Dengue and chikungunya virus diseases are becoming an increasingly important global health threats and are continuously expanding their geographical range. The study aims to investigate knowledge and diagnostic practice of dengue and chikungunya fever among healthcare workers in Moshi Municipality.Most of healthcare workers heard of chikungunya and dengue 146 (71.2%) and 203 (99%) respectively. Ninety-five (46.3%) and 152 (74.1%) had good knowledge regard chikungunya and dengue respectively. One hundred and twenty-two of HCWs 122 (59.5%) reported that there is no vaccination for dengue virus. Most HCWs 199 (97.0%) reported that the absence of diagnostic tool for dengue virus lead to difficult in managing the infection. The finding of this study showed that there is insufficient knowledge regarding chikungunya while knowledge regarding dengue is relatively fair. This calls for training regarding these infections.The online version of this article (10.1186/s13104-019-4074-x) contains supplementary material, which is available to authorized users. Togaviridae and genus Alphavirus and dengue fever is caused by the virus of the genus Flavivirus the family Flaviviridae. Both infections are transmitted between human by the bite of infected Aedes aegypti and to a less extent Aedes albopictus [Chikungunya and dengue are currently among the important Arbovirus. Chikungunya fever is caused by the chikungunya virus of the family bopictus –3. Dengubopictus . It is ebopictus , with prbopictus . Notwithbopictus , with 2 bopictus .Given the time when dengue-like illness were detected, one would expect adequate knowledge on diagnostic practices of this disease among health care workers (HCWs). However, studies indicate that understanding and diagnosing dengue vary considerably. For example, a study done in government and private hospitals as well as clinics in Karachi indicated that 100% of the doctors were aware about dengue viral infection but lack knowledge about its diagnosis (72%) and management, while 50% of doctors wanted to isolate the patient . AnotherAlthough dengue and chikungunya are reported to prevail in Tanzania –14, chalTanzania has seen a surge of dengue infections since 2010, therefore, there is increasing need for skilled HCWs in managing dengue. Understanding how HCWs manage suspected cases of dengue is crucial to improving patient outcomes. HCWs who interact directly with patients have an important role in both treating and preventing the spread of dengue . TherefoA descriptive cross sectional was conducted in Moshi Municipality in Kilimanjaro region from March to May 2017. Moshi municipality is one of the districts in Kilimanjaro region located at the lower slopes of Mount Kilimanjaro in Northern part of Tanzania. There are 56 health facilities in Moshi Municipal. A total of 15-health facilities both public and private were randomly selected. Public health facilities are Bondeni dispensary, Chuo Cha Polisi health centre, Majengo health centre, Njoro dispensary, Shirimatunda dispensary, Pasua Health centre, Rau dispensary and Mawenzi hospital. Private health facilities are Mary land health centre, Moshi-Arusha hospital, Shanti Town Dispensary, St Joseph hospital, Upendo Health centre, YMCA dispensary and CRCT Kilimanjaro First Health.The sampling technique for this study was non-probability convenience sampling. In this technique the HCWs that were present in particular health facility at the time of data collection were interviewed after consultation with the head of heath facility.Face to face interviews were conducted using structured questionnaires with questions specifically designed for HCWs. Interviews were conducted in English and Swahili language.The study instrument was developed following an extensive review of the literature. The questionnaire was pretested among two separate groups of 5 residents in Kilimanjaro Christian Medical University College and nurses who were excluded from the main study. Inputs from the residents were then used to refine the questionnaire. The questionnaire covered the following areas: (1) demographic information , (2) health information relating to whether the respondent had heard chikungunya and dengue disease or not, (3) knowledge about chikungunya and dengue symptoms, signs, and transmission modes, (4) diagnosis and treatment of chikungunya and dengue.Knowledge of dengue and chikungunya was quantified using knowledge score as described by Itral et al. and Al-zurfi et al. with few modifications , 23. Fort test. Univariate analysis was performed by logistic regression. A significance level of ≤ 0.05 was used throughout.Data were analysed using Statistical Package for Social Sciences 20.0 software . Descriptive statistics are presented as proportions for categorical data. Mean knowledge score was calculated using student A total of 205 HCWs were included in the study. Half of the HCWs were from the health centres (105/205: 51.2%), while majority  were male. A hundred and five (51.2%) were aged between 31 and 40 years, (82/205:40.0%) were nurses. More than half of HCWs (112/205: 54.6%) had a work experience of 5 to 10 years.Aedes mosquito and  knew that the infection is transmitted through Culex mosquito. Few participants , knew that the mosquito which chikungunya virus bite during the day time. A number of the HCWs correctly reported that chikungunya virus infections are transmitted by the same vector as yellow fever virus,  while  correctly answered that chikungunya virus infections are transmitted by the same vector as Zika virus. About half of HCWs knew that the drug that is used to treat patients infected with chikungunya virus is Anti-pain  Table Of 205 HCWs, majority  had heard chikungunya fever virus, and among these, only  heard through other people (such as friends and neighbours). Few HCWs  reported that chikungunya is caused by virus while others  wrongly reported that chikungunya is caused by parasites. Seventy-six (37.1%) of the HCWs answered that the main symptom of chikungunya is muscle pain and almost all HCWs  reported that not all mosquitoes transmit chikungunya. Eighty-five (41.8%) reported the vector that transmitting this virus is Aedes mosquito transmit dengue while  wrongly reported that the infection is transmitted through Culex mosquito. Few HCWs , knew that the mosquito which transmit dengue virus bite during the day time. A number of the HCWs correctly reported that dengue virus infections are transmitted by the same vector as yellow fever virus,  while  correctly answered that dengue virus infections are transmitted by the same vector as Zika virus. More than half of HCWs knew that the drug that is used to treat patients infected with dengue virus is Anti-pain  Table Almost all HCWs  heard dengue fever, among these  heard through media. Half of HCWs  correctly reported that dengue fever is caused by virus while  wrongly reported that dengue fever is caused by parasites. One hundred and sixteen (56.6%) of the HCWs answered that the main symptom of dengue fever is muscle pain and almost all HCWs  reported that not all mosquitoes transmit dengue virus. More than half  correctly reported that Among the HCWs, few participants  had good knowledge with regarding to chikungunya fever (knowledge score of 40 and above) with the mean knowledge score of (Mean ± SD) 31.7 ± 18.9. Also 152 (74.1%) had good dengue knowledge score with the mean knowledge score of (Mean ± SD) 43.6 ± 13.2 Fig. HCWs were asked several questions concerning diagnostic practices of chikungunya and dengue in the health facilities. Most of HCWs  didn’t know if there is vaccine for chikungunya virus while (77/205 37.6%) reported absence of vaccine for chikungunya. Two (1.0%) reported that there diagnostic tools for chikungunya and almost all HCWs  reported that the absence of diagnostic tools for chikungunya virus lead to difficulties in managing the infection (Table One hundred and twenty-two of HCWs (59.5%) reported that there is no vaccine for dengue virus. One hundred and five (51.3%) reported that there are diagnostic tools for dengue and almost all HCWs 199/205; 97.0%) reported that the absence of diagnostic tools for dengue virus lead to difficulties in managing the infection (Table 05; 97.0%The results of univariate analysis of the selected predictors and chikungunya or dengue knowledge score are shown in Additional file This study aimed to assess knowledge and diagnostic practices regarding dengue and chikungunya among HCWs in Moshi Municipality. This study identified knowledge gaps among HCWs that should be targeted to improve the HCWs ability to practice and manage dengue and chikungunya infections.For control and management of diseases a good knowledge about aetiology, mode of transmission, management and control is needed. This study found that the good knowledge regarding chikungunya was 46.3%. This level of knowledge was not satisfactory among HCWs. HCWs are expected to be knowledgeable about diseases as this knowledge could have been obtained in medical schools. Findings of this study was higher as compared with the study conducted in other District but the same Kilimanjaro region, which reported a overall knowledge to be 2.4% . SlightlIt is important to note that while majority of the HCWs were aware of dengue, a proportion of the respondents hold wrong notions about the cause, symptoms, transmissions and treatment of dengue. The HCWs didn’t know the treatments of the infections as most of them reported the treatments of infections as antiviral or antimalarial drugs. In a study of hospitalised febrile patients in northern Tanzania, most febrile illnesses like dengue and chikungunya were treated as malaria and some cases with antibacterial .There is insufficient knowledge regarding chikungunya fever among HCWs in Moshi Municipality. In view of this result, government and other non-government organizations should revisit the medical school curriculum especially in areas where vector borne diseases are prevalent. Refresher training of HCWs should be conducted regularly to improve their technical skills and build their capability.Our results have some limitations. It is possible that some respondents might have provided desirable responses to some questions, since the study was a face to face interview. The study included a number of HCWs from Moshi Municipality, Small sample size which is limited only to HCWs from Moshi Municipality, hence finding are not nationally representative and cannot be generalized across the country.Additional file 1: Table S1. Factors associated with good or poor knowledge regarding chikungunya and dengue infection.


We introduce and study the computational power of Oritatami, a theoretical model that explores greedy molecular folding, whereby a molecular strand begins to fold before its production is complete. This model is inspired by our recent experimental work demonstrating the construction of shapes at the nanoscale from RNA, where strands of RNA fold into programmable shapes during their transcription from an engineered sequence of synthetic DNA. In the model of Oritatami, we explore the process of folding a single-strand bit by bit in such a way that the final fold emerges as a space-time diagram of computation. One major requirement in order to compute within this model is the ability to program a single sequence to fold into different shapes dependent on the state of the surrounding inputs. Another challenge is to embed all of the computing components within a contiguous strand, and in such a way that different fold patterns of the same strand perform different functions of computation. Here, we introduce general design techniques to solve these challenges in the Oritatami model. Our main result in this direction is the demonstration of a periodic Oritatami system that folds upon itself algorithmically into a prescribed set of shapes, depending on its current local environment, and whose final folding displays the sequence of binary integers from 0 to  Understanding molecular folding will not only shed light on the origin and functions of the molecules existing in nature, it will also enable us to control the process more finely, and engineer artificial molecules with a wide range of uses, from performing missing functions inside living organisms, to producing precisely targeted drugs.The process by which one-dimensional sequences of nucleotides or amino-acids acquire their complex three-dimensional geometries, which are key to their program molecular self assembly in a computer science way.Biomolecular nano-engineering includes DNA self-assembly, which gave rise to an impressive number of successful experimental realizations, from arbitrary 2D shapes  to molecseed, kinetic tile assembly [Since then, many models have been designed to refine different features of experiments: hierarchical self-assembly ,7, modelassembly ,8, 3D anassembly , among oannealing: Namely, by heating the molecules up to high temperatures in a precisely controlled environment and then cooling them down at a precisely controlled rate. This  assembly paradigm allows researchers to design DNA structures with amazing precision by taking advantage of computational techniques to optimize the thermodynamics of strand-strand interactions, see for instance [However, the potential applications of this type of DNA technology inside cells are limited by the high thermal stability of the DNA duplex, where consequently DNA nanostructures are typically formed through the process of instance .linearly and expressed regardless of the environment. This  contrasts with more classical programming models such as Turing machines, or even tile assembly, which are able to jump to different parts of the program depending on the input. Another important difficulty is that even predicting the final folded shape of a biopolymer given the sequence is still the center of active research, especially for proteins [However at the same time the method requires precise control over temperature and other variables that are not always possible to control in every environment, such as for example within living cells. Other biopolymers such as RNA and proteins do not share this limitation, as nature uses both mediums to produce exquisite structures on a continuous basis. Researchers have successfully assembled functional nanoparticles and scaffolds out of these alternatives ,12,13,14proteins ,17,18,19hydrophobic-hydrophilic (HP) model [configuration) of a sequence is NP-complete [In particular, a large body of computer science literature is focused on energy optimization, one of the main drivers of folding. For example, in different variants of the P) model , it has complete ,24,25,26kinetics of folding, which is the step-by-step dynamics of the reaction, has been demonstrated by biochemists to play a major role in the final shape of molecules [prevalent role at the heart of the mechanism of RNA switches in biology [control this mechanism to engineer various things out of RNA represented by the RNA origami architecture for single-stranded rectangular tiles [The olecules , and eve biology . In recear tiles , and folar tiles  and RNA-ar tiles .co-transcriptional nature of RNA folding, whereby molecules fold concurrently while being transcribed . In oritatami, only the latest produced beads of the molecules are allowed to move in order to adopt a more favorable configuration. The folding is driven by the respective attraction between the beads.In our model, called Oritatami, we consider a sequence of “beads”, which are abstract basic components, standing for nucleotides or even sequences of nucleotides  to design a single sequence that folds into the target shapes in the given environments.As Winfree’s thesis  on the vThe oritatami system has received considerable attention since its proposal in the conference abstract of this paper . As mentThe FPT algorithm (Theorem 3) has brought about useful modules not only for the binary counter but also for subsequent oritatami systems including the above-mentioned Heighway dragon fractal assembler, Satisfiability (SAT) tautology checker , and theOther variants of the rule design problem have been investigated by ,44. An aGiven two words B of bead types, using an attraction rule , on the triangular lattice graph Oritatami is about the folding of finite sequences of beads, each from a finite set configurationc of a sequence w in w. We denote by i-th bead of c. A partial configuration of a sequence w is a configuration of a prefix of w. For any partial configuration c of some sequence w, an elongation of c by k beads is a partial configuration of w of length w (the index w will be omitted when the context is clear). We denote by k beads of a partial configuration c of a sequence w and by c.A oritatami systemtranscriptp, which is a sequence of beads, of a type chosen from a finite set B, (2) an attraction rule, which is a symmetric relation delay time.An  and a configuration c of a sequence w, we say that there is a bond between two adjacent positions c in number of bonds in a configuration c of w, written c: formally, Given an attraction rule dynamics for a sequence w is a function S of partial configurations of length ℓ of w, S : the First Half-AdderModule B : the Left-Turn moduleModule C : the Second Half-AdderModule D : the Right-Turn moduleModule The molecule is semantically divided into 4 parts, called Encoding. The current value of the counter is encoded in standard binary with the most significant bit to the left. Each bit is encoded into a specific folding of the modules A and C of the molecule in the rows corresponding to a zag pass: namely folding A0 and C0 for 0, and A1 and C1 for 1. During the zig pass, the value of the carry is encoded by the position of the molecule when it starts to fold Module A or C: A or C starts to fold in the top row; A or C starts to fold from the bottom row.In the zig pass (←), modules A and C “read” from the row above the value encoded into the folding in the row above during the previous zag-phase (or in the seed configuration for the first zig pass), and fold into a shape  output by the preceding module A or C to the next.B to adopt the Left-turn shape which reverses the folding direction and starts the next zag pass.When the zig pass reaches the leftmost part of the row on top, the beads there forces the module In the zag pass (→), modules A and C “read” the bricks above ACAy or Cy where B and D fold into the same brick B2 or D2 in this pass.D to fold into the Right-turn brick which reverses the folding direction and starts the next zig pass.When the molecule reaches the rightmost part of the row on top of it, the special beads there force the module Let’s run the first passes of the 3 bits counter to get acquainted with the process.The seed configuration is shown in B, encodes a sequence that will trigger the carriage return at the end of the next zig pass.The first part k repetitions of the same sequence of 4 patterns, plus an extra repetition of the first pattern at the end . Each zig pass starts with a carry equal to 1, i.e., starts folding from the bottom row. In the first zig pass, the first module A ; and C00 and A00 meaning that the (input) carry is 0 and the bit read from the seed is 0.Then, as illustrated in B, of the zig pass binds to the 3-beads long carriage-return pattern at the leftmost part of the seed and folds into the shape BT conducing the molecule to go down by 6 rows, reverse direction and start the following zag pass. Note that the bottom of the shape BT contains the exact same carriage-return pattern.Finally, as illustrated in The first zag pass (→). The zag pass is mostly a normalization pass as illustrated in A00 and A11 as C0, C00 and C11 as A0, A10 and A01 as C1, and C10 and C10 as A0. Shapes A0 and C0 encode 0, and Shapes A1 and C1 encode 1, both to be read during the upcoming zig pass. Modules B and D just fold into the shapes B2 and D2 respectively, encoding nothing but padding.D, of the zag pass binds to the 3-beads long carriage-return pattern in the rightmost part of the seed and folds into the shape DT conducing the molecule to go down by 6 rows, reverse direction and start the next zig pass. Note that, as for the shape BT, the bottom of the shape DT contains the exact same carriage-return pattern.Finally, as illustrated in A11 in the second zig pass. A11 corresponds to reading a 1 with a carry 1 which propagates the carry: indeed, the folding ends at the bottom row which propagates the carry to the next module C which folds into C01 as it reads a 0 from above with carry 1. Note that shape A11 is then rewritten as C0 in the following zag pass below.A, B, C or D implements various “functions” that are “called” when the molecule is folded. Which function is called depends on two things:the current “state” of the molecule:state is whether the carry is 0 or 1. As mentionned earlier this is encoded in the position of the molecule when the module starts to fold: it starts in the top row if the carry is 0; in the bottom row if the carry is 1.here, the the local environment of the molecule:environment, i.e., the beads already placed around the current area where the folding takes place, acts as the memory in the computation.the Each module function of the module is called; the function then reads the input encoded by the beads already placed in the environment.The position where the folding of a module starts, determines which beads of a given module will be exposed to and interact with the environment. Then, by creating bonds (or not) with the environment, each module will adopt a specific shape. Therefore, the possible binding schemes will be different depending on this initial position. Similarly, depending on the beads already placed in the environment, the part of the module exposed to it will adopt one form or another depending on how many bonds it can create with the environment. Adopting the language of computer science: the position at which a module starts to fold, determines which C are implemented in the zig pass. As the zig pass goes from right to left, the figure is meant to be read from right to left. In the zig pass, Module C implements two functions: (1) Add 1 to the bit above and propagate the carry if needed, or (2) Copy the bit above unchanged. Add is called if the carry is 1 at the beginning of the folding and Copy is called if the carry is 0. The following step-by-step description of the folding explains how:Beads 30–33  if the bit read above is 0; or the beginning of a “switchback” pattern  if the bit read is 1.Bead 34 is attracted by beads 9 and 10 encoding a bit 0 above which allows beads 36 and 37 to bind with 31 as well, but prefers to bind with 31 together with 35 otherwise. This induces two different shapes: the beginning of a “wave” pattern :in these three cases the folding of the beads 38–41 starts from the same position. As the environment is different for each of them, we could design the rule so that this part of the module prefers to adopt the same shape, climing along the part already folded to the top row to start the next module with a carry Beads 38-41, with carry propagation (carry = 1 and bit read = 1):because the switchback pattern is upside down in this case, bead 37 stays low and bead 38 can firmly attach to the top with beads 5, 6 and 33, and the tip of the module folds downwards as 40 and 41 are attracted by 37. This ensures that the folding of the module ends at the bottom row, propagating the carry C01, 39–38–33–32 for C00, 39–38–37–36 for C10, and 41–36–35–30 for C11. This will allow to distinguish between them in the following zag pass to write the correct bit for the new value of the counter.Note that the bottom rows of the four resulting foldings differ significantly: 39–38–37–30 for A (beads 0–11): the First Half-AdderModule B (beads 12–29): the Left-Turn moduleModule C (beads 30–41): the Second Half-AdderModule D (beads 42–59): the Right-Turn moduleModule As mentioned earlier, each 60-period of the molecule is semantically divided into four modules:bricks the various configurations each module will adopt in the final folding, as they are the bricks upon which the whole folding is built.Depending on the environment in which they fold upon themselves, each of these modules may adopt the following different configurations. We call A and C:ACAx or Cx) and the (input) carry from the preceding module (Bc or Dc);Ay and Cy in the zag pass where y is the bit written: namely ACThere are six bricks for Modules B and D:BDCAB2 and D2 in the zag pass.and There are four bricks for Modules y, x, c and folding certificate, which shows the correctness of each step of the folding in each possible environment. It consists in displaying in a compact but readable way, all the possible extensions of the current configuration, and displaying for each of them the number of bonds created (the number in the north-east corner); the maximum bonding configurations are displayed in bold. For readability, we group together extensions when they share a common path up to their last bond (the number of paths grouped is then displayed in the south-east corner for cross-checking). The resulting enumeration is displayed as a tree where only the maximum bond configuration (in bold) gives birth to configurations at the next level. Following the bold path in the tree certificate shows the folding adopted by the molecule. See A01 in the surrounding consisting of the bricks B2, C0, D2 and D1, and for Brick A11 in the surrounding consisting of the bricks B2, C1, D2 and D1. A reading a 0 from above (Brick C0) and a carry from the right (Brick D1); whereas A reading a 1 from above (Brick C1) and a carry from the right (Brick D1). Note that the output configuration is exiting the region from the top for Brick A01 (no carry is propagated) and from the bottom for Brick A11 (a carry is propagated).The proof works in three stages: (1) we describe the targeted final folding and show that this target folding implements correctly a binary counter: i.e., that the bricks implement correctly the relationships between the In the next subsections, we use our new tools to prove the correctness of the Oritatami System presented in A, First Half-Adder (beads 0–11):Module B, Left-Turn module (beads 12–29)Module C, Second Half-Adder (beads 30–41)Module D, Right-Turn module (beads 42–59)Module Let us first describe each of the possible bricks for each module:A and C in their folding. Using two similar but different modules A and C allow to avoid interference and simplify the design.Note the similarities between the two half-adders The final configuration. In order to program the molecule, we have partitioned the triangular grid into regions that will be populated each by a module adopting one of the brick configurations above. The regions and the bricks populating them are displayed in -The leftmost column consists in -The rightmost column consists in The rightmost and leftmost columns have width 3:Zig- and Zag-rows to be read from right to left and from left to right, respectively. The rows i in binary from the row jth lowest weight bits of i: i: pth inner row, qth inner column, -*r is even, and r is odd;*if -r is even, or D2 if r is odd.if -r is odd and Cr is even.if -*r is even, and r is odd;*if The The seed row on top consists in the bottom row of the brick sequence BT,C0,.output nascent configurations of the previous time step, or the input nascent configurations of the present time step. In this section, we list all the possible input/output nascent configurations according to our design.Recall that the inertial dynamics extends only the favored nascent configurations from the previous time step . We call this set of configurations, the output nascent configurations for all bricks as illustrated in d is not fixed.The lemmas in Proof of Correctness. The proof of Theorem 1 proceeds by induction: it is enough to show that each module folds into the expected brick in each region. The folding of each module depends on its environment, i.e., on the bricks already folded nearby and on the current minimum energy configurations output by the previous step. First, Our second main result concerns the design of a rule for achieving a set of given foldings depending on the environment.The rule design problem (RDP) consists in the following:Input: disjoint sets of bead types B and k seed configurations k target configurations n, and a dynamics Two Output: , A rule We begin by showing that the rule design problem is NP-complete:Theorem 4.For any positive delay and transcript length, the rule design problem is NP-complete.Proof. q variables m 3-clauses p folds into We reduce from 3-SAT with p. This reduces to the delay 1 case. □Finally, extending this proof to a larger delay time k) and not on the length of the transcript (n). As Theorem 3 will show next, the problem is indeed linear-time in the length n of the transcript when the number of desired configuration is a constant, and exponential-time only in k and fixed parameter tractable (FPT) as it can be solved in polynomial time when some parameters  with k seed-target configurations, each of length n, and delay time δ is fixed-parameter tractable, as it can be solved in time and space complexity The bounds given on the memory use seem to indicate that the algorithm might be impractical even if its time complexity is linear in the length of the transcript. However, we implemented lazily  and then, its memory usage remains modest because the number of output nascent configurations remains small. We have used this procedure successfully to solve key parts of our design. Indeed, note that in our design, there are only very few input configurations (most of the time just one) which makes the potentially heavy extension step very fast in practice, at least for this design.Algorithm 1 Rule Design Problem FPT Algorithm—Oblivious dynamics1:functionExtendOblivious2:This procedure computes all the possible extensions of rule R s.t. bead     ▹3:    Let 4:p in any of the     Let 5:for all partial rule do    6:        Let 7:try        8:fordo           9:               let 10:               let 11:ifthen               12:throwrejectRule     ▹ The rule                    13:           14:catchrejectRule: continue                         ▹ Do not add         15:return    16:functionProjectOblivious)17:This procedure retains only one representative partial rule for every possible subrule of the last     ▹18:    Let 19:i-prefixes of the target configurations     Let 20:for all partial rule do    21:R in         Let 22:ifthen        23:           Pick one rule 24:return    25:functionFindRuleOblivious26:k seed configurations     Let 27:    Initialize 28:fordo               ▹ Extend the rule to place correctly the bead     29:        Let 30:for all partial rule do        31:ExtendOblivious           32:ProjectOblivious2:This procedure computes all the possible extensions of rule R s.t. bead     ▹3:    Let 4:p in any of the 1-elongations of the favored nascent configurations in     Let 5:for all partial rule do    6:        Let 7:try        8:fordo           9:               let 10:ifthen               11:throwrejectRule     ▹ The rule                    12:           13:catchrejectRule: continue        ▹ Do not add         14:return    15:functionProjectInertial16:This procedure retains only one representative partial rule for every possible subrule and resulting favored nascent configurations of the last     ▹17:    Let 18:    Let 19:for all partial rule and all subsets do    20:R in         Let 21:ifthen        22:           Pick one rule 23:return    24:functionFindRuleInertial25:k seed configurations     Let 26:    Initialize 27:fordo    ▹ Extend the rule to place correctly the bead     28:        Let 29:for all tuple do        30:ExtendInertial           31:ProjectInertial or not , or if this is undefined (⊥). We denote by domain of R. Two subrules compatible, denoted by matches withmergingFormally, a i of a configuration c. Algorithm 1 solves RDP for the oblivious dynamics in time linear in the length of the sequence as follows. It incrementally constructs a set of rules that place each bead at its desired position in each of the k environments. It proceeds by maintaining a set i beads in every of the k environments. At the i-th step, the main procedure FindRuleOblivious first extends each candidate subrule R in ExtendOblivious which scans all the possible attraction rule extensions of R for the new nascent bead (the k configurations, and retains only the ones that place the i-th bead at its correct position for all k configurations. Note that this extension of the rule does not change the positioning of the FindRuleOblivious calls procedure ProjectOblivious which retains only one representative of each subset of rules that define the same attractions for the For all Algorithm 2 works similarly for the inertial dynamics A variant of the rule design problem was studied by Ota and Seki  with oneThe uniqueness of the bead types used in the transcript is thus crucial for the FPT algorithm to exist. Thus, it is not clear whether their variant admits an FPT algorithm or not. Note that other algorithms were proposed, such as the heuristic rule set optimization algorithm by Han and Kim .kind of problem that need to be solved in order to engineer RNA shapes, and later, even proteins.The purpose of our new model is not to be entirely accurate with respect to phenomena observed in nature, but instead to start developing an intuition about the thermodynamics and molecular agitation. Using existing works in molecular dynamics [In the future, a number of extensions of this model seem natural. In particular, extending it with a more realistic notion of dynamics , would adynamics  for the


Drosophila data with a comprehensive search of transcriptomic sequences and selected meta-transcriptomic datasets to identify a completely new lineage of segmented positive-sense single-stranded RNA viruses that we provisionally refer to as the Quenyaviruses. Each of the five segments contains a single open reading frame, with most encoding proteins showing no detectable similarity to characterised viruses, and one sharing a small number of residues with the RNA-dependent RNA polymerases of single- and double-stranded RNA viruses. Using these sequences, we identify close relatives in approximately 20 arthropods, including insects, crustaceans, spiders, and a myriapod. Using a more conserved sequence from the putative polymerase, we further identify relatives in meta-transcriptomic datasets from gut, gill, and lung tissues of vertebrates, reflecting infections of vertebrates or of their associated parasites. Our data illustrate the utility of small RNAs to detect viruses with limited sequence conservation, and provide robust evidence for a new deeply divergent and phylogenetically distinct RNA virus lineage.Metagenomic sequencing has revolutionised our knowledge of virus diversity, with new virus sequences being reported faster than ever before. However, virus discovery from metagenomic sequencing usually depends on detectable homology: without a sufficiently close relative, so-called ‘dark’ virus sequences remain unrecognisable. An alternative approach is to use virus-identification methods that do not depend on detecting homology, such as virus recognition by host antiviral immunity. For example, virus-derived small RNAs have previously been used to propose ‘dark’ virus sequences associated with the Drosophilidae (Diptera). Here, we combine published  Pioneered by studies of oceanic phage , since tShi et al. 2018aShi et al. 20Despite the successes of metagenomic virus discovery, there are clear limitations to the approach. First, ‘virus-like sequences’ from a putative host need not equate to an active viral infection of that species. They may represent integrations into the host genome, infections of cellular parasites or other microbiota, infections of gut contents, or simply contaminating nucleic acid , which has since been shown to constitute two divergent segments of an insect-infecting Partitivirus RNA viruses. The protein encoded by segment 5 of these viruses shares a small number of conserved residues with the RNA-dependent RNA polymerases (RdRps) of Picornaviruses, Flaviviruses, Permutotetraviruses, Reoviruses, Totiviruses, and Picobirnaviruses, but is not substantially more similar or robustly supported as sister to any of these lineages—suggesting that the new lineage may warrant recognition as a new family. We find at least one homologous segment in publicly available transcriptomic data from each of forty different animal species, including multiple arthropods and a small number of vertebrates, suggesting these viruses are associated with a diverse range of animal taxa.Here, we combine data from Drosophila . In brief, about 5,000 flies were collected in 2010 from Kenya (denoted pools E and K), the USA (pool I), and the UK (pools S and T). Ribosome-depleted and double-stranded nuclease normalised libraries were sequenced using the Illumina platform, and assembled using Trinity  and inferred their protein sequences for similarity searching by translating all long open reading frames present in each contig. We used these to build a database for Diamond , and used tblastn  using blastp  as part of an experimental evolution study  and parasitoids were collected after eleven (adults) or fourteen (larvae) generations of experimental selection. Poly-A enriched cDNA libraries were constructed using the Illumina TruSeq RNA kit (adults) or the Illumina TruSeq Stranded mRNA kit (larvae). Libraries were sequenced in single-end, 100 bp cycles on an Illumina HiSeq2500 (sequence data available under NCBI PRJNA290156). Trimmed and quality filtered reads were assembled de novo using Trinity . The subsequent differential expression analysis identified several highly expressed fragments that were not present in the L. fabarum draft genome nor in transcripts from the host aphid (A. fabae), and were not identified in the whole-transcriptome annotation using blastn. Subsequent protein-level searches  revealed sequence similarity in four of the fragments to putative ‘dark’ virus sequences from Drosophila , for re-assembly with Trinity , which artificially enriches for polyadenylated RNAs such as mRNA-like expression products. We therefore sought relatives in a strand-specific meta-transcriptomic dataset that had been prepared without poly-A enrichment.Strand-specific RNA libraries can be used to identify strand-biases in viral RNA, providing a clue as to the likely genomic strand of the virus and evidence for replication. Viruses with +ssRNA genomes tend to be very strongly biased to positive-sense reads, replicating double-stranded (dsRNA) viruses are weakly biased towards positive-sense reads, and replicating negative-sense (−ssRNA) viruses are weakly biased towards negative-sense reads. This is because mRNA-like expression products of replicating viruses have an abundance approaching that of the genomic strand. Unfortunately, much RNA sequencing is strand-agnostic . Briefly, between one and twelve adults  of each of sixteen different species were collected from Penryn  and Buckfastleigh  in July and September 2017, respectively. Total RNA was extracted from each individual using Trizol-Chloroform extractions according to the manufacturer’s instructions, and a strand-specific library prepared from the combined pool using an Illumina TruSeq stranded total RNA kit treating samples with Gold rRNA removal mix. This was sequenced by the Exeter University Sequencing service using the Illumina platform. The reads were assembled  Trinity , and the Trinity .We then used an RT-PCR screen to confirm the identity of the host, and to confirm that the five putative segments co-occurred in the same individual. RNA was reverse-transcribed using GoScript reverse transcriptase (Promega) with random hexamer primers, then diluted 1:10 with nuclease free water. PCRs to amplify short regions from the five viral segments (S1–S5) were carried out with the following primers: S1F ATGCATCTCGTTCCTGACCA and S1R GCCCCTTCAGACAGCTCTAA; S2F CACCACCAAGAACGGACAAG and S2R TGCCACCACTCTAACCACAT; S3F AGCAATTCAACGACCACACC and S3R GATAGGGGACAGGGCAGATC; S4F ATGAACGAGAGGTGCCTTCA and S4R CTCCATCACCTTGACATGCG; S5F TGCACTGTTCAGCTACCTCA and S5R CCGTGTCGTTCGATGAAGTC, using a touch-down PCR cycle  30 s, 72 °C 1 min; for 10× cycles followed by; 95 °C 30 s, 52 °C 30 s, 72 °C 1 min; for a further 30× cycles). As a positive control for RT we used host Cytochrome Oxidase I amplified with LCO/HCO primers   and 41 (segment 5) protein sequences were aligned, depending on level of sequence conservation. Regions of low conservation at either end of the alignments were selected by eye and removed. However, no internal regions were trimmed, as trimming leads to bias towards the guide tree and gives false confidence  between the new viruses and previously described virus families, we selected for phylogenetic analysis the RdRp sequences from representatives of major lineages of +ssRNA viruses. We aligned a core RdRp sequence of 215–513 residues for a total of 255 viruses, using two different methods; T-coffee ‘Expresso’ , which uDrosophila to be associated into complete virus genomes. Using similarity searches we initially identified six fragments from Lithobius forficatus . Motivated by this discovery of four homologous sequence groups across these taxa, we performed a new search of the Drosophila-associated sequences formed two groups (four sequences from drosophilid pool E and four from drosophilid pool I) encoding proteins that ranged between 40 and 60 per cent amino acid identity . However, as this dataset contained a large number of reads from the Jasmine whitefly Dialeurodes kirkaldyi, we think it unlikely that the plant is the true host.Subsequent searches later identified homologues in fourteen other arthropod transcriptomes, including six from Hymenoptera, five from Hemiptera, two from Coleoptera, and one each from Lepidoptera and Odonata . We alsoDrosophila, all segments were sources of 21 nt small RNAs from along the length of both strands of the virus, demonstrating that the virus is recognised as a double-stranded target by Dicer-2  in JRR Tolkien’s invented language Quenya  and submitted the sequences to GenBank under accession numbers MN264686-MN264690.Based on the high abundance and the clear pattern of co-occurrence, we searched for other wasp-associated contigs displaying the same properties, reasoning that these were likely to be additional segments of the same virus. This search identified a candidate fifth segment of about 2 kbp, again containing a single open reading frame . We thenNetë is Quenya for another one, one more) and submitted them to GenBank under accession numbers MN264681–MN264685.To determine whether these virus genomes are likely to be dsRNA, +ssRNA, or −ssRNA, we identified a related virus in a strand-specific meta-transcriptomic dataset that had been prepared without poly-A enrichment from several species of Lepidoptera . All five segments were detected , and as Ceraphron sp. transcriptome. However, we were unable to identify any 5′ pattern or motif shared among the segments.Overall, this virus accounted for 3 per cent of the reads in the metagenomic pool, giving around 10,000-fold coverage of the genome . An analCrocallis elinguaria individual , collected at latitude 50.169, longitude −5.125 on 23 July 2017. RT-negative PCR showed that viral segments were not present in a DNA form.An RT-PCR survey of the individual moth RNA extractions used to create the metagenomic pool showed that all five segments co-occur in a single Bufo gargarizans) and a lizard . Five of these pools also contained homologues of segment 1 (the second most conserved protein), and one also contained segment 4 (the third most conserved protein). These sequences have been submitted to GenBank under accession identifiers MN371231–MN371254; see After identifying the complete (five segments) virus genomes in transcriptomic datasets from 12 different arthropods, and incomplete genomes (between one and four segments) in a further fifteen arthropod datasets , we soug20L. fabarum sample, to 0.16 RPKM (six read-pairs) for Zhanggezhuang virus from a metagenomic pool of Branchiopoda, with a median of 16.9 RPKM (Lysiphlebus) as true hosts, but does not support or refute that the virus may infect vertebrates . Second, for two high-coverage low species-complexity vertebrate metagenomic pools (the B. gargarizans lung sample and C. versicolor gut sample) we searched raw assemblies for Cytochrome Oxidase I sequences of contaminating invertebrates. This found that <0.5 per cent of the RNA from B. gargarizans (Dongxihu virus RPKM of 94.6) and <0.01 per cent of RNA from C. versicolor (Bawangfen virus RPKM of 338.5) derived from contaminating invertebrates, strengthening the possibility that the vertebrate is the true host. Third, for segment 5 (which was available for most taxa) we examined the deviation in dinucleotide composition from that expected on the basis of the base composition, as this is reported to be predictive of host lineages (The finding that these virus sequences can be associated with both vertebrates and invertebrates may indicate that they are broadly distributed across the metazoa (note the only non-metazoan associated sequence came from a plant transcriptome contaminated with insects). However, metagenomic data alone cannot confirm this, as such datasets can include contamination from gut contents or parasites of the supposed host taxon. We therefore explored four sources of evidence that could be used to corroborate the targeted taxon as the true host. First, we examined the viral read abundance, as very high abundance is unlikely for viruses of contaminating organisms. Abundance ranged from over 37,124 Reads Per Kilobase per Million reads  for Sina virus in one 6.9 RPKM . This stlineages . HoweverFinally, we also analysed the phylogenetic relationships for all of the segments, as (except for vectored viruses) transitions between vertebrate and invertebrate hosts are generally rare . This shE-value > 1), and only a weakly-supported 110 amino acid region of the segment 1 alignment with similarity to methyltransferase/mRNA capping enzymes . However, in contrast to searches using blastp, the alignment of segment 5 displayed a more strongly-supported 300 amino acid region with similarity to the RdRp of Norwalk virus . This sequence was approximately equally matched to around twenty-five different reference structure or motifs, including RdRps from both +ssRNA viruses such as Picornavirales, Flavi-like viruses, and Permutotetraviruses, and dsRNA viruses such as Reoviruses, Picobirnaviruses, and Totiviruses. Notably, this region of similarity included a very highly conserved GDD motif that is shared by many viral polymerases, supporting the idea that segment 5 encodes the viral polymerase. Raw HHpred output is provided via doi:10.6084/m9.figshare.11341982.Having identified 1:1 homologues in multiple datasets, we were able to use the aligned protein sequences to perform a more sensitive homology search for conserved protein motifs using HHpred . This stThe new virus lineage described here has a distinctive genome structure comprising four 1.6 kbp +ssRNA segments each encoding a single protein of unknown function, and one 2 kbp +ssRNA segment encoding an RdRp. The putative RdRp is substantially divergent from those of characterised +ssRNA and dsRNA virus families, to the extent that similarity cannot be detected using routine blastp. On this basis we propose the informal name ‘Quenyaviruses’, reflecting the naming of the four founding members, and suggest that they may warrant consideration as a new unplaced family.To explore their relationships with other RNA viruses using an explicit phylogenetic analysis, we selected a region of 215-513 amino acid residues of the core RdRp region from 11 representative Quenyaviruses and 244 other +ssRNA viruses, representing most major lineages. We excluded birnaviruses and permutotetraviruses, which have a permuted RdRp that cannot be straightforwardly aligned . PhylogeAll methods found the Quenyavirus RdRps to form a monophyletic clade, supporting their treatment as a natural group . Two of Drosophila, purely on the basis of the characteristic small RNA signature created by the host antiviral RNAi pathway transcriptomic datasets, primarily of arthropods. Four of these segments had initially been identified as ‘dark’ viruses of  pathway . Now, byDrosophila virus sequences of As with other metagenomic studies of virus diversity, this work raises two important questions. First, how well have we truly sampled the virosphere? Metagenomic studies often contain sequences lacking detectable homology, and it has been suggested that these include many ‘dark’ viruses . This mavez061_Supplementary_DataClick here for additional data file.


Sunitinib is an oral tyrosine kinase inhibitor (TKI) commonly used in the treatment of renal cell carcinoma (RCC). Among a broad range of toxicities, anemia and macrocytosis are common in patients treated with sunitinib. Correlation between sunitinib-associated macrocytosis and cobalamin deficiency has been reported in small case series and retrospective analyses, although others have not found an association. Here, we present a case of transfusion-dependent macrocytic anemia with cobalamin and folate deficiency in a patient with RCC treated with sunitinib. Sunitinib is an oral inhibitor of tyrosine kinases including vascular endothelial growth factor receptor (VEGFR), and has classically been used as a first-line treatment in advanced RCC. Due to off-target effects, the VEGFR-targeted TKIs have a number of class-specific side effects including hematologic toxicity. In a pivotal phase III trial, anemia 71%), neutropenia 72%), and thrombocytopenia 65%) were frequent events in patients treated with sunitinib for metastatic RCC [%, neutro%, and th% were frA 71-year-old male presented with metastatic clear-cell RCC. In November 2016, he was incidentally found on imaging to have a left renal mass but declined further workup and was lost to follow-up. The patient was subsequently noted on routine laboratories in September 2017 to have creatinine elevation to 1.45 mg/dL from an unknown baseline. Renal ultrasound revealed a solid left kidney mass measuring up to 10 cm. Systemic imaging with computed tomography of the chest, abdomen, and pelvis showed a 13.5 x 7.6 cm enhancing, exophytic mass of the left kidney and innumerable bilateral pulmonary nodules concerning for metastatic malignancy. Cytoreductive nephrectomy was performed in November 2017 and pathology showed clear-cell RCC with sarcomatoid features. Approximately five weeks later, he was initiated on systemic treatment with sunitinib 50 mg daily, six-week cycles with a two-weeks on, one-week off schedule.Laboratories at baseline showed hemoglobin 9.1 g/dL and MCV 88.1 fL. Imaging with computed tomography after three cycles of sunitinib showed a partial response; however, serial laboratories showed the development of worsening macrocytic anemia with hemoglobin 6.6 g/dL and MCV 106.9 fL. Further laboratory workup showed total bilirubin 2.6 mg/dL, direct bilirubin 0.2 m/dL, lactate dehydrogenase 210 U/L, and haptoglobin 27 mg/dL. Direct antiglobulin testing was negative and iron studies, thyroid function tests, and liver tests were normal. Notably, the patient was found to have significant deficiencies in cobalamin  and folate  and peripheral smear showed numerous hypersegmented neutrophils. Testing for antibodies against parietal cells and the intrinsic factor was negative. There were no prior values of cobalamin or folate for comparison. On review of history, the patient endorsed a balanced diet with adequate intake of sources of folate and cobalamin, had no history of gastrointestinal surgeries, and denied significant alcohol use. Treatment of nutritional deficiencies was initiated with folic acid one milligram by mouth daily and cobalamin intramuscular 1000 mcg for one dose followed by cobalamin one gram by mouth daily. The patient was transfused with two units of packed red blood cells. Although there was some concern that these findings represented toxicity from sunitinib, given evidence of clinical benefit the decision was made to continue treatment with close monitoring of hematologic parameters.Repeat laboratories during cycle four of sunitinib showed stable blood counts and normalization of cobalamin (813 pg/dL) and folate (>20 ng/mL) levels, however during cycle five the patient complained of worsening fatigue and exertional dyspnea, and laboratories showed worsening macrocytic anemia with hemoglobin 7.1 g/dL and MCV 108.0 fL. Cobalamin and folate levels remained normal. The patient was transfused with two units packed red blood cells and sunitinib was held.After four weeks of sunitinib, laboratories showed hemoglobin 9.2 g/dL and MCV 100.5 fL, and the patient reported a marked improvement in energy. Sunitinib was restarted at a reduced dose of 37.5 mg daily on two weeks on and one week off schedule. The patient was also started on a regimen of parenteral cobalamin  and continued on oral folate. Following one cycle, hemoglobin was relatively stable at 8.6 g/dL and MCV had improved to 93.9 fL. Labs for several cycles thereafter showed stable levels of hemoglobin, folate, and cobalamin. Current treatment plans are to continue sunitinib with close hematological monitoring and ongoing supplementation with oral folate and parenteral cobalamin.p < 0.001) at three months. Additionally, among 10 patients in the analysis who had laboratories 2-3 months after discontinuation of sunitinib, MCV decreased by a median of 9.7 pL (p = 0.002) [Macrocytosis is a well-documented side effect of sunitinib. Retrospective analyses have shown that macrocytosis develops in a large proportion of patients after approximately three months of treatment, worsens with continued treatment, and resolves with discontinuation of sunitinib . In the = 0.002) . Sunitinib-associated macrocytosis has been hypothesized to occur via off-target inhibition of the stem cell growth factor c-Kit, leading to impaired erythrocyte maturation . This cop = 0.2) [Although sunitinib-associated macrocytosis and anemia may occur secondary to the inhibition of erythrocyte maturation, there have also been, to our knowledge, at least three reports of an association with cobalamin deficiency. In 2007, two small case series were published successively in The New England Journal of Medicine. Gilleson et al. reported on six patients who developed macrocytosis after treatment with sunitinib. All patients had deficiencies in cobalamin and five had reduced levels of holotranscobalamin. Folate was normal in all patients and none developed worsening anemia or symptoms of cobalamin deficiency . Shortlyp = 0.2) .n = 12; median, 422 pg/mL; normal, 221-700) or folate  [Other reports have contradicted these findings. In a retrospective analysis of 21 patients with sunitinib-related macrocytosis, Price et al found cobalamin deficiency in only two of seven patients for whom data were available, a smaller proportion than other studies. Folate deficiency was not found in eight patients . AdditioA theoretical mechanism for sunitinib-associated cobalamin deficiency has not been clearly elucidated. In general, cobalamin deficiency can occur via reduced absorption, reduced release from stores, or increased cellular consumption . GillesoMacrocytosis and anemia are common effects in patients treated with sunitinib and likely occur via inhibition of c-Kit. Data from several small studies suggest that a subset of patients can also develop megaloblastic anemia secondary to cobalamin deficiency. The mechanism for this effect is not clear. Patients with more severe macrocytosis may be more likely to have evidence of cobalamin deficiency. In a small case series, parenteral cobalamin replacement therapy appeared to mitigate these effects. Our patient had evidence of severe, transfusion-dependent megaloblastic anemia which persisted despite oral cobalamin replacement but has improved over a short interval of parenteral replacement. While our patient also had decreased levels of folate, evidence for an association with folate deficiency is less convincing and has been seen in only one small case series. Given good evidence for cobalamin deficiency as a rare side effect of sunitinib, this should be added to materials describing the possible toxicities associated with sunitinib in order to promote physician awareness. Further studies would be useful, and clinicians should consider monitoring cobalamin levels in patients who develop macrocytosis while on treatment with sunitinib.


While one of the biggest problems we are facing today is water scarcity, enormous quantities of water are still being used in irrigation. If contaminated, this water can act as an effective pathway for the spread of disease-causing agents, like viruses. Here, we present a novel, environmentally friendly method known as cold atmospheric plasma for inactivation of viruses in water used in closed irrigation systems. We measured the plasma-mediated viral RNA degradation as well as the plasma-induced loss of viral infectivity using potato virus Y as a model virus due to its confirmed water transmissibility and economic as well as biological importance. We showed that only 1 min of plasma treatment is sufficient for successful inactivation of viruses in water samples with either high or low organic background. The plasma-mediated inactivation was efficient even at markedly higher virus concentrations than those expected in irrigation waters. Obtained results point to reactive oxygen species as the main mode of viral inactivation. Our laboratory-scale experiments confirm for the first time that plasma has an excellent potential as the eukaryotic virus inactivation tool for water sources and could thus provide a cost-effective solution for irrigation mediated plant virus transmission. The outstanding inactivation efficiency demonstrated by plasma treatments in water samples offers further expansions of its application to other water sources such as reused wastewater or contaminated drinking waters, as well as other plant, animal, and human waterborne viruses, ultimately leading to the prevention of water scarcity and numerous human, animal, and plant infections worldwide.The online version of this article (10.1007/s12560-019-09388-y) contains supplementary material, which is available to authorized users. The availability of clean water is in continued decline due to the increasing global population and food demand, along with higher standards of living and climate change WWAP . Water sNTN group are the most devastating and cause mosaic, chlorotic, and necrotic lesions on leaves as well as necrotic ringspots on tubers  light, ozonation], time consuming (slow filtration), require large infrastructure , frequent maintenance , produce undesirable side components , or need additional decontamination steps  , reactive species, UV photons, and neutral particles . Due to some of these components, with the emphasis on the reactive chemical species, CAP has great antimicrobial potential . The less complex infected water samples contained only purified PVYNTN (Online Resource 1a) diluted in nutrient solution. Ultracentrifugation-purified virus particles had lower concentration (henceforth referred to as ‘low concentration pure virus’) than chromatography-purified virus particles (henceforth referred to as ‘high concentration pure virus’). Untreated samples of infected homogenate and both types of pure virus were used as positive controls.We used two sources of viruses in nutrient solution to create an approximation to water samples of different complexities that represent those used in closed irrigation systems. Plants infected with PVYSolanum tuberosum cv. ‘Pentland Squire’) grown in vitro cultures and infected with PVYNTN. This was then mixed with 20 mL nutrient solution that consisted of tap water with added minerals   or CAP (various times).Each infected homogenate was prepared by grinding 88 ± 3 mg of the green parts  of potato plants (NTN-infected tobacco and potato tissues from plants grown in the soil were prepared by grinding them in chilled (to 4 °C) grinding buffer. PVYNTN was then purified using either a standard purification method that included saccharose and CsCl gradient ultracentrifugation (low concentration pure virus) or convective interaction media (CIM) monolithic chromatography (high concentration pure virus) , or CAP (various times). The high concentration pure virus particles stayed untreated (positive control) or underwent CAP treatments (various times).For isolation of the pure viruses, PVYThe viral RNA for all of the virus preparations was quantified prior to their treatments using reverse-transcription droplet digital PCR (RT-ddPCR). RT-ddPCR was performed with One-Step RT-ddPCR advanced kit for probes  as described by Mehle et al.  with minNTN in the infected samples  was used to observe the light emitted by the plasma during the treatments. An Avantes AvaSpec-3648 optical spectrometer with a resolution of 0.5 nm from 200 to 1100 nm was used. The integration time was set to 1 s. We measured spectra during CAP treatments of various samples: nutrient solution, low concentration pure virus, and infected homogenate. Additionally, as a control, the OES spectrum of CAP treatment without sample (CAP in the air) was recorded .We used a series of control treatments to confirm that any effect on the PVYNTN RNA from samples. RNeasy plant minikit  was used for RNA extractions from infected homogenates according to the manufacturer`s instructions, with minor modifications, namely, without using mercaptoethanol. RNA extractions from the pure virus were carried out with QIAmp Viral RNA minikit (Qiagen), according to the manufacturer’s instructions, with minor modifications, namely, luciferase RNA (2 ng/sample) was added to the carrier RNA prior to extraction as an external control and the final elution step was performed with 45 µL of RNAse-free water. Sterile water was used as negative control of the extraction to monitor for potential contaminations during all extractions.To examine the effects of different treatments on degradation of viral RNA, we first extracted the PVYNTN genes using reverse-transcription polymerase chain reaction (RT-PCR): P1 and P3 (which code for proteins involved in virus replication), NIa (which codes for a serine-like cysteine protease), and CP (which codes for the coat protein)  plants with nutrient solution (negative control), positive controls or treated samples. The inoculation process and growth conditions for the tobacco plants were as described in Mehle et al. , and confirmed viral infectivity and systemic spreading using reverse-transcription real-time (quantitative) PCR (RT-qPCR). We sampled two developed non-inoculated upper leaves 14 ± 1 days and 32 ± 1 days post-inoculation, and pooled together all of the plant material from the plants inoculated with the same sample. We then extracted the RNA with RNeasy plant minikit according to the manufacturer`s instructions, with minor modifications i.e., without using mercaptoethanol and the final RNA elution was carried out with 150 µL of RNAse-free water. After that we performed RT-qPCR using AgPath-ID One-Step RT-qPCR mix , as described by Mehle et al. . Minimum time needed for inactivation of viruses in infected homogenate was 5 min, whereas only 1 min was needed for inactivation of pure virus both in high and low concentration. We speculated that difference in inactivation times originated from the amount of organic matter present in the infected homogenate, which can absorb the plasma irradiation and as such might ‘protect’ viruses from it i.e., viruses might become less accessible to the irradiation. The most obvious proof that CAP interacts with plant organic matter was a discoloration of samples in the first few minutes of treatments (Online Resource 4). Additional cause for the difference could be varied initial amount of virus in the samples, which was determined using RT-ddPCR. Concentrations of PVYNTN in the infected homogenate ranged from 4.2 × 105 to 6 × 106 RNA copies/µL of sample, while for the low and high concentration of pure virus, average determined concentrations were 3.6 × 104 and 2.7 × 105 RNA copies/µL of sample, respectively  was achieved in 17 out of 18 CAP treatments Table . Only onNTN RNA was successfully degraded by the CAP treatments after 15 min for the infected homogenate, and after 5 min for low concentration pure virus  of the pure virus for 15 min effectively reduced the PVYNTN infectivity. However, the same treatment did not affect the PVYNTN infectivity in the infected homogenate  were observed for all CAP treatments (Online Resource 5). The presence of OH emission system and Balmer H emission line in the OES spectra proves that water vapor is dissociated in the plasma. The intensities of spectral features did not change during the treatments, regardless of the sample type. A new spectral feature, N2 emission bands, can be seen only in the OES spectrum of CAP in the air. These are present due to the diffusion of the ambient air into the plasma stream. The OH is also present in the free air CAP because of the humidity in the ambient air. However, the OH intensity is much smaller compared to the submerged CAP, thus indicating that the water from the samples is evaporated and dissociated in the plasma. We did not detect any response in the range between 200 and 300 nm, the wavelengths at which UV radiation damages nucleotides in different ways  and in the pure PVYNTN form. The inactivation was efficient even though the PVYNTN genome concentrations were significantly higher than those expected in irrigation waters Supplementary material 2 (AVI 1331 kb)Below is the link to the electronic supplementary material.


Parapoxvirus to understand worldwide distribution of and infection by orf virus, especially transmission between goats and sheep.Zoonotic transmission of parapoxvirus from animals to humans has been reported; clinical manifestations are skin lesions on the fingers and hands after contact with infected animals. We report a human infection clinically suspected as being ecthyma contagiosum. The patient, a 65-year-old woman, had 3 nodules on her hands. She reported contact with a sheep during the Aïd-el-Fitr festival in France during 2017. We isolated the parapoxvirus orf virus from these nodules by using a nonconventional cell and sequenced the orf genome. We identified a novel orf virus genome and compared it with genomes of other orf viruses. More research is needed on the genus  Parapoxvirus is a genus of double-stranded DNA viruses (family Poxviridae) that contains 4 virus species: orf virus, bovine papular stomatitis virus, parapoxvirus of red deer, and pseudocowpoxvirus. Recently, a complete genome from a gray seal infected by a parapoxvirus was reported and constituted a putative novel virus in this genus  in which >300 genomes are available. One of the reasons for limited availability of parapoxvirus genomes is that many of these viruses are not cultivable on most diagnostic laboratories, cell lines  studies, or electron microscopy. At least 11 complete orf virus genomes are available, and at least 19 are available for the entire genus In 2017, we identified a 65-year-old woman in France who had 3 nodules on her hands. She was given a diagnosis of ecthyma contagiosum. Genomic and electron microscopy data confirmed the initial diagnosis as infection with orf virus and identified this virus as the etiologic agent. We also isolated this virus on OA3.Ts cells.A 65-year-old woman came to North Hospital  because of 3 nodules on her hands. She reported contact 3 weeks earlier with the carcass of a dead sheep during the Aïd-el-Fitr festival  in the Bouches-du-Rhones Department in southern France. Clinically, she had 3 painless, well-delimited erythematosus nodules on her fingers with an erythematosus halo . On the We performed a parapoxvirus PCR on the cutaneous biopsy sample by using primers forward 5′-CGGTGCAGCACGAGGTC-3′, reverse 5′- CGGCGTATTCTTCTCGGACT-3′, and 6FAM-5′-GCCTAGGAAGCGCTCCGGCG-3′. These primers are specific for the B2L gene, which encodes the major membrane protein of parapoxvirus.https://www.thermofisher.com). We then inoculated 400 μL of this sample onto 2 shell vials   containing 1 mL of OA3.Ts testes cells from Ovis aries sheep  at a concentration of 106 cells/ mL. We incubated 1 vial at 32°C and 1 vial at 37°C in an atmosphere of 5% CO2 and observed daily by inverted microscopy to detect any potential cytopathic effect.For virus culture, we crushed a biopsy sample and resuspended it in Hanks’ balanced salt solution  containing OA3.Ts cells and Dulbecco’s modified Eagle medium (Thermo Fisher Scientific) plus 10% fetal bovine serum and 1% glutamine. We then incubated the cells, and when they reached a confluence of 80% of confluence, we removed the medium and inoculated the monolayer with 5 mL of virus suspension at a multiplicity of infection of 0.01. We incubated the flasks at 37°C for 1 h to enable adsorption. We then added 20 mL of Dulbecco’s modified Eagle medium to the flasks and incubated them for 3 days. On the third day, we discarded the supernatant, washed the cell monolayer 3 times with phosphate-buffered saline, and removed the monolayer by using a scraper. Once all the flasks were scraped and washed twice to collect cells, we transferred all contents to a 50-mL tube and kept the tube on ice.For virus production, we prepared 15 flasks , and incubated this suspension for 10 min on ice. We performed mechanical lysis by using a sterile douncer device (80 cycles on ice). In parallel, we filtered the entire supernatant by using a 0.45-μm polyvinylidene difluoride membrane  and centrifuged the supernatant. Finally, we added 10 mL of 25% sucrose to a plastic centrifugation tube, and slowly transferred the virus mixture from the filtrate to avoid mixing with the sucrose solution . We centrifuged the tube at 60,000 × g for 1 h at 4°C, collected the pellet, and stored the pellet at −80°C in small aliquots before genome sequencing.We then centrifuged the cells at 500 × 2) at 24 h postinfection.We maintained OA3.Ts cells in culture containing minimal essential medium plus 10% fetal bovine serum. We inoculated the cell monolayer with parapoxvirus at a multiplicity of infection of 0.01 and incubated. We then collected the contents after scraping the flask (T-25cmhttp://www.agarscientific.com). In brief, we fixed cells for 1 h with 2.5% glutaraldehyde in a 0.1 mol/L sodium cacodylate buffer and washed with a mixture of 0.2 mol/L saccharose/0.1 mol/L sodium cacodylate. We then postfixed cells for 1 h with 1% OsO4 diluted in 0.2 mol/L potassium hexacyanoferrate (III)/0.1 mol/L sodium cacodylate solution. After washing the cells with distilled water, we gradually dehydrated them with ethanol, then gradually replaced the ethanol with LR white resin. We performed polymerization for 24 h at 60°C. We obtained ultrathin, 70-nm sections by using a UC7 ultramicrotome  and placed the sections onto HR25 300-mesh copper/rhodium grids . We colored the sections with Reynolds solution. We obtained electron micrographs by using a Tecnai G2 transmission electron microscope  operated at 200 keV and used ImageJ software (https://imagej.nih.gov) to determine particle size.We used the protocol of cell embedding as described in Bou Khalil et al. (https://www.illumina.com) and the paired-end strategy. We barcoded sequences and compared them with 19 other genomic projects prepared from Nextera XT DNA Sample Prep Kit (Illumina). We quantified genomic DNA by using the Qubit Assay and the High-Sensitivity Kit  at a concentration of 43 ng/µL. To prepare the paired-end library, we performed a dilution to obtain 1 ng of each genome as input to prepare the paired-end library. The tagmentation step (Illumina) fragmented and tagged the DNA. Limited cycle PCR amplification (12 cycles) completed the tag adapters and introduced dual-index barcodes. After purification by using AMPure XP Beads , we normalized the libraries on specific beads according to the Nextera XT protocol (Illumina). We pooled normalized libraries into a single library for sequencing, then loaded the pooled single-strand library onto the reagent cartridge and then onto the instrument along with the flow cell. We performed automated cluster generation and paired-end sequencing with dual index reads in a single 39-h run for 2 × 250 bp.We sequenced genomic DNA of the parapoxvirus by using MiSeq Technology . Within this run, we determined the index representation for the parapoxvirus to be 5.45%. We filtered the 1,078,648 paired-end reads according to the read qualities.We obtained total information of 10.2 Gb from 1,140,000 clusters of density/mmWe assembled paired-end reads by using the Hybrid spades program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&) of all predicted proteins against the nonredundant database at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) and performed annotation by using delta-blastp results (https://www.ebi.ac.uk). To determine average nucleotide values, we compared close phylogenetic strains by using the OrthoANI algorithm .We used Prodigal software for gene prediction (https://www.megasoftware.net) to delete inverted repeat regions nonaligned at both ends. We conserved 208,216 positions to build a tree by using the general time-reversible model on the PhyML version 3 program .Genome organization of orthopoxviruses are known to be conserved and follow the typical structure with inverted terminal repeat variations and a conserved central core genome Figure 1Mucor circinelloides , and 1 hit with Ovis aries sheep, the natural host. The protein showing the best hit with O. aries sheep was annotated as the interleukin-10 precursor. The gene for this protein is found in parapoxvirus and was probably acquired from mammals and known as a potential keystone protein that reduces inflammation during the infectious cycle   in the genus Conversely, phylogenetic analysis of the entire virus genome showed clustering of orf viruses depending on the host (sheep or goats). This result was similar to those of previous analyses performed on different complete genomes by Chi et al. . However, implication of the host immune system in the severity of orf virus disease and in its evolution have been demonstrated (Our results highlight the necessity of obtaining more complete genomes for parapoxviruses and retracing the route of infection when humans are infected. Further investigations of parapoxviruses should address the difficulties in isolating and cultivating this fastidious virus by using nonconventional cells for diagnostic analysis. However, recent description of a seal parapoxvirus (Additional information on human infection with orf virus and description of its whole genome, France, 2017.


Humans cannot synthesize vitamins but obtain vitamins through dietary intake. Cobalamin, or vitamin BOnly a limited number of bacteria and archaea generate cobalamin de novo . MoreoveMammalian herbivores—including rabbits, mice, rats, and nonhuman primates—practice coprophagy, or eating of feces, to obtain cobalamin because these organisms do not obtain sufficient cobalamin from vegetarian dietary sources. Notably, animal products from ruminants, such as cattle, are excellent dietary sources of cobalamin for humans despite being noncoprophagic herbivores. Ruminants are heavily colonized with cobalamin-producing microbes in the rumen, a specialized organ that allows fermentation of ingested feed, and this cobalamin is later absorbed in the small intestine . Therefo3 bacteria/mL in the small intestine increases to 105 to 106 bacteria/mL, causing chronic diarrhea and malabsorption [Bacteroides that are common intestinal bacteria outcompete IF for binding to cobalamin [The ileum harbors a low level of bacteria compared to the colon, and an abnormal expansion of small intestinal bacteria may result in direct host–bacterial competition for cobalamin. Small intestinal bacterial overgrowth (SIBO) is a condition in which the typically low level of 10sorption . CobalamB. thetaiotaomicron expresses three transporters that uptake distinct corrinoids [B. thetaiotaomicron levels in the gut only occurred in the absence of bacteria from the phyla Firmicutes and Actinobacteria [In the colon, cobalamin availability is determined by two sources—host dietary intake (approximately 50% is not absorbed by the host)  and cobarrinoids . Even sobacteria , which ieut operon that enables growth on ethanolamine (EA), a ubiquitous metabolite in the human body. EA metabolism influences pathogen growth and/or virulence in the intestinal tract , at sites of extraintestinal dissemination , and in the urinary tract (uropathogenic Escherichia coli) [eut operons of the Firmicutes and Enterobacteriaceae also carry distinct regulatory elements that require cobalamin to drive eut expression. In the Firmicutes, EA is the signal that initiates phosphorylation of the sensor kinase EutW. This phosphate is then transferred to the noncanonical response regulator EutV . In the absence of cobalamin, the eut-encoded small RNA (sRNA) EutX/Rli55 sequesters EutV, thereby inhibiting EutV antitermination activity. The sRNA contains an AdoCbl-binding domain. AdoCbl binding causes a structural change in EutX/Rli55 that results in transcription termination and production of a truncated sRNA that cannot sequester EutV [eut expression requires the transcription factor EutR. Although EutR binds the eut promoter in the absence of EA and AdoCbl, both molecules are required for transcription initiation [E. coli O157:H7 (EHEC) and Salmonella, EutR regulates expression of virulence factors required for host infection and dissemination [eut expression, transcriptional activation requires EA and AdoCbl. In an analogous manner, cobalamin is required for enzymatic activity of propanediol dehydratase, which breaks down 1,2-propanediol, a metabolite that enhances Salmonella growth [Citrobacter rodentium virulence during infection of the mammalian gastrointestinal tract [C. rodentium is a murine pathogen frequently used to model EHEC and enteropathogenic E. coli infections).Although growth within a host is a requisite for pathogenesis, cobalamin influences the ability of a pathogen to infect a host and cause disease, independent of its role as a cofactor for MetH and MCM. For example, many bacteria encode the ia coli) –17 (Fig ter EutV , 18. In itiation , 19. In mination , 13, 14,a growth . Additioal tract  (C. rodePropionibacterium acnes and Mycobacterium tuberculosis, respectively. For example, in P. acnes, cobalamin supplementation results in decreased expression of genes encoding cobalamin synthesis and increased production of porphyrins [M. tb infects the human lung. Cobalamin transport and synthesis impact host survival and M. tb growth, respectively, although none of the three cobalamin-dependent M. tb enzymes are required for growth in vivo or in vitro [P. acnes and M. tb infection.Furthermore, cobalamin has been correlated with influencing gene expression and host survival of rphyrins , which hin vitro . FurtherSalmonella and C. rodentium infection. In this study, mice with a genetic deletion in IF production displayed worsened pathology compared to wild-type mice and increased mortality during Salmonella and C. rodentium infection [It is clear that cobalamin impacts the ability of a pathogen to establish infection and influence disease progression; however, the contribution of cobalamin to the host’s ability to defend against infection is less understood. Recently, Mottram and colleagues reported that IF mediates a protective effect against nfection . These dRecent studies have built on our understanding of the critical role of cobalamin as an enzymatic cofactor to include newly identified roles in gene expression, pathogenesis, and immune function. Elucidating the vast influence of cobalamin in host–microbiota–pathogen interactions will expand our understanding of host–microbial homeostasis and lead to creative new opportunities to disrupt pathogenesis.


A total of 118 sera were collected during 2016 from two groups of dromedaries from Kebili and Medenine governorates in the south of Tunisia. The aim of this study was to provide the first serological investigation of four emerging vector-borne diseases in two groups of dromedaries in Tunisia. Sera were tested by ELISA and serum neutralisation test to identify West Nile virus (WNV), bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and Rift Valley fever virus (RVFV). In the first group, the seroprevalence for BTV was 4.6%, while in the second group, it was 25.8% for WNV and 9.7% for BTV. Only serotype 1 was detected for BTV in the two groups. No evidence for circulation of RVF and EHD viruses was revealed. Results indicated that dromedaries can be infected with BTV and WNV, suggesting that this species might play a significant role in the epizootiology of these viral diseases in Tunisia and neighbouring countries. The economic value of the dromedary as a source of meat and transportation with the renewed interest around the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the Arabian Peninsula raises the question of the impact of the dromedary on new and emergent diseases in North Africa.The effective control of West Nile (WN), bluetongue (BT), epizootic haemorrhagic disease (EHD) and Rift Valley fever (RVF) depends on a thorough understanding of the possible implications of dromedaries in the replication or the dissemination of the West Nile virus (WNV), bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and Rift Valley fever virus (RVFV).Tunisia is located at the interface between the sub-Saharan region and Europe. It has encountered several episodes of emerging vector-borne diseases such as WN, BT and EHD. Tunisia has experienced three major WN epidemics that have particularly affected humans in 1997, 2003 and 2012 . For EHD, all the sera were tested by a blocking competitive ELISA  to detect the specific anti-EHDV VP7 antibodies. Sera were screened for the presence of group-specific BTV antibodies by using the c-ELISA IDEXX Bluetongue Competition® assay . The Usutu virus (USUV)- and WNV-specific antibodies were detected using a commercially available competition ELISA, . Serum neutralisation (SN) test for 26 BTV serotypes and for WNV–USUV was used on positive samples  where the dromedaries usually circulate in the arid desert. The second lot of 31 sera was collected from individual dromedaries in contact with sheep herds in the oases of Kebili, which are characterised by traditional irrigation where the main practice is flooding : Culicoïdes jumineri, Culicoïdes sahariensis and Culicoïdes submarimitimus although Culicoïdes imicola is considered the main vector of BTV  confirms this finding . This study confirms the circulation of serotype 1 in Tunisia that was detected for the last time in 2011 (Sghaier et al. V Mellor . AccordiV Mellor . For WNVV Mellor , where aThis study, for the first time, reports infection of dromedaries with WNV and BTV in Tunisia, confirming the reported infections of dromedaries with BTV and WNV in North Africa and the Middle East. A larger serosurvey with an entomological monitoring and a syndromic surveillance in a One Health concept is needed to understand the implications of dromedaries in the transmission of emerging arthropod-borne viruses which can be very useful for the purposes of control and planning surveillance activities not only in Tunisia but also for all the regions of North Africa.