntwetwe village, and developed a fever, a headache, and abdominal pain . She wasAt the time of arrival at Mulago hospital, the patient had been ill for 2 weeks. She presented in a coma  with prolonged, generalized convulsions that did not respond to 2 doses of first-line anticonvulsants. She had a temperature of 39.2°C, a blood pressure reading of 137/93 mmHg, a heart rate of 198 beats/minute and a respiratory rate of 40 breaths/minute. She displayed decorticate posturing, had absent corneal reflexes, and had papilledema on funduscopy.3, and normal glucose and protein levels. A malaria blood slide was negative, and the bacterial CSF culture remained negative after 7 days.On laboratory examination, her haemoglobin level was 13.3 g/dL. CSF analysis showed less than 5 total cells/mmThe patient was admitted to the High Dependency Unit and treated with amlodipine, intravenous (IV) ceftriaxone, IV metronidazole, and IV phenobarbitone. Nonetheless, she did not regain complete consciousness and left the hospital. She died at home 2 weeks after leaving the hospital.S. pneumoniae, N. meningitidis, and H. influenzae were performed. These tests also were negative.The severity of outcome elicited a further investigation into the cause of disease. The clinical syndrome of decreased consciousness, fever, and seizures in absence of malaria parasitaemia  were suggestive of an encephalitis . ConsequTo test for an unexpected or unknown virus, a viral metagenomics assay was performed on CSF and plasma, which initially generated 6,947 sequence reads. No known viruses were identified, yet 2 sequence reads  showed some, although low, amino acid identities (the percentage of matching amino acids) to several orthobunyavirus L-segments. Using genome walking, the L-segment sequence was extended to 936 nucleotides. A PCR developed to target this sequence confirmed the presence of this sequence in both CSF and plasma of the patient.To acquire additional sequence information and also confirm that no other viral infection occurred in this patient, 2 Illumina MiSeq libraries were subsequently prepared on separate CSF and plasma samples. Among the total of 4.5 million reads, no known virus was found, yet several sequences of the M and S segments of the novel virus were found. Consequently, sequence information from all 3 orthobunyavirus segments was obtained. All showed large genetic variations to their closest relative, the Tataguine virus . Hence, To determine the phylogenetic clustering of Ntwetwe virus with known orthobunyaviruses, reference sequences from related serogroups were used . For all2 RNA copies/mL in CSF and 5 × 103 RNA copies/mL in plasma.To determine the virus concentration, a reverse transcription quantitative polymerase chain reaction was performed, which provided a viral load of 3 × 10To provide additional support for a causative relationship between Ntwetwe virus infection and encephalopathy, a clinically-relevant Ntwetwe virus infection was hypothesized to elicit a host immune response similar to CNS infections with known viral pathogens. A panel of 11 inflammatory biomarkers was measured in CSF and plasma. The concentrations in plasma were compared to those of reference patients with encephalopathies of varying etiologies see .For plasma, the biomarker profile of the Ntwetwe virus patient was most similar to those of patients with a CNS infection .For CSF, an insufficient availability of sample material of reference cases restricted similar comparisons of biomarker profiles, as described above for plasma. Therefore, the individual inflammatory biomarker concentrations were compared to reference ranges  7]. Ele. Ele7]. Ntwetwe virus is a novel orthobunyavirus identified in the CSF of a Ugandan child with a suspected CNS infection for which, despite extensive conventional investigations, no known pathogen could be identified. Partial sequence information from all  Ntwetwe virus segments was obtained, and showed considerable genetic distance to currently known viruses. According to the International Committee on Taxonomy of Viruses, the amino acid sequences of the nucleocapsid proteins differ by more than 10% for distinct orhobunyavirus species . The NtwThe clinical presentation and host response of the blood and CNS comparments of the described patient were highly suggestive of a viral encephalitis. The decorticate posturing and autonomic dysregulation were likely suggestive of a severe brain impairment that may have been caused by brainstem herniation or cerebral haemorrhaging, which both are known to occur during severe forms of encephalitis . AlternaAnopheles mosquitoes [Anopheles mosquitoes are highly prevalent in Uganda [Thus far, the patient described here is the only patient identified with a Ntwetwe virus infection. The prevalence of Ntwetwe virus infection, therefore, currently remains unknown. Phylogenetically, Ntwetwe virus clusters with the Anopheles-Tataguine clade, of which all viruses are transmitted by squitoes , making n Uganda , and mayn Uganda . Westernn Uganda . Besidesn Uganda , increasn Uganda . ConsequClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table 1Click here for additional data file.Supplementary Table 2Click here for additional data file.Supplementary Table 3Click here for additional data file.Supplementary Table 4Click here for additional data file.Supplementary Table 5Click here for additional data file.Supplementary Table 6Click here for additional data file.Supplementary Table 7Click here for additional data file.Supplementary MethodsClick here for additional data file.Supplementary ResultsClick here for additional data file.Supplementary Figure 1Click here for additional data file.


The authors are testing the hypothesis that hybridization is more common between species that are sufficiently genetically similar so that they can cross, but also genetically distinct enough to prevent homoeologous chromosome pairing and multivalent formation during meiosis in offspring, which can lead to sterility or reduced fertility in the gametes  were employed for detection of hybridization. To provide a time frame for the evolutionary events, a molecular clock was calibrated using *Beast (Heled & Drummond, Artemisia.To assess genomic divergence and traces of hybridization, Leucanthemum are clearly more divergent among each other than those in Rhodanthemum. The data also showed that the diploid species of Leucantheumum carried more genomic signatures of past interspecific hybridization events than did those of Rhodanthemum. The importance of genetic divergence as a stimulus for hybridization had been proposed by Darlington (et al., et al., The data revealed that the diploid species of rlington , and it Leucanthemum hybridize so frequently? A map of present distribution of the genus reveals localization in many of the previous refugial areas in Europe during the Pleistocene. As Rhodanthemum southward in the Atlas mountains of Morocco would have been much less affected by Pleistocene climatic changes, and this, plus the lower genetic divergence among species, may explain the absence of polyploids in this lineage.The remaining question was why did the species of The important conclusion by