Aceria tosichella, and its relationship with wheat. WCM is a major pest of wheat and other cereals and a vector of at least four damaging plant viruses . The WCM–virus pathosystem causes considerable yield losses worldwide and its severity increases significantly when mixed-virus infections occur. Chemical control strategies are largely ineffective because WCM occupies secluded niches on the plant, e.g., leaf sheaths or curled leaves in the whorl. The challenge of effectively managing this pest–virus complex is exacerbated by the existence of divergent WCM lineages that differ in host-colonization and virus-transmission abilities. We highlight research progress in mite ecology and virus epidemiology that affect management and development of cereal cultivars with WCM- and virus-resistance genes. We also address the challenge of avoiding both agronomically deleterious side effects and selection for field populations of WCM that can overcome these resistance genes. This report integrates the current state of knowledge of WCM–virus-plant interactions and addresses knowledge gaps regarding the mechanisms driving WCM infestation, viral epidemics, and plant responses. We discuss the potential application of molecular methods  to understand the chemical and cellular interface between the wheat plant and WCM–virus complexes.Wheat production and sustainability are steadily threatened by pests and pathogens in both wealthy and developing countries. This review is focused on the wheat curl mite (WCM),  Triticum aestivum L., is the most abundant source of calories and protein in the human diet  which belongs to the superfamily Eriophyoidea , phyoidea . WCM is phyoidea . Moreovephyoidea , short dphyoidea  enable gThe greatest economic impact of WCM results from its ability to transmit at least four damaging plant viruses to several different cereal crops. In this review we integrate the current state of knowledge of WCM–virus-plant interactions and address knowledge gaps regarding the mechanisms driving WCM infestation, viral epidemics, and plant responses.Figure 2B) that promotes a humid environment generally preferred by WCM. WCM feeding also reduces photosynthetic capacity  and uncultivated grasses . WCM hascapacity .Wheat streak mosaic virus . The mite was also shown to transmit High plains wheat mosaic virus   by WCM was verified by Triticum mosaic virus .The WCM has been shown to be the only transmitter of four distinct viruses to wheat and numerous other grass hosts . These v HPWMoV) . TransmiOf these viruses, WSMV is the most widely distributed and studied and it has been identified from every major wheat growing region around the world . The greWheat streak mosaic virus infection of wheat results in a light and dark green mosaic pattern on the youngest emerged leaves  and nuclear  DNA sequences, differing also in host preference . Some liIn North America these two lineages have been shown to transmit WSMV . HoweverMT-8 and MT-1 have been found coexisting in mixed populations in wheat-producing areas in North America, Australia, and Europe, where plants from a single wheat field contained both MT-1 and MT-8 , furtherWsm1 gene from Thinopyrum intermedium (Host) Barkworth & D.R. Dewey to the short arm of chromosome 4D in wheat  to both the mite and viruses by introgressing favorable traits from resistant germplasm into advanced breeding lines , in addiin wheat .Wsm1 gene. This gene has demonstrated resistance to both WSMV and TriMV  of wheat origin. Four varieties have thus far been released with the Wsm2 gene: ‘RonL’  was translocated to wheat from rye  (Cmc1) translocated from Aegilops tauschii (Coss.) Schmal. to wheat (Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey, contributed with gene Cmc2 (A. tauschii (Cmc4) was found to be independent of Cmc1  . It was eale L.) . Howevereale L.) , 1997. Ato wheat  has beento wheat  and the to wheat . A thirdene Cmc2 . A secon of Cmc1  and has  of Cmc1 . Additio of Cmc1 , rye  revealed a wide range of intracellular physiological changes induced by this mite although the specific source(s) of induction  are not known (Proteomic analyses of rice leaves from control plants and those infested with ot known . Similarot known , to asseot known . ComplemRegarding the ability of mites to transmit WSMV, a genotyping-by-sequencing study e.g.,  incorporDifferent strains of WSMV have also been detected that are differentially transmitted by individual WCM genotypes . MutatioThe spread of WCM and its associated plant viruses to cereal-producing regions worldwide is of increasing scientific and economic importance . ColonizAS, BR, and GH designed the conception, wrote the manuscript, and read and approved the submitted version with equal contribution.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Universitat Autònoma de Barcelona  and Universidad de León .Diarrhoea is a major cause of death in neonate pigs and most of the viruses that cause it are RNA viruses. Next Generation Sequencing (NGS) deeply characterize the genetic diversity among rapidly mutating virus populations at the interspecific as well as the intraspecific level. The diversity of RNA viruses present in faeces of neonatal piglets suffering from diarrhoea in 47 farms, plus 4 samples from non-diarrhoeic piglets has been evaluated by NGS. Samples were selected among the cases submitted to the Veterinary Diagnostic Laboratories of Infectious Diseases of the Kobuvirus and Rotavirus were more commonly reported, with Sapovirus, Astrovirus 3, 4 and 5, Enterovirus G, Porcine epidemic diarrhoea virus, Pasivirus and Posavirus being less frequently detected. Most sequences showed a low identity with the sequences deposited in GenBank, allowing us to propose several new VP4 and VP7 genotypes for Rotavirus B and Rotavirus C.The analyses identified the presence of 12 virus species corresponding to 8 genera of RNA viruses. Most samples were co-infected by several viruses. Kobuvirus and Sapovirus may also have an aetiological role. Even most animals were co-infected in early life, the association with enteric disease among the other examined viruses was unclear. The NGS method applied successfully characterized the RNA virome present in faeces and detected a high level of unreported intraspecific diversity.Among the cases analysed, Rotaviruses were the main aetiological agents of diarrhoea in neonate pigs. Besides, in a small number of cases  Escherichia coli, Clostridium perfringens, etc.) and viral pathogens . Less commonly neonates can suffer from diarrhoea caused by parasites [Porcine epidemic diarrhoea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine deltacoronavirus (PDCoV) or Rotavirus A, B, C or H  [Rotavirus A, B and C are a major cause of diarrhoea in pigs worldwide, having a significant impact in health and productivity, plus the potential of zoonotic transmission to humans [Rotavirus infection. In contrast, other RNA viruses including Kobuvirus, Astrovirus, Sapovirus, Sapelovirus, Teschovirus, and Torovirus, have been detected in pig faeces but its role as causative agents of neonatal diarrhoea has not so far been fully elucidated [Kobuvirus (KobuV) has been found frequently (16–99%) in faecal samples of pigs worldwide [KobuV were present concomitantly with other viruses making difficult a precise assessment of its role as agent of disease [Diarrhoea in neonatal piglets is a common problem in many pig herds. Usually, this condition is seen either in the offspring of gilts or as an epidemic problem affecting litters from sows of any parity. In the first case, the problem is usually attributed to an inadequate acclimation protocol for gilts while in the latter, the most often cause is the introduction of a new enteric pathogen. These patterns reflect the immune status of sows against the causing agents and the transfer of colostral and lactogenic immunity to piglets. The agents involved in neonatal diarrhoea of piglets are diverse and include bacterial  –9. Amongo humans . The virucidated –14. For orldwide . In some disease , 16.Next Generation Sequencing (NGS) allows for an in-depth characterization of the genetic diversity among rapidly mutating virus populations by directly sequencing viral strains at the interspecific as well as the intraspecific level , 18. TheKobuvirus, Rotavirus , Sapovirus (SAV), Mamastrovirus , Alphacoronavirus (PEDV), Enterovirus , Pasivirus (PasiV) and Posavirus (PosaV). The most commonly reported RNA virus was KobuV, followed by RVA, RVC, SAV, RVB and AstV3  presented more than one virus and in one sample seven RNA viruses were identified: KobuV, RVC, AstV 3, 4 and 5, SAV and PEDV. Rotavirus  plus KobuV was the combination present in 12 out of the 14 (86%) samples infected with 2 viruses. RVA, RVB, RVC or a combination of two or three Rotavirus were also present in 41 out of the 47 (87%) diarrhoeic samples analysed. For the 6 Rotavirus-negative samples KobuV was present in all 6; being the single virus present in 4 of them. In the other two samples Astrovirus 3, 4, and 5, SAV, PasiV, PosaV and EntVG were detected . In all 47 cases, one virus represented at least 45% of the total viral reads. Interestingly, when mixed rotavirus infections were detected (RVA-RVC or RVB-RVC), the proportion of one virus clearly predominated against the other. When coupled with the viral load, the relative frequencies per virus , RVB (6) or RVC (9) –, with KobuV and SAV being much less frequent . Besides, few cases (4 KobuV and 1 RVA) represented simultaneously high proportions with viral reads below 10E3.The NGS approach used allowed the comparison of the relative frequencies of each viral species in the examined samples. Thus, when the number of reads for a given virus was compared to the total number of reads attributable to a mammalian RNA virus in the sample, it was evident that Rotaviruses, particularly ent Fig. . In onlyrus Fig.  showed tFabavirus, Bromovirus, Fijivirus, Luteovirus) and fungi .In the four non-diarrhoeic samples, the number of viral reads obtained and indexed was lower compared to those of the diarrhoeic samples, none of the mammalian virus detected surpasses 10E0 to 10E1 reads  than in KobuV (n = 17). The full-length sequences obtained – 17 KobuV, 23 RVA, 11 RVC, 8 RVB, 4 SAV and 2 AstV3 – were deposited in GenBank with the Accession Numbers MH238075 to MH238338, MK936372 to MK936426, MK953017 to MK953236, and MK962320 to MK962342. Considering the segmented nature of the Rotavirus genome, every segment for every genome was uploaded separately. For the remaining RNA viruses, every complete genome was uploaded in a single file.The filtering results included both whole genome (complete) and partial (less than 90% of the genome covered) sequences of the virus listed above  with the closest GenBank sequences ranged from between 74 and 90%. In fact, according to the proposed nucleotide cut-off values for VP7 (80%), two undescribed VP7 genotypes were reported in nearly all (7 out of 8) the RVB positive samples. Similarly, for VP4, 3 samples could harbour a new RVB genotype (tree not shown). The RVB genotypes reported and its frequency would be: GX2P [The genotyping of the 23  was G9P  , AstV4 (89%), SAV (87%), EntVG (82%) and PasiV (86%) except for PosaV (96%), AstV5 (97%) and PEDV (99%) which were very similar to other European sequences reported. Figures SAV and AstV3, respectively.For other viruses, nucleotide identities with the closest sequences deposited were low: %), SAV 8%, EntVG The de novo assembler and virus detection analyses performed did not detect contigs that could be related to the presence of an unknown RNA virus, as all of them where positively indexed against the Reference RNA sequences dataset and/or the RefSeq Genome Database available at the BLAST resource of the NCBI website.Diarrhoea is the most common infectious cause of neonatal death in pigs . PreventClassically, infectious neonatal diarrhoea has been seen as a problem caused by single aetiological agents. However, with the development of molecular techniques, a growing number of evidences indicated that many agents can be found in the faeces of affected animals , 21. TheIn this work, we examined the RNA virome present in faecal samples from 47 cases of diarrhoea plus four non-diarrhoeic negative controls in which specific bacteriological agents were excluded. The approach taken using NGS did not include any previous enrichment or PCR step; hence, the results obtained represented not only the RNA viruses present in a sample analysed but also their relative abundances. This may contribute to our understanding of the role of each virus in a given case.KobuV and SAV may have a role in this process. The examination of the relative proportions of viral reads versus the number of reads showed that for any given case always one virus predominated, representing more than 45% of the mammalian viral reads obtained. This value can be tentatively proposed as a cut-off for the assignment of an etiological agent. In contrast, for all the other viruses examined, the relative abundance of their genomes would be more consistent with a subclinical infection. Whether this could be the consequence of a limited virulence, because of some level of passive immunity, or other causes, cannot be addressed in the present study.In the samples examined, the predominance of Rotaviruses reads reinforce the notion of these viruses as primary agents of neonatal diarrhoea; although, occasionally, Since the NGS filtering method used in the present study could only detect known agents, additional analyses to screen for potential viral motifs were performed. The de novo assembly and virus detection analyses were not able to identify any contig that could be related to the presence of an unknown RNA virus.Interestingly, in the non-diarrhoeic samples analysed the number of mammalian reads was very scarce, but a higher number of plant and fungi viruses was reported. This is somewhat surprising, since the examined animal were suckling piglets (less than 1 week of age) that do not eat feedstuff. It is difficult to explain the origin of those viruses, since samples were taken from the rectum and thus, environmental contamination is little likely.RVA-RVC and RVB-RVC was very common, suggesting the need for simultaneous testing all three agents in cases of neonatal diarrhoea. Regarding KobuV, our results also agree with an increased prevalence of this agent observed in cases of diarrhoea in suckling piglets worldwide: Brazil [KobuV infection between diarrheic and healthy piglets in several European countries [KobuV was present: one with high proportion of KobuV reads and high number of viral reads, and others where these two circumstances were not fulfilled. The first pattern could correspond to clinical cases caused by KobuV, while the second could correspond to secondary or concomitant infections by this agent. A similar pattern would apply to SAV. Regarding the other agents, since they were always in combination and their reads were not preeminent, its role must be seen as secondary at best. For instance, no differences in Astrovirus prevalence between diarrheic and non-diarrheic piglets were reported [Enterovirus prevalence between healthy and diarrheic pigs [PEDV was seldom found in our samples and when found only traces could be detected. Prior to this, a higher frequency for this pathogen could be expected. However, while PEDV has caused recently severe epidemics in America [Porcine Respiratory Coronavirus [Our results agree with a number of studies that reported different Rotavirus species as agents of neonatal diarrhoea (reviewed in ). One in: Brazil , Korea [: Brazil  and Viet: Brazil ; despiteountries , 16, 31.reported , 33, noreic pigs . Interes America , in Euro America . Of noteonavirus .RVA was present. This is compatible with its assumed role as primary agent. We have recently described the introduction of a new RVA strain that rapidly spread across pig farms in Spain and that was also present in the samples of this study as well [In any case, it is somewhat surprising to detect that many different viruses (up to seven in a single sample) in so young animals, even though the pattern was already observed in diarrhoeic piglets . While i as well .RVA (discussed above). This, together with the depth of the corresponding branches in the trees, suggested a long evolutionary history on a local basis. Certainly, we cannot discard the existence of other clusters for any of the reported viruses. However, the detection of several new genotypes for RVB and RVC plus the relative low nucleotide identity with other available sequences in GenBank reinforces the idea of local evolution at least for these two viruses. Moreover, with the results obtained we propose to define for RVB one new VP4  are more relevant with regards to the clinical expression of the disease. Similarly, for SAV no differences were seen, and all isolates clustered together within genogroup III, the most commonly reported porcine SAV [It is worth noting that the phylogenetic analyses for all the examined viruses indicated the existence of local clusters except for w VP4 (P ) and twod region . Regardicine SAV .Kobuvirus was the most common virus detected, probably it was the primary agent of diarrhoea only in a small number of cases. Similarly, Sapovirus would be responsible of a single diarrhoea case. For the other examined viruses, the results indicated that many animals were infected in early life but the association with enteric disease was unclear. The NGS approach applied permit not only the detection RNA viruses present in a sample, but also to determine their relative abundances. This approach can be used for a more comprehensive diagnosis of neonatal diarrhoea and is useful for the examination of the RNA virome in faeces.The results of this study suggested that based on the abundance among the cases analysed, Rotaviruses were the main RNA viruses involved in neonatal diarrhoea in pigs. Although Universitat Autònoma de Barcelona  and Universidad de León . Four samples from healthy non-diarrhoeic piglets were also included to be used as negative controls. In the cases studied, a bacteriological cause for the diarrhoea had been excluded after a microbiological culture for aerobes and anaerobes and molecular analyses (PCR for E. coli virulence factors and C. perfringens and C. difficile toxins). Only cases apparently unrelated (different farms with no evident connection) were included. Finally, 47 faecal samples, each one representing an outbreak of neonatal diarrhoea in 47 farms from Catalonia, Castile and Leon, Aragon, Galicia and Valencia were analysed  routinely submitted for diagnosis during 2017 to the Veterinary Diagnostic Laboratories of Infectious Diseases of the Faecal samples were kept frozen at − 80 °C until used. When needed, samples were thawed and diluted 1:5 in sterile distilled water. Then, to remove debris, diluted samples were sequentially clarified for 2 min at 2000 g, 5 min at 5000 g and 10 min at 10,000 g. Total RNA extraction was performed using the TRIzol reagent (ThermoFischer Scientific) following the manufacturer’s instructions. For each reaction, 250 μL of the diluted faeces was used.The assessment of the diversity of viral RNA within each sample was directly characterised from the total RNA extracted, without any previous amplification step (no primers were required), applying a NGS approach previously applied to faeces . BrieflyIn the cases where a presumptive aetiological agent could not be clearly established, further analysis to detect unknown RNA viruses were undertaken with the program rnaSPAdes , an RNAsAdditional file 1: Summary of the 51 samples analyzed: name, origin, RNA viruses detected and number of reads indexed.Additional file 2: Summary of the RNA viruses and Accession Numbers of the strains used to filter the quality reads in the step iv) of the NGS approach, used together with step iii), to identify the RNA virus species present in a given sample.


Dioscorea spp.). The full diversity of viruses infecting yam, however, remains to be explored. High-throughput sequencing (HTS) methods are increasingly being used in the discovery of new plant viral genomes. In this study, we employed HTS on yam to determine whether any undiscovered viruses were present that would restrict the international distribution of yam germplasm. We discovered a new virus sequence present in 31 yam samples tested and have tentatively named this virus “yam virus Y” (YVY). Twenty-three of the samples in which YVY was detected showed mosaic and chlorotic leaf symptoms, but Yam mosaic virus was also detected in these samples. Complete genome sequences of two YVY viral isolates were assembled and found to contain five open reading frames (ORFs). ORF1 encodes a large replication-associated protein, ORF2, ORF3 and ORF4 constitute the putative triple gene block proteins, and ORF5 encodes a putative coat protein. Considering the species demarcation criteria of the family Betaflexiviridae, YVY should be considered as a novel virus species in the family Betaflexiviridae. Further work is needed to understand the association of this new virus with any symptoms and yield loss and its implication on virus-free seed yam production.To date, several viruses of different genera have been reported to infect yam ( Dioscorea spp.) is a preferred staple food for over 90 million people in West Africa, with this region contributing over 95% of the world’s total yam production [Yam , Yam mild mosaic virus  and Dioscorea bacilliform viruses  are widespread in West Africa and YMV has been shown to cause important diseases in yam [Despite its importance and high value, yam productivity is compromised severely by the impact of yam viruses and the unavailability and associated high costs of high-quality clean seed yam . To dateotyvirus ,4,5,6,7 s in yam ,8,9,10. s in yam .The use of virus-free (“clean”) planting material is the only efficient method of controlling these virus diseases. However, production and distribution of clean seed yams is hampered by the absence of a formal seed yam certification system ,12 and tBetaflexiviridae, extending our knowledge on the diversity of viruses present in yam. Our study shows that YVY could be detected in several West African yam plants also infected with YMV and displaying symptoms associated with mosaic disease. Further work is needed to understand the biology of YVY, its association with any symptoms and importance on yield, and implications for seed production.In the present study, we describe and characterize the complete genome sequence of a new virus species present in yam. The new virus, tentatively named “yam virus Y” (YVY), represents a putative new species of the family D. rotundata cv. Danicha sample yielded 48,934,725 reads. After quality trimming and removing reads corresponding to the yam host, RNA-seq datasets were de novo assembled for genome reconstruction. A total of 43,710 contigs were obtained. Near-complete and complete genomes of known Yam mosaic virus (YMV), Dioscorea mosaic-associated virus (DMaV) RNA1 and RNA2, and yam badnavirus were obtained by directly mapping assembled contigs to a custom-made database containing viral reference sequence genomes publicly available from the NCBI GenBank.The HTS of Nerine latent virus . At the amino acid level and using BLASTx, the predicted protein sequence showed homologies with members of the Betaflexiviridae family. Conserved domains of a viral methyltransferase (Met), a helicase (Hel), and a RNA-dependent RNA polymerase (RdRp) were identified with amino acid sequence identity levels of 46% (58% sequence coverage) to Apple stem pitting virus (GenBank accession ARQ84116).Contigs that did not match to any known viruses were annotated using BLASTn/x. Using BLASTn, one contig (7324 nucleotides (nt) long) showed a nucleotide identity score of 69% (12% sequence coverage), corresponding to the partial viral replicase region of The draft assembly was then further extended using the Geneious iterative assembler with ten iterations. A single continuous sequence of 7557 nt was obtained representing the complete genome sequence of a new virus, hereafter named “yam virus Y, isolate Danicha”  . The rawTo confirm that the new viral sequence was not an artefact of the HTS analysis and further prove the presence of YVY-Dan sequence in yam, we performed a long reverse transcription polymerase chain reaction (RT-PCR) using primer “YVY-RdRp1-PF”  and oligo-dT (18 mer) as reverse primer. A fragment of the expected size (~3.5 kb) from the partial RdRp region to the poly-A 3’ end was obtained .D. rotundata cv. Makakusa tissue culture plant showing mild mosaic and chlorotic symptoms obtained by Bömer et al. [Betafexiviridae family identified one contig (7584 nt long) displaying conserved domains of a viral Met, Hel, RdRp and Flexi-CP and with amino acid sequence identity levels of 46% (56% sequence coverage) to Apple stem pitting virus (GenBank accession ARQ84116). This sequence represents another new complete genome of YVY referred to “yam virus Y isolate Makakusa” . YVY-Mak represents 6.6% (2.3 million reads) of the total RNA-seq reads and is covered at a depth of 42X.To investigate the possible existence of another YVY isolate, RNA-Seq data from a six weeks old r et al.  was analr et al. . A targeBetaflexiviridae. ORF1 encodes a putative viral replicase protein with an estimated mass of 208 kDa. Three conserved domains typical for Betaflexiviridae [The genomes of YVY-Dan and YVY-Mak are 7557 nt and 7584 nt long, respectively, excluding the 3’ polyA tail. Genomes encode for five putative ORFs, including a triple gene block (TGB)  characteiviridae  were ideiviridae . ORF5 eniviridae .Betaflexiviridae is less than 72% nucleotide (nt) or 80% amino acid (aa) identity in either the complete CP (ORF5) or replicase (ORF1) genes [Apple stem pitting virus (ARQ84116) with 46% aa identity (79% sequence coverage). For the CP gene, both isolates shared 33% aa sequence identity (88% sequence coverage) to Cherry twisted leaf-associated virus (AHA59520). The amino acid sequence identity level of both YVY-Dan and YVY-Mak is well below the demarcation criteria. Similar results are obtained with the nucleotide sequences of both replicase and CP genes. Based on these results and when comparing the nucleotide and amino acid sequences of different genes between the two isolates , the demarcation criteria for isolates of different species in the family 1) genes . For theisolates , YVY shoBetaflexiviridae. Phylogenetic analysis indicated that isolates obtained in this study (YVY-Dan and YVY-Mak) form a well-supported clade and group together with members of unassigned species in the family Betaflexiviridae, the closest relative being Sugarcane striate mosaic-associated virus (SCSMaV). YVY-Dan and YVY-Mak share the highest replicase amino acid and nucleotide identity  with SCSMaV. Phylogenetic analysis based on the full-genome sequences (Betaflexiviriade. No recombination events were detected for the two YVY isolates (data not shown).Phylogenetic trees were generated based on the deduced amino acid sequences of the entire replicase protein a of YVY-equences b resulteBased on the complete genome sequences of YVY, new specific PCR primers targeting a 790 bp region of the RdRP gene and a 788 bp region comprising the CP-3′UTR were designed . These primers were used to screen the presence of the new virus by RT-PCR in 55 yam breeding lines and landraces actively growing in screenhouses at the Natural Resources Institute , the International Institute of Tropical Agriculture  and the Council for Scientific and Industrial Research -Crops Research Institute  . ResultsEight RT-PCR products from the CP-3′UTR region were sequenced and deposited in GenBank (MK895044-MK895051). A multiple sequence alignment was performed with these sequences using ClustalW in Geneious software. Before further analysis, the CP-3′UTR region was trimmed to the CP coding region (711 nt), not including primer sequences. Nucleotide identity sequencing analysis of the CP gene showed that the different isolates share identities between 84% and 97% with YVY-Dan and 83% to 85% with YVY-Mak .Phylogenetic analysis based on the CP gene  indicateD. rotundata yam samples. The complete genome sequences of two isolates, YVY-Dan and YVY-Mak, were obtained by HTS analysis. The genome of YVY contains five ORFs. ORF1 encodes large replication-associated protein, ORFs2, 3, and 4 constitute the putative triple gene block proteins associated with viral cell-to-cell movement in plants [In this study, we identified a novel virus, tentatively named “yam virus Y” (YVY), in n plants  and ORF5n plants ,20. Neven plants ,22.Betaflexiviridae. The degree of similarity (30% nt and 47% aa on the replicase gene) is well below the demarcation criteria thresholds recommended for all members of the family Betaflexiviridae (less than 72% nt or 80% aa identity in either the complete CP or replicase genes), suggesting that YVY should be considered as a new virus species in the family Betaflexiviridae. Phylogenetic analysis based on the CP gene indicate that YVY isolates are grouped in at least two different groups. More isolates are needed to better understand the importance of the diversity of YVY isolates.Sequencing and phylogenetic analysis showed that the virus sharing the highest pairwise identity to both YVY isolates was SCSMaV, an unassigned species of the family Yams are a major staple and play a critical role in food security and income generation for small holder farmers in West Africa. Nigeria and Ghana are the major producers and exporters of yam, respectively, and as a consequence several efforts have been made to boost yam production in these countries. The Ghanaian government recently introduced a policy that is geared towards increasing food productivity and ensuring food security for the country, which resulted in a higher demand for high-quality seed yam. Also, the “Yam Improvement for Income and Food Security in West Africa” (YIIFSWA) project  will devThe limited survey of viruses in yam collections in the United Kingdom, Nigeria and Ghana showed that YVY is widespread in yam. The new virus was generally found in symptomatic plants that were YMV positive  to enable us to investigate these widespread viruses further in the future.D. rotundata) used in this study were obtained from the Natural Resources Institute , the International Institute of Tropical Agriculture  and the Council for Scientific and Industrial Research—Crops Research Institute  collections.Yams (D. rotundata (n = 15) actively growing in a quarantine aphid-proof glasshouse as described by Mumford and Seal [D. rotundata landrace cv. Danicha showing viral symptoms was used for HTS analysis.The NRI samples consisted of YMV-infected and uninfected  breedingand Seal  and fromand Seal . The YMVand Seal ,24,25. DD. rotundata from IITA (n = 25) and CSIR-CRI (n = 15) were collected and analyzed for the presence of YVY.Yam breeding lines and landraces of D. rotundata cv. Danicha using a modified cetyltrimethyl ammonium bromide (CTAB) method combined with the RNeasy Plant Mini Kit  [For HTS analysis, total RNA was extracted from leaf tissues of Germany) . Total RD. rotundata [RNA-seq reads were trimmed to remove low quality sequences using Trimmomatic version 0.36 . Trimmedotundata  using Maotundata . Trinityotundata  was usedFor YMV and YVY detection by RT-PCR, total nucleic acids were extracted from 100 mg leaf tissue using a modified CTAB method previously described by Abarshi et al. .The presence of YMV was confirmed by RT-PCR using the primer pair YMV-CP-1F/YMV-UTR-1R , which aThe RT-PCR protocol was set up in 20 µL reactions containing 1XDreamTaq buffer , 0.25 mM of each dNTP , 0.2 µM of each primer, 1 U DreamTaq DNA polymerase , 2.5 U AMV Reverse Transcriptase  and 1 µL of purified RNA as a template. Tubes were then subjected to thermal cycling consisting of a RT step at 50 °C for 10 min, followed by 95 °C for 4.5 min and 35 cycles of 95 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min, and one final cycle of 72 °C for 10 min. RT-PCR amplification products were analyzed by agarose gel electrophoresis, purified using the GeneJET PCR Purification Kit  and Sanger sequenced by the Source BioScience sequencing service .https://www.ncbi.nlm.nih.gov/orffinder/). Conserved domains of the putative gene products were searched using the conserved domain tool (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The full-length genome and partial nucleotide and deduced amino acid sequences of YVY were aligned with other viral sequences from Betaflexiviridae family obtained from GenBank database. Multiple sequence alignments were performed using Geneious software v10.2.6 . Phylogenetic analysis for full genome, replicase and CP genes were performed in MEGA7 [Putative open reading frames (ORFs) were identified using the NCBI ORF finder (in MEGA7  using thin MEGA7 .Our study describes the presence of a novel virus of unassigned taxonomic status that is indicated to be widespread in West African yams. YVY was detected in both symptomatic and asymptomatic plants, but symptomatic plants were always positive to YMV, an endemic virus known to be responsible for mosaic disease in West Africa. Yams infected by YVY alone were generally asymptomatic. This data will be seminal for further studies regarding the biological and epidemiological features of this novel virus. It will be paramount to strengthen the yam seed certification system in order to prevent the spread of viral diseases that might threaten food and income security to small holder farmers in West Africa.


In this paper, we demonstrate that 20 μm microbeads can be preferentially assembled into substrate trenches of similar width by employing a polymer (depletant) that induces the depletion volume effect (depletion attraction). In previous works, we proved that this strategy is useful to assemble mesoscale parts in a site-specific manner. Here, we show that it is also applicable to assemble functional parts, such as fluorescent particles, into trenches engraved on the surface of two- and three-dimensional template components. A convenient advantage of this strategy is that it is independent of material properties for assembling mesoscale functional components into desired patterns. The assembly of small parts or components, which are not compatible with standard photolithography-based fabrication technologies, onto a substrate to form desired patterns is a difficult task that has been receiving considerable attention in recent years. The main widespread techniques to assemble small components  into regular patterns are microstamping ,2 and caWith recent advances of three-dimensional manufacturing techniques and apparatus, a development of versatile methods for assembling small components onto three-dimensional surface will be of particular interest in the fabrication of highly sophisticated devices ,19,20,21In the field of colloid science, control of dispersion and aggregation of colloidal particles is important, and interactions between them and with other macro-objects have been studied extensively . The vanThe excluded volume effect is unique in a sense that it does only depend on the number and spatial arrangement of molecules/components present in the system. Up to now, the thermodynamic characteristics of this effect have been verified experimentally to assemble components up to several micrometers. Dinsmore and Yodh  investigIn the past literature, polymers with molecular weight of 10,000 or less and gyration radius lower than 4 nm, or nanoparticles of up to 100 nm in size, have been employed as nano-objects (depletants) for assembling components of ~1 μm ,26,27,28In this study, using the same principle, we show that functional exogenous components with the size of several tens of μm can be specifically allocated inside grooves engraved on a template component. The components were immersed into a PEG aqueous solution, which was stirred for a long time. The extent of the bonding force can be readily controlled by adjusting the concentration of the polymer. Using this approach, we show that fluorescent particles, as a functional model component, can be assembled into trenches engraved on a sub-millimeter-sized 3D template fabricated with micro stereolithography. As this method does not depend on material properties of the assembled elements, we expect that it can be applied in the allocation of various hard and soft components such as semi-conductor, LED, cells, and gel-particles on 3D templates.Vdep) effect. In the environment where the depletant is present at a high concentration around spherical particles, two or more particles aggregate, creating regions of overlapping Vdep. The extent of reduction of Vdep (ΔVdep) is represented in the figure by the cross hatched area. On the other hand, for the interaction between a sphere and a flat surface . First, an appropriate PEG concentration for the present assembling system is identified by using a simple trenches’ pattern: Four straight trenches of 22 μm width engraved into a ~0.3 mm × 0.3 mm × 0.1 mm template a. Based 5 polystyrene beads (20 μm), into an aqueous solution of 4MDa PEG and molar concentrations of 0, 5, 50, or 500 nM  in a 0.2 mL plastic PCR tube. Sodium dodecyl sulfate (SDS), an anionic amphiphile, at a concentration of 0.5% (w/w), is included in the solution to avoid hydrophobic interaction among beads and templates. This system is agitated by stirring the tube with a rotational mixer  at 30 rpm for designated time periods. The assembly of beads in trenches is, afterwards, inspected by an inverted microscopy  and placing the flat face of the PCR tube down on the stage. The flow of the experimental procedure is illustrated in The assembling procedure is carried out by immersing, one or several template parts together with ~10cPEG). cPEG varying from states where trenches are not fully filled . Meanwhile, at cPEG = 50 nM, the average number of confined beads reaches ~29 (81% of the complete packing) and C.V. is reduced to ~5%, indicating reproducible results. We decided to employ cPEG = 50 nM in all of the following experiments.The average number of beads confined in the trench region per template is plotted in Using the experimental condition determined in the previous experiment, new assembly of the same beads are conducted onto the template of more complex designed patterns. In a similar experiment, we can confine beads into trenches forming a game character shape after ~5 days of agitation c. After Using an identical experimental condition to previous experiments, we conducted the assembly of the same beads onto a template with trenches in five faces of a 0.4 mm cube to prove the applicability of this approach into 3D templates. During agitation, the cube illustrated in In this and previous experiments, we conducted agitation for a long time (>1 day) to confirm that all trench area is filled by beads. However, we found that more than 50% of the trench area is already filled at 1.5 h in the separate experiment . This obHere, we demonstrated the assembly of 20 μm fluorescent beads into the trenches formed on template components. In all cases (both for simple and complex 2D design patterns as well as 3D surfaces), the resulting images have shown good quality of the process. This feature’s main advantage is the surface-independent characteristic that arises from changing in the allocating mechanism from surface tension/electrostatic forces to the depletion volume effect. The method can also be applied to components made of different material properties, since any solid with non-specific interaction can be assembled by simply adding depletant in the environment. The assembly can also be controlled by altering the concentration of depletant. The present approach can be applied to weak and reversible assemblies in a material-independent manner, such as assembly of soft materials and biological cells. Although we expect that these goals could be achieved in the near future at a phenomenological level, detailed understanding of thermodynamics and depletion effect of extremely large polymers (1 MDa) is needed to further expand the possible unexpected applications .


Severe fever with thrombocytopenia syndrome virus (SFTSV) can be transmitted between humans. We describe a case of severe fever with thrombocytopenia syndrome in which SFTSV RNA was detected in semen after its disappearance from serum. Our findings indicate possible sexual transmission of this emerging virus. Banyang virus, family Phenuiviridae). Recently, the person-to-person transmission of SFTSV has been described (Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening emerging infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV), a tickborne virus . Body temperature was 39.0°C, and he was disoriented; Glasgow coma scale score was 9. He had no jaundice, signs of meningeal irritation, or apparent tick bites. Laboratory tests at admission had the following results: leukocytes 2.4 × 108 copies/mL by real-time reverse transcription PCR (RT-PCR) analysis (Because of the patient’s low platelet count and other suggestive signs and symptoms, we suspected SFTS. Serum SFTSV RNA level was 2.03 × 10analysis . We confWe considered this case severe, with multiple poor prognosis factors, such as disturbance of consciousness, laboratory data, and high viral load in serum (5 copies/mL in his semen that day. On day 44, we could no longer detect semen SFTSV RNA, and he was discharged on day 51 after onset (We observed restoration of platelet count 10 days after symptom onset. Other abnormal laboratory findings recovered 7–13 days after symptom onset. The viral load in serum began to decrease from day 8 after onset and became negative on day 30 after onset. Although the patient’s general status was gradually improved and laboratory tests recovered to almost normal levels by day 30, we detected SFTSV RNA at 2.4 × 10er onset . In this study, SFTSV RNA was detected in semen, and SFTSV persisted longer in semen than in serum. It is well known that some viruses, such as Zika virus and Ebola virus, can be sexually transmitted; these viruses have been detected in semen for a prolonged period after symptom onset (Compared with that of Zika and Ebola viruses, the clinical significance of potential sexual transmission of SFTSV is unknown. However, this possibility should be taken into consideration in sexually active patients with SFTSV. Our findings suggest the need for further studies of the genital fluid of SFTS patients, women as well as men, and counseling regarding sexual behavior for these patients.Additional information about a case of severe fever with thrombocytopenia syndrome virus detected in semen, Japan.


Interestingly, sequence alignment of the structural polyproteins belonging to arthritogenic alphaviruses revealed conserved regions which share homology with human proteins implicated in rheumatoid arthritis (RA). These conserved regions were predicted to exhibit binding to HLA class II alleles, showcasing their potential to incite T cell help. Molecular docking of the viral peptide and the corresponding homologous region in the human protein onto HLA-DRB1 revealed strong similarities in their binding patterns. Linear and conformational B cell epitope prediction analyses showed that these potential mimics have high propensity to elicit an efficient B cell response. We thus propose that the origin of polyarthritis post-arthritogenic alphaviral infections may also be mediated through a hitherto unknown autoimmune response due to the presence of cross-reactive epitopes between viral and human proteins.Alphaviral infections are foremost in causing debilitating clinical outcomes in humans characterized by rheumatic arthritis like conditions. Though the presence of virus in joints and associated inflammation has been implicated as one of the reasons for the acute and chronic polyarthritis post alphaviral infections, the basis for rheumatic like outcomes is not clear. Through an  The members of the old world viruses include Chikungunya Virus (CHIKV), Ross River Virus (RRV), Mayaro Virus (MAYV), O’nyong nyong virus (ONV), Semiliki Forest Virus (SFV) and the Barmah Forest Virus (BFV). These mosquito transmitted viruses are globally distributed and are known to cause acute febrile illnesses, malaise, maculopapular rashes, myalgia, and severe arthralgia in humans5. Most often the infection remains endemic, but some viral strains are also associated with large epidemics such as the Chikungunya virus (CHIKV) out-break which was spread across 40 countries with 1.4–6.5 million reported cases globally6. Several studies also report a simultaneous increase in alpha virus associated arthritis lasting even after decrease in viral load7.Alphaviruses belonging to the group IV togoviridae family are positive sense, single stranded RNA viruses. These enveloped viruses are classified as old world and new world. While new world viruses are encephalitogenic, members of the old world are known to induce polyarthritis10. Production of a broad range of pro-inflammatory cytokines  and chemokines (MCP-1 and RANTES) post alpha viral infections has also been reported by Lidbury et al.11. Interestingly, a comparative study between CHIKV infection induced gene expression and rheumatoid arthritis induced gene expression changes in mouse models showed similarity in induced cytokine  profiles12. Another study by Morrison et al.13, demonstrated the presence of activated complement products in the serum of RRV infected mice, drawing similarities with the pathogenesis of rheumatoid arthritis13.While there is a resurgence of alphavirus associated arthritis cases, the knowledge about the molecular level events during infections involved in these conditions and about the direct cause and effect of the phenomenon is very sparse. For instance, macrophages, natural killer cells, CD4+ and CD8+ T lymphocytes have been shown as the main components of the inflammatory cellular infiltrate in animal models of CHIKV and RRV infections14. In these scenarios, initiation of autoimmune disease by an infectious pathogen is hypothesized to involve immune recognition of self-peptides by way of “molecular mimicry”15 thereby activating autoreactive T cells and generation of autoantibodies. Previous studies17 hypothesized that microbial antigens like pulD from Klebsiella sp., nuclear antigen-1 from Epstein-Barr virus and OSP-A from Borrelia sp. may have a possible association with autoimmune diseases like ankylosing spondylitis, Systemic Lupus Erythematosus (SLE) and Lyme arthritis, respectively. A significant sequence similarity between P2-C protein of Coxsackie virus and glutamate decarboxylase of humans has been proposed as a trigger for Type 1 diabetes18. The similitude in clinical manifestation of an alphaviral infection induced arthralgia and rheumatoid arthritis at both phenotypic and molecular levels suggests that alphaviral infections could be a causative link for rheumatoid arthritis.Resemblance between proteins of pathogens and host components has been examined as a trigger for many autoimmune diseasesin silico both at sequence and structural level for their immunogenic potential in terms of their ability to act as T and B cell epitopes. Overall, our results posit that polyarthritis associated with alpha viral infections may also involve an autoimmune component due to cross reactivity between viral epitopes and host proteins.Overarching goal of this study is to explore the possibility of existence of peptides in alphaviruses which may set off an autoimmune response resulting in rheumatoid arthritis like symptoms in alphavirus infected patients. Structural polyproteins of the alphaviruses were scanned for the presence of conserved stretches of amino acids that might share homology with sequences in human proteins implicated in rheumatoid arthritis. These conserved regions were validated 6. However, molecular mechanisms contributing to this pathology are not very well understood. In the present study, we show a possible role of autoimmune process mediated through molecular mimicry as a likely cause for arthritic like conditions post alphaviral infections. The overall workflow of the study is depicted in Fig. Arthritogenic alpha viruses such as Chikungunya virus (CHIKV), Ross river virus (RRV), Semiliki forest virus (SFV), Mayaro virus, O’nyong-nyong virus (ONV) and Barmah forest virus (BFV) are generally associated with rheumatic diseases in humans, primarily characterized by polyarthralgia and polyarthritis. Although most patients recover within few weeks, the clinical conditions last for 6 months to more than 3 yearsAlphaviral infections share a common feature of inducing RA like conditions, but the commonality in the alphaviruses leading to such a rheumatic outcome is not known. We hypothesized that the commonality could lie in the structural polyprotein of these viruses. Therefore, we have analyzed the structural polyproteins of six arthritogenic alpha viruses for the presence of conserved regions amongst them. As described in the methods section, amino acid sequences of the structural polyproteins of CHIKV, RRV, SFV, ONV, BFV and Mayaro virus were retrieved from UniProt database and subsequently multiple sequence alignment was performed using MUSCLE. Conserved regions identified are shown in Supplementary Fig. The structural polyproteins of arthritogenic alphaviruses are about 71% identical at the primary (amino acid sequence) level. A total of 41 conserved regions were identified in the structural polyproteins, with E1 glycoprotein region exhibiting highest conservancy with 18 conserved regions, followed by p62 and capsid proteins harboring 15 and 8 conserved regions respectively.To deduce the possibility of autoimmune reactions involved in mediating post alphaviral polyarthralgia through the presence of molecular mimics, we performed a sequence similarity search between the conserved regions among arthritogenic alpha viruses and the human proteome using the standard BLASTp program. Each of the 41 conserved regions was scanned against the human proteome. Very interestingly, 24 regions showed varying degrees of homology (52.7% to 100%) with human proteins that have been implicated in arthritis like conditions in humans (Table Human leukocyte antigen (HLA) is the most widely distributed molecule with high level of polymorphism and are the major governing factors in initiating cellular immune responses. It is thus important that immunogenic peptides bind to multiple HLA alleles to elicit a strong immune response. The 24 conserved regions of the structural polyprotein  and its corresponding homologous region KPLANVTLM (human derived) to bind to a class II HLA allele was assessed by docking the peptides in the binding groove of HLA-DRB1, an allele known to predispose individuals to rheumatoid arthritisThe docked p-MHC complexes were analyzed for the hydrogen bonding interactions between the peptides and HLA-DRB1. The binding interactions between the viral and human peptide with HLA-DRB1 were visualized using LigPlot+. Post docking analysis of the peptides revealed significant interactions with HLA-DRB1. In viral peptide-HLA-DRB1 complex, ten hydrogen bonds within a distance of 3.5 Å suggested the stability of the peptide in the binding groove of the pocket. Similarly, a set of eleven hydrogen bonds were formed between human peptide and HLA-DRB1 within a distance of 3.5 Å. Furthermore, it can be seen in Fig. To further strengthen the case of alpha viral induced molecular mimicry, we have analyzed the 24 conserved regions for the presence of both linear and structural B cell epitopes which in turn is a measure of their ability to act as stimulators of B cells and generate an autoimmune reaction against its homologous region in the human protein leading to polyarthralgia. Linear B cell epitope prediction was performed using three different prediction algorithms namely Bepipred linear epitope prediction, Emini surface accessibility scale and Kolaskar and Tongaonkar antigenicity scale. Prediction results are summarized in Table Out of the 24 conserved regions analyzed, 5 regions were predicted to be plausible B cell epitopes by all the three algorithms. 6 regions were predicted to be plausible B cell epitopes by at least 2 of the 3 algorithms tested. 12 regions were predicted to be B cell epitopes by only one of the algorithm. Only one out of the 24 conserved regions was not predicted as B cell epitope by all the three algorithms.Interestingly, 9 out of the 24 conserved regions shared homology with human proteins which were experimentally proven to have antibodies developed against them in patients diagnosed with rheumatoid arthritis  suggests that the origin of commonality in the clinical features may lie in the amino acid sequence of the structural polyprotein of the viruses. On comparative analysis of these regions of consensus with the human proteome, we have identified proteins that share stretches of homologous regions with the structural polyprotein. Further, very interestingly we found that presence of auto antibodies specific to some of these proteins in rheumatoid arthritis (UniProt ID: Q8JUX5), the Ross river virus (strain NB5092) (UniProt ID: P13890), the Semliki forest virus (UniProt ID: P03315), the Mayaro virus (strain Brazil) (UniProt ID: Q8QZ72), the O’nyong-nyong virus (strain SG650) (UniProt ID: O90369) and the Barmah forest virus (UniProt ID: P89946) were investigated for the presence of conserved regions. Amino acid sequences of these proteins were retrieved from the UniProt database34.We carried out the bioinformatic analysis using protein sequences available for the clinical isolates of alphaviruses known to cause polyarthralgia35 in MEGA 7.036. A stretch of amino acids was scored as conserved region if it is present in at least four out of the six alpha viruses.Conserved regions within the structural polyproteins were identified by multiple sequence alignment using MUSCLE alignment algorithmhttps://blast.ncbi.nlm.nih.gov/Blast.cgi)37 was employed to expound the existence of sequence homology between the conserved regions identified in the alpha viruses and the human proteome. The search set was limited to Homo sapiens (taxid: 9606) in the UniProtKB/Swiss-Prot database. Default BLASTp algorithm parameters were used and the results were limited only to the top 100 hits.BLASTp program  was retrieved from Protein Data Bank database41. The resident peptide in the crystal structure was stripped off using PyMol to make the binding groove of HLA-DRB1 available for docking the viral peptide and its human homologue.Peptide-MHC docking was performed using GalaxyPepDock42. The analysis was performed using three different prediction algorithms namely, Bipipred, Emini surface accessibility and Kolaskar and Tongaonkar with default algorithm parameters.Shortlisted conserved regions from the structural polyprotein were explored for their ability to act as B cell epitopes. Full length amino acid sequence of structural polyprotein of Chikungunya virus (strain S27-African prototype) (UniProt ID: Q8JUX5) was subjected to linear B cell epitope prediction analysis available at Immune epitope database (IEDB)43 to predict the regions on the structural polyprotein of the Chikungunya virus (PDB ID: 3J2W). ElliPro accepts a PDB structure as input and uses three different algorithms based on protrusion index of the residues, protein shape approximation and neighboring residues clustering to predict linear antibody-based epitopes. Default parameters of the ElliPro program were used for prediction analysis.Further, to validate if the conserved regions act as potential linear B cell epitopes at the structural level, we employed ElliPro serverSupplementary information


Digramma interrupta remains disputable as it was raised by Cholodkovsky from Ligula alternans. This study aimed to survey the evolutionary relationships and the taxonomic position of D. interrupta and L. intestinalis. It also intended to support or reject the validity of D. interrupt as an independent genus and its correlation with L. intestinalis on the basis of their morphological characteristics and a study on molecular data.The position of Alburnoides bipunctatus and 418 Abramis brama, were collected from north and north-western parts of Iran. A. bipunctatus samples were obtained from fresh water sources of the Maragheh dam (northwest) and the Ramesar Lake (north). Moreover, samples of A. brama were captured from the Aras Dam (northwest) and the Bandar-e-Anzali lagoon (north). PCR was used to generate a fragment spanning two independent ITS-inclusive parts: ITS1-5.8S and ITS2 with two pairs of primers.Overall, 1301 fish varieties, including 883 L. intestinalis and D. interrupta samples amounts to about 3% to 7%. Between samples of L. intestinalis and GenBank data, and also between D. interrupta specimens and GenBank data, the diversity was seen for about 1% to 3%. Moreover, about 1% to 4% nucleotide variation was seen only in L. intestinalis samples caught from the same host, which could be supplementary to the presence of a species and/or strains in this genus.Nucleotide variation between D. interrupta was just a rare diplogonadic form of the Ligula species, not a different genus and not synonymous with the Ligula genus, but only another species of the Ligula genus.Maybe  Ligula intestinalis, and Digramma interrupta is distributed all around the world.Members of diphyllobothridae are the most significant cestoda that infect fish as the intermediated host in the plerocercoid phase . This faD. interrupta in diphyllobothridae family remains disputable since it was raised by Cholodkovsky from L. alternans. Morphological differences, with one set of reproductive organs in L. intestinalis and two sets of this in D. interrupta, present the most important differences between them. However, the morphological features are not completely reliable to distinguish genus from each other  that contains ITS1- 5.8S and ITS2. In addition, this study intends to assess the genetic diversity of L. intestinalis or D. interrupta from different hosts and geographical regions.One of the aims of this study was support or reject the validity of Alburnoides bipunctatus (A. bipunctatus) and 418 Abramis brama (A. brama), were collected from north and north-western parts of Iran. A. bipunctatus samples were obtained from fresh water sources of the Maragheh dam (northwest) and the Ramesar Lake (north). Moreover, samples of A. brama were captured from the Aras Dam (northwest) and the Bandar-e-Anzali lagoon (north)  . The samL. intestinalis and D. interrupta by using characters that are suitable for species identification according to taxonomic keys  was extracted by using a DNA isolation kit  according to the manufacturer’s instructions.PCR was used to generate a fragment spanning two ITS-inclusive independent parts, namely ITS1-5.8S and ITS2 with two pairs of specific primers. These primers were designed by Vector NTI11. The first for the 891bp fragment comprised ITS1-5.8S, and second for the 421bp sequence-long inclusive ITS2 locus . The PCRA. bipunctatus were distinguished from L. intestinalis, and all specimens of A. brama were determined to be D. interrupta. From 883 specimens of A. bipunctatus and 418 collected samples of A. brama, 558 (63.19%) and 67 fishes (16%) were infected, respectively (All isolates morphologically derived from ectively .D. interrupta and 10 samples were determined L. intestinalis. All the molecular results were coordinated with the morphological outcome.After blast, the data, according to the ITS locus, 13 isolates were determined from D. interrupta samples were registered in the GenBank under accession numbers KC900982-KC900994. Ten samples were referred to as L. intestinalis registered in the Gen-Bank under accession numbers KC900972-KC900981. Nucleotide variation between the L. intestinalis and D. interrupta samples was about 3% to 7%.All 13 L. intestinalis samples in this survey and the GenBank samples, and also between D. interrupta samples and the GenBank samples, nucleotide variation was about 1% to 3%. Finally, L. intestinalis samples were caught in Iran from the same host and nucleotide variations of about 1% to 4% were seen among them wonderfully.Between L. intestinalis and D. interrupta samples from present study with five isolates of this other L. intestinalis and D. interrupta that registered in GenBank, while another clade contains genus Schistocephalus solidus that is available in GenBank. All samples caught in this study, including 10 isolates of L. intestinalis and 11 isolates of D. interrupta, along with five isolates from GenBank were located in the same clade and so we could call them monophyletic. S. solidus is located in a different clade (The results of the genealogy tree based on the ITS2 locus display that two clades were obvious. One containing nt clade , Fig. 6.D. interrupta and L. intestinalis in Iran showed that most such reports have been from the northwest and western parts, while only a few reports came from the northern area (L. intestinalis is remarkably host-specific remarkably in Kenya (Studies on the distribution of ern area , 5. In tern area . Moreovein Kenya . AccordiClimatic conditions in the north-western part of the country are more conducive to these parasites, while another probable reason could be the neighborhood of Turkey. Since Turkey is a good source of pollution by Ligulidae plerocercoid, maybe water imported from Turkey can be attributed to infection of water by plerocercoid in northwest Iran . AnotherD. interrupta was detected only from A. brama, in Russia (L. intestinalis from A. bipunctatus. However, the morphological difference could be used to detect the genus and species from each other, but it is not enough and an accurate meter is needed for distinguishing the species and lineage from each other. Therefore, over the last two decades, molecular studies have helped scientists to improve and revise their information about the taxonomical status of parasites and their phylogeny.Host specificity probably exists in Iran because n Russia . In addin Russia , resultsDespite various studies on the epidemiological aspect of this Cestoda, any molecular characterization is not found in Iran and also in the entire Middle East zone. Thus, there is lack of sufficient information about the molecular characteristics of this Pseudophyllidean Cestoda in this zone. It confirms the necessity of a study on molecular and morphological features of this genus, and another genus of this family, as well as their true taxonomical station in this region.Digramma the transitional reproductive organ structure. A single set of reproductive organs was located at the anterior end of the larva in the carassian intermediate hosts and also two rows of this were located at the posterior end. Sexual dimorphism was reported in D. interrupta in A. brama the reproductive organs are found in one row, whereas two rows occur in those from carp (D. interrupta was just a rare diplogonadic form of the Ligula species (L. intestinalis dimorphism with some of them debating on the potential existence of this species in the Ligula genus. For example, based on ITS region and 28S rRNA, verified that plerocercoid of L. intestinalis specimens from Rutilus and Gobio may represent different strains or species (D. interrupta validity because of the transitional shape of reproductive organs in the proglottids of these two genera.Liao and Liang  watched rom carp . Maybe D species . In L. i species . Despite species , the morD. interrupta exhibited identical sequences with L. intestinalis with about 1% heterology based on the ITS1-5.8S regions analysis, whereas a high degree of genetic diversity of about 3% to 7% has seen based on the ITS2 locus. The present study also suggests that ITS2, unlike ITS1-5.8S sequences, can act as a useful genetic marker.In the present study, D. interrupta is almost different from L. intestinalis. Therefore, with nucleotide variation levels between L. intestinalis and D. interrupta specimens, besides morphological differences, it cannot be verified whether D. interrupta is a synonym of the genus L. intestinalis, against the theory of some researchers (There is not any yardstick for identifying species or genus borders by using the result of the DNA-sequence distinction, with a value of divergence based on ITS2 sequences, earchers , 11.L. intestinalis and D. interrupta is present only in some global locations (D. interrupta was just a rare diplogonadic form of the Ligula species (Ligula genus, but only another species of the Ligula genus. Phylogenetic tree results have displayed differences between clade A and clade B, verifying that the effect of geography in speciation and its effect on molecular structures. L. intestinalis and D. interrupta samples were different from samples registered in GenBank. Eleven samples of D. interrupta and eight samples of L. intestinalis were located in a common cluster so that it may be support the analogy of this genus. Moreover, the discrepancy amount of 1% to 4%, shown in the sequence of L. intestinalis samples, were compared with each other in the same host that could supplement the presence of a species and/or strains in this genus much like results derived by other researchers (On the other hand, they are different genera because of low levels of nucleotide variation and unstable morphological difference between them. Maybe polymorphism seen in ocations , 12. Besearchers .L. intestinalis from D. interrupta. It can also be used to detect the variations within L. intestinalis. Further, D. interrupta may be another species of the Ligula genus. On the other hand, they could not be different genera because of low levels of nucleotide variation arising from the possibility of polymorphism and therefore D. interrupta is another species of the Ligula genus. Its possible presence as a species and/or strains in the Ligula genus is based on host specificity.ITS2 may be one of the most useful markers used to distinguish


An isolated Rift Valley fever (RVF) outbreak was reported in 2018 in Free State Province, South Africa. Phylogenetic analyses based on complete genome sequences of 3 RVF viruses from blood and tissue samples indicated that they were related to a virus isolated in 2016 from a man returning to China from Angola. Rift Valley fever (RVF) is endemic to sub-Saharan Africa; major outbreaks were reported in South Africa during the 1950s, the 1970s, and 2008–2011  (2). LinOn April 28, 2018, an outbreak of suspected RVF was reported on a sheep farm in the Jacobsdal area of the Free State Province of South Africa. The illness rate was ≈55.8% and the case-fatality rate 100% (35 sheep died) (http://www.copanusa.com) to pierce and swab the tissue pools and then placed the swabs into Eppendorf tubes containing 700 μL phosphate-buffered saline (pH 7.0). After agitation, we removed 200 μL buffer for total nucleic acid extraction. We used either whole blood in EDTA (RV2613-1/RSA/2018) or a combination of tissue swab specimens from liver, spleen, and kidney for extractions (RV2613-2/RSA/2018 and RV2613-3/RSA/2018) using the MagNA Pure 96 . We detected the presence of RVFV RNA using real-time reverse transcription PCR (RT-PCR) (https://www.thermofisher.com) in a 20-μL reaction with 0.25 μmol/L of each primer (https://www.inqababiotec.co.za), for Sanger sequencing, using the primers incorporated during the generation of the amplicons and 9 additional primers   Table 1.h primer  Table 1  primers  Table 1.https://beast.community) under the Hasegawa-Kishino-Yano substitution model, a strict molecular clock, and a constant population size estimated that RV2613/RSA/2018 and BIM-01/2016 had a common ancestor ≈7 years ago that shared a common ancestor with Kakamas/2009 ≈28 years ago. Virus RV2613/RSA/2018 had a higher sequence identity with the original Entebbe-44 isolate than the SNS vaccine or vaccine-derived Ken Rintoul-57  were similar to one another: no nucleotide differences in the large segment, 2 in the medium segment, and 1 in the small segment. The high sequence identity among these 3 viruses and the lack of segment reassortment, together with the isolated geographic distribution of the outbreak, indicate a single introduction. After phylogenetic analysis, we clustered the 3 viruses into lineage K, with their closest known relatives BIM-01/2016, isolated from a worker from China in Angola, and the virus RVFBJ01/2016 derived from cell culture . We assentoul-57  Table 2.The sequence data imply that this outbreak was likely the result of a single introduction of virus that probably remained localized to 1 farm because of the onset of colder winter temperatures and a decline in rainfall. The phylogenetic relationship of this virus to known others suggests a persistent, yet largely unnoticed, low-level spread of RVFVs in southern Africa. This finding reemphasizes the importance of active disease surveillance programs with diligent reporting of suspected cases, as well as suitable vaccination regimens.Additional information regarding genomic characterization of Rift Valley fever virus, South Africa, 2018.


The steep decline in Zika cases since 2016 has led to a perception that the threat posed by the virus has diminished. Recent outbreaks and global spread underline the need for continued vigilance. Tatum Anderson and Gary Humphreys report. On 11 September 2018, a 78-year-old resident of Shastri Nagar, an area to the north of Jaipur in Rajasthan, India, checked into the Sawai Man Singh hospital reporting joint pain and weakness.Tests for dengue and influenza came back negative. Samples of the woman’s blood were sent to the National Institute of Virology in Pune, Maharashtra state, where it was determined that she was infected with the Zika virus.State health authorities were notified and initiated a response that included boosting surveillance and control efforts in the Shastri Nagar area.Aedes aegypti mosquitos being caught, three of which tested positive for Zika. According to Dr Neena Valecha, Director of the National Institute for Malaria Research in New Delhi, vector surveillance in and around Shastri Nagar resulted in about 200 “This is the first time the Zika virus has been isolated in the mosquito population in India, and it suggests that the disease is being transmitted locally rather than being brought in from outside,” she says.A second outbreak, located in the neighbouring state of Madhya Pradesh, also started in November. As of 20 November 2018, 160 people had been confirmed to be infected with Zika virus in the Jaipur outbreak, including 63 pregnant women. In the Madhya Pradesh outbreak, 120 people, including 34 pregnant women, had been confirmed infected.“This is the first time the Zika virus has been isolated in the mosquito population in India.” Neena ValechaThese outbreaks are just the latest reminders of two important facts. First, the risk of becoming infected with the Zika virus is global. The Zika virus is present in more than 84 countries. Second, transmission is ongoing and the virus can be expected to spread.Aedes mosquitoes, the Zika virus was first reported in humans in 1952. It was not associated with severe disease until an outbreak in French Polynesia in 2013–2014 and to this day only around 20% of people infected suffer any symptoms at all. Most of those infected experience mild symptoms such as fever, rash, conjunctivitis, muscle and joint pain, or headache.Transmitted primarily by Zika infection can, however, be followed by Guillain–Barré syndrome, a serious neurological condition in which the immune system attacks the peripheral nerves, but what has put the virus under the spotlight in recent years, most is its association with birth defects.Zika infections in pregnant women can be transmitted to the developing fetus and can cause severe malformations that collectively make up congenital Zika syndrome: microcephaly , limb contractures and eye abnormalities.The 2015–2016 outbreak in the Region of the Americas brought the Zika virus, until then a little known viral infection, to the fore, revealing unknown complications and triggering a public health emergency.At the height of the 2016 emergency, 216 207 probable cases of acute Zika virus disease were reported in Brazil, the focus of the epidemic, and an estimated 8604 babies were born with malformations.In response, on 1 February 2016, the World Health Organization (WHO) declared a Public Health Emergency of International Concern, prompting a comprehensive response, including the development of surveillance networks, vector-control measures and risk communication strategies.WHO also encouraged the development of diagnostic technologies and included Zika among the list of priority diseases targeted by WHO’s global strategy and preparedness plan, designed to facilitate research and development during epidemics, called the R&D Blueprint.But just as the international response was ramping up, Zika cases started to decline globally.“The most recent available data show that there has been a substantial decline in the number of reports of suspected cases of Zika in Brazil since 2016,” says Dr Márcio Henrique de Oliveira Garcia at the Secretariat of Health Surveillance, Ministry of Health of Brazil, who notes that there have been similar declines throughout the Americas.Following this decline, WHO declared the end of the Public Health Emergency of International Concern in November 2016.For Dr David Heymann, chair of the International Health Regulations Emergency Committee on the Zika Virus, which advises WHO on its Zika response, the decision reflected both the evolving epidemic and the response that had been initiated.“Bringing the public health emergency to a close was recommended by the emergency committee to transition the program from an emergency response to a long-term commitment for prevention and control,” he says.Three months later, the then Director–General of WHO, Dr Margaret Chan, said that WHO and affected countries needed to manage Zika not on an emergency footing, but in the same way they respond to other established epidemic-prone pathogens, like dengue and chikungunya, which in her words “ebb and flow in recurring waves of infection.”The tidal metaphor raises compelling questions. Why did Zika ebb? Where and how will it re-emerge?For Dr Eve Lackritz, the Zika taskforce lead in the high-threat pathogens unit of WHO’s Health Emergencies Programme, the Zika epidemic likely waned due to an increase in population-level immunity. The more that populations have been exposed to the virus, the more they have developed immunity, hence the decline in cases.Answering the second question is harder. Some epidemiologists have suggested that populations with no previous exposure to the virus are most likely to experience outbreaks in the future. Lackritz points out that even in areas severely affected by the epidemic, there remain large numbers of people who were not exposed and therefore have not developed immunity.“Smaller outbreaks continue to occur in the Americas and elsewhere as infection spreads to pockets that had previously been unaffected and where the population remains susceptible,” she says, pointing to ongoing case reports. Evidence of endemic transmission has been reported in Indonesia, Lao People's Democratic Republic, Singapore, Thailand and Viet Nam. “Transmission is likely ongoing, but remains poorly understood,” she says, emphasizing the need to strengthen surveillance systems worldwide to improve our understanding of the extent and patterns of Zika virus transmission.“Evidence of the global spread of the epidemic (Brazilian) strain of Zika virus continues to accumulate.” Eve LackritzA question of considerable importance for Valecha, is whether new outbreaks in India and elsewhere will result in cases of congenital Zika syndrome. Here too, the picture is unclear.In addition to congenital malformations caused by the Brazilian Zika strain, Lackritz points out there have also been babies born with microcephaly after their mothers were infected with the endemic Asian strain reported in Thailand and Viet Nam.In addition, there is evidence that the Brazilian Zika strain is spreading. “Evidence of the global spread of the epidemic (Brazilian) strain of Zika virus continues to accumulate,” she says. “Angola, for example, has reported a cluster of microcephaly cases and recently identified the introduction of the Brazilian Zika strain.”Are India’s Zika virus outbreaks part of the pattern of smaller, localized outbreaks or the beginning of something bigger?Prior to the current outbreaks in Rajasthan and Madhya Pradesh, there had been only four confirmed cases of Zika virus infection in the entire sub-continent, three of them in Ahmedabad Gujarat, which is also adjacent to Rajasthan. Low levels of population immunity might therefore be expected.“The situation is a matter of concern,” says Valecha, but she is hopeful that the government’s vigorous response, which has included increased surveillance and monitoring and vector control efforts, will be sufficient to stop the outbreaks in their tracks or reduce transmission to a minimum.Aedes mosquitos thrive in urban environments, and breed in small collections of water in trash, used tyres and artificial containers.However, she insists on the importance of focusing control efforts, ranging from larval control to personal protection measures, particularly in urban areas. “In the past, vector control has tended to focus on rural areas,” she says. “It is important to adapt vector control programmes to increasingly urbanized populations.” Lackritz concurs, noting that Going forward, new vector control methods will give health authorities more options in the way they deal with Zika outbreaks. There are several in the pipeline, including Wolbachia-based biocontrol and OX513A transgenic mosquitoes.A safe and effective Zika virus vaccine would also be helpful. Since 2016, research and development projects on 45 Zika vaccine candidates have been initiated. Several vaccine candidates are currently in phase 1 and 2 of clinical trials. However, many challenges remain, including the low number of cases occurring globally, which limits the ability of studies to evaluate the protective effects of vaccines in phase 3 trials.Monitoring for congenital malformations will be an important focus of India’s Zika response in the coming months, as public health officials seek to determine the extent of the harm caused by the outbreaks. Says Valecha: “The true picture will only be visible when we follow these women who got the infection in the first trimester.”