First cases of clinically uncommon African swine fever (ASF), caused by virus genotype II are described in this article. These cases occurred in Armenia, Tavush region, Dilijan municipality in 2011. The aim of this study was to identify and describe the new pathogenic forms of ASF in Armenia.The isolation and identification of ASF virus (ASFV) were carried out using conventional techniques. Clinical signs of infection were recorded daily. Gross anatomical pathology characteristics were observed during routine postmortem examinations. Blood and serum were obtained by puncture of the jugular vein using a vacutainer system.The presence of ASFV DNA in the spleens was confirmed by polymerase chain reaction. Sequenced sections of p72 showed phylogenetic identity to genotype 2. The pathology exhibits unusual manifestations of the main disease. The unusual form of ASF demonstrates characteristics of a subacute form of the disease, with the possibility of conversion to a chronic form. Decreased lethality, low level of hemorrhages, and absence of severe pancytopenia in smears from spleen, lymph nodes, and blood are common features of the new form of ASF. Unlike severe thrombocytopenia in the typical ASF, the unusual form exhibited moderate or minor decrease of this feature. Despite a moderate decrease in hemadsorption titers, the unusual pattern of the disease was characterized by viremia and the presence of the virus in the visceral organs, including the brain.Our data allow assuming that new nosological form of ASF (genotype II) may present as a transitional form of the disease with the possibility of chronization. African swine fever (ASF) is the main threat to the porcine industry in the world. Depending on viral and host factors, ASF virus (ASFV) infection of domestic swine can be expressed in several disease forms, ranging between highly lethal up to 100%) and subclinical. Manifestations in 2007 ASF affecting domestic pigs and wild boars have been reported in the Caucasus region for the first time. The virus strain involved was related to isolates of genotype II. Almost all cases of ASF caused by genotype II can be described as peracute, acute, and/or subacute forms [00% and sInitially, ASF cases in Dilijan district were recorded in 2007, while the first cases of atypical ASF in Armenia, in Dilijan district, evolved in 2011 [It is well known that transmission of ASF virus can occur through direct contact between sick and healthy pigs or by contact with infectious excretions and secretions. Indirect transmission can also occur if healthy animals ingest infected meat products or have contact with contaminated fomites . In the Biological samples collection was approved by the Institutional Review Board/Independent Ethics Committee of the Institute of Molecular Biology of NAS RA (reference number IRB00004079).Biological samples were collected on November 30, 2011, and December 15, 2011. The investigated pigs were free from known porcine viral diseases and vaccinated against the classical swine fever. Blood and serum were obtained by puncture of the jugular vein using a vacutainer system. Samples of liver, brain, bone marrow, heart, kidney, spleen, lymph nodes, and lung were taken and held in separate, disposable plastic containers.Clinical signs of infection were recorded daily. Gross anatomical pathology characteristics were observed during routine postmortem examinations.Laboratory analyses were conducted at the Laboratory of Cell Biology and Virology, IMB of National Academy of Sciences of Armenia. All stages of processing and sample preparation for the diagnosis of ASF were performed in a biosafety level 3 laboratory by qualified staff. Blood clots from dead pigs were used to obtain serum samples, and kidney, lung, brain, bone marrow, liver, spleen, and lymph node biopsies were used for tissue samples.DNA was extracted from 200 μl of heparinized whole blood samples and spleen samples by using a 5 PRIME Archive Pure DNA Cell/Tissue kit. Specific oligonucleotide primers and the fluorogenic probe were designed to target a highly conserved region within the B646L (p72) and B602L (chaperon) open reading frames. For phylogenetic analysis of ASFV, a portion of the highly conserved p72 gene was amplified and sequenced. The amplified products were characterized by nucleotide sequencing and compared to those obtained from published sequences.Before third epidemic wave in Armenia, all ASF cases were documented as well-known peracute (rare) and acute and/or subacute  forms . IsolatePostmortem examination at autopsy of the pigs infected by Georgia 2007 and Dilijan 2011. IMB isolates revealed that infection with new isolate leads to a reduction in both the number and size of hemorrhages in all visceral organs. However, swelling of the spleen and liver still persisted. Most of the lymph nodes were not affected. Specifically, unusual ASF was characterized by insignificant lymphopenia in the blood, bone marrow, and spleen. In lymph nodes, lymphopenia was absent. In all studied tissues, leukocyte population demonstrated a slight left shift. It is also necessary to state about marked (approximately 1.5-2.0 log) reduction of ASFV hemadsorption titers (compared to Georgia 2007 isolate) in porcine sera, despite the verified finding of virus in all the viscera of pigs.In general, the atypical form of ASF (caused by Dilijan 2011 IMB isolate) described above demonstrates the characteristics of a subacute form of the disease, with the possibility to conversion to a chronic form. The new isolate developed an uncommon clinical manifestation of ASF - unlike the common subacute form, it demonstrated partially reduced the ability for hemadsorbtion and decreased frequency and intensity of hemorrhages. It also showed a less pronounced pathology of blood clotting system compared to the typical ASF induced by Georgia 2007 isolate . LeukopePresumably, such phenomenon can be either a result of penetration of a new type of ASF virus with lower virulence into the zone of previous ASF outbreaks or variation in the same virus, with the arising of new, less virulent mutant strain.et al. [It is known that the ASFV is capable of changing quickly enough to result in a transition from acute/subacute disease to a chronic form. It is reported that the ASF virus is inclined to modification in the chronic form of the disease . There cet al. . Howeveret al. .In Armenia, like in all Caucasus region, the majority of the swine production (over 90%) can be classified as backyard and where very scarce information is available on pig trade patterns . This tyMost likely cause of uncommon ASF is the change in the virus genome, which is consistent with a typical evolution pattern of viruses. Although DNA viruses have a significantly lower rate of mutations, compared to RNA viruses, their variability is still much higher than that in cellular organisms. Emergences of new, less virulent mutant of ASF virus has high importance for the survival of the virus, as the chances of its transmission are being increased. It is well documented that DNA viruses have a tendency to establish prolonged, chronic, and inapparent (latent) persistent infections ,18. The In the current study, we show for the first time formation of the unusual or transient form of ASF during the third epidemic wave in Armenia.The new strain of ASF virus isolated from pigs in northeast Armenia caused distinct pathological changes (disease has turned into a more chronic infection during late autumn 2011), compared with acute form ASF (Georgia 2007). Nucleotide sequences obtained from the p72-based PCRs of Dilijan 2011 IMB virus clustered with p72 genotype II. So we can conclude arising of the unusual or transient form of ASF during the third epidemic wave in Armenia.ZAK and EMK designed the experiment. MAS, LHH, and HEV conducted the experiment. ZAK did technical writing and revision of the manuscript. ZAK and EMK prepared the manuscript. All authors have read and approved the final version of the manuscript.


Flaviviridae family, is a mosquito-borne virus known to cause microcephaly through vertical transmission. Infection presents with mild, self-limiting symptoms. Currently, a Zika virus outbreak has spread across most of South and Central America. Travel-related and sexually transmitted cases have been reported across the United States. However, the vector-borne transmission has been limited to Florida and Texas. We present seven cases of Zika virus infection that presented at a single institution in South Florida.Zika virus, an arbovirus of the Patients were included that had real-time polymerase-chain reaction (RT-PCR) for Zika virus RNA in urine or serum or enzyme-linked immunosorbent assay (ELISA) for Immunoglobulin M (IgM) antibody against Zika virus in serum.All seven patients reported recent travel or employment in areas of active Zika virus transmission and at least two of the four most commonly reported symptoms  with a rash present in all patients. All patients had positive RT-PCR for Zika virus RNA in urine. RT-PCR for Zika virus RNA in serum was negative in four of five patients that were tested, indicating that these patients likely presented one to two weeks after symptom onset.The future of Zika virus outbreaks in other cities in the United States is still uncertain. However, it is clear that prevention and control policies are urgently needed. We have presented seven confirmed cases of Zika virus infection in South Florida. In addition to conducting research concerning both the diagnostic and therapeutic aspects of the virus, there is a need for public awareness of its presentation, methods of transmission, and subsequent clinical outcomes. Zika Virus (ZIKV) is an arbovirus of the Flaviviridae family that is most commonly transmitted via Aedes aegypti and Aedes albopictus mosquito vectors . Other mIn 1947, ZIKV was first discovered in rhesus monkeys in the Zika Forest of Uganda. The first human cases were reported in Nigeria in 1954. ZIKV spread to Malaysia by 1969 and Indonesia by 1977. The first major ZIKV outbreak spread by mosquitos was recognized in 2007 in the Yap Islands of Micronesia, followed by a second larger outbreak in French Polynesia between 2013-2014 ) and dengue (IgM) virus serology were negative. Body fluid samples were analyzed at an offsite laboratory; therefore, the results were not available until after discharge. A pregnancy test was negative. The patient was treated with one dose of each of the following medications: morphine 2 mg IV, ondansetron 4 mg IV, and methylprednisolone 80 mg IV. She was also given 1 L of normal saline IV. On discharge, the patient was instructed to follow up with her primary care physician.A 33-year old female presented to the emergency department complaining of a gradually worsening rash localized to her chest, neck, and right side of her face for two days. Associated symptoms were fatigue, sore throat, nasal congestion, “itchy, red, watery eyes,” and myalgia. She denied fever, chills, and arthralgia. She had returned to the United States from the Dominican Republic one week prior, but she did not recall being bitten by a mosquito. She reported that her son had a positive test for ZIKV. On physical exam, the patient was afebrile and hemodynamically stable. She had an erythematous posterior pharynx and a trace fine papular rash on the chest, neck, and right face. Blood chemistry tests were all within normal limits. Hematologic tests showed mild anemia , mildly increased lymphocyte percentage , and mildly increased monocyte percentage . RT-PCR for ZIKV RNA was positive in urine and negative in serum. The patient was not tested for chikungunya or dengue virus infections. The ZIKV blood and urine samples were analyzed at an offsite laboratory; therefore, the results were not available at the time of discharge. A pregnancy test was negative. The patient was treated with one dose of each of the following medications: diphenhydramine 25 mg IV, acetaminophen 975 mg IV, and ondansetron 4 mg IV. She was also given 1 L of normal saline IV. On discharge, the patient was instructed to follow up with her primary care physician.3/mL, normal range: 4.5–13.5 × 103/mL) and a mildly increased hemoglobin  and hematocrit . Lymphocyte percentage was decreased . RT-PCR for ZIKV RNA was positive in urine and negative in serum. Serology was positive for dengue virus  and negative for chikungunya virus (IgM and IgG negative). Body fluid samples were analyzed at an offsite laboratory; therefore, the results were not available at the time of discharge. The patient received no treatment during this visit. On discharge, the patient was instructed to follow up with his primary care physician and to avoid sexual contact.A 17-year-old male presented to the emergency department due to his father’s concern that the patient was infected with ZIKV. He had no complaints or symptoms at the time of the visit. The patient reported experiencing joint pain, pruritic generalized rash, headache, and fever on the previous day. He denied any ocular complaints. He had returned from Puerto Rico on the previous day. While in Puerto Rico, he was in contact with a friend who experienced the same symptoms. Per the patient, the friend’s physician told him he had a viral condition. On physical exam, he was afebrile and hemodynamically stable. There were no positive relevant findings on physical exam. Blood chemistry tests were all within normal limits. Hematologic tests showed a mildly decreased white blood cell count  from a 12-day trip to Saint Martin and Saint Barthélemy: territories with known vector-borne ZIKV transmission . The patA 49-year-old female presented to her primary care physician with fever and pruritic rash for four days. She denied symptoms of conjunctivitis and joint pain. Three days before the onset of symptoms, she returned with her husband (see Case 6 above) from a 12-day trip to Saint Martin and Saint Barthélemy: territories with known vector-borne ZIKV transmission . The patThese seven cases illustrate the common clinical picture of a ZIKV infection. All patients had risk factors for acquiring the infection – whether by working in an active vector-borne area, person-to-person transmission, or traveling to endemic areas. The patients presented with classic signs and symptoms of ZIKV infection, including fever, chills, fatigue, maculopapular rash, arthralgia, headache, and conjunctivitis. Five patients (71%) had fever, arthralgia, and rash. All seven had rash, and at least one more of the common ZIKV infection symptoms. Only two patients (29%) had less than three of the common symptoms. One of these two patients presented with other less common symptoms . Six of the seven patients were symptomatic at the time of presentation, and all of their symptoms were self-limiting.RT-PCR of ZIKV RNA was positive in urine for all seven cases. Four patients (57%) were negative for ZIKV RNA in serum Table . These fThe patients in cases 6 and 7 were most likely infected while traveling, but local vector-borne infection cannot be ruled out. With the lower end of the range of the incubation period being two days after exposure , it is pSevere disease requiring hospitalization is uncommon, and case-fatality rates are low . HoweverAlthough local transmission of ZIKV has not been reported in recent months, it remains a concern. As summer months arrive, Aedes aegypti populations have historically grown up to 10 times larger than in winter. Therefore, there is a need for public awareness of its presentation, methods of transmission, and subsequent clinical outcomes. Efforts in these areas now could serve to help prevent or limit local transmissions from returning to South Florida in the warmer months.


Picornavirales order of viruses. PSAV1 is closely related to Biomphalaria virus 2 (BV2).We present the complete genome sequence of a virus found in raw sewage collected in Pittsburgh, PA, USA. Pittsburgh sewage-associated virus 1 (PSAV1) encodes one large open reading frame with conserved domains typically found in the  Picornavirales order contains several virus families, such as Picornaviridae, Dicistroviridae, and Secoviridae, that infect mammals, insects, and plants. These viruses are small and nonenveloped and contain a monomeric single-strand positive-sense RNA genome of 7 to 10 kb in length. Usually they contain one or two large open reading frames (ORFs), which generate a polyprotein that is subsequently cleaved by a viral protease into functional polypeptides. The global diversity of these and other RNA viruses continues to be revealed through deep sequencing studies (–The  studies . Raw sew studies –5.Dicistroviridae. The termini of the genome were completed with 5′ and 3′ rapid amplification of cDNA ends (RACE), which added 49 and 201 bp to the 5′ and 3′ ends, respectively. The 3′ RACE revealed that the genome is poly(A) tailed. The 8,995-bp genome sequence was confirmed by Sanger sequencing of staggered PCR amplicons across the genome.Viral nucleic acid from Pittsburgh raw sewage was extracted and sequenced . After nBiomphalaria virus 2 (BV2) (GenBank accession number KY024322), an unclassified Picornavirales virus  binding and ATP hydrolysis (4D), B (PXG), and C (GDD), which are characteristic of a picornavirus RdRp (A BLASTN search showed 78% identity between PSAV1 and es virus , includidrolysis . PSAV1 pdrolysis . The RdRrus RdRp . We werePicornavirales clade remain to be discovered.The phylogenetics of PSAV1 show that it is closely related to BV2, a snail virus. Since PSAV1 was isolated from raw sewage, its host is unknown. Both viruses share no significant homology to other viruses. These findings suggest that more members of this distant MG552116.The genome sequence of PSAV1 has been deposited in GenBank under the accession number


LRR-RLK genes have been investigated in flowering plants; however, no comprehensive studies have been performed for these genes in more ancestral groups. The subfamily classification of LRR-RLK genes in plants, the evolutionary history and driving force for the evolution of each LRR-RLK subfamily remain to be understood.Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases in plants and play crucial roles in development and stress responses. The evolutionary relationships among LRR-RLK genes in the Physcomitrella patens moss genome, 67 LRR-RLK genes in the Selaginella moellendorffii lycophyte genome, and no LRR-RLK genes in five green algae genomes. Furthermore, these LRR-RLK sequences, along with previously reported LRR-RLK sequences from Arabidopsis thaliana and Oryza sativa, were subjected to evolutionary analyses. Phylogenetic analyses revealed that plant LRR-RLKs belong to 19 subfamilies, eighteen of which were established in early land plants, and one of which evolved in flowering plants. More importantly, we found that the basic structures of LRR-RLK genes for most subfamilies are established in early land plants and conserved within subfamilies and across different plant lineages, but divergent among subfamilies. In addition, most members of the same subfamily had common protein motif compositions, whereas members of different subfamilies showed variations in protein motif compositions. The unique gene structure and protein motif compositions of each subfamily differentiate the subfamily classifications and, more importantly, provide evidence for functional divergence among LRR-RLK subfamilies. Maximum likelihood analyses showed that some sites within four subfamilies were under positive selection.We identified 119 LRR-RLK genes was established in early land plants. Positive selection contributed to the evolution of a few LRR-RLK subfamilies.Much of the diversity of plant The online version of this article (doi:10.1186/s12862-017-0891-5) contains supplementary material, which is available to authorized users. All living organisms sense and conduct signals through cell surface receptors. In plants, many such cellular signaling transductions are mediated by receptor-like kinases (RLKs). The largest group of plant RLKs is the leucine-rich repeat RLK family (LRR-RLK) . LRR-RLKLRR-RLK genes have dual roles in development and defense due to cross-talk between these two pathways or recognition of multiple ligands by the same receptor [LRR-RLK genes have been extensively studied and the results show that they have crucial roles in plant development and stress responses. However, there are numerous LRR-RLK genes, and the functions of the vast majority of them are largely unknown.LRR-RLKs function in a wide array of plant processes. Some LRR-RLKs are involved in the control of plant growth and development; for example, CLV1 is involved in controlling meristem development , 7, RUL1receptor . For exaLRR-RLK genes, investigations have been only performed in flowering plants [LRR-RLK genes remain to be answered. First, how many LRR-RLK gene subfamilies can be classified in plants, and when did each subfamily originate? Based on the phylogenetic relationships of kinase domains and the arrangement of LRR motifs, LRR-RLK genes were classified into 15 groups in Arabidopsis thaliana [Oryza sativa [Populus trichocarpa [LRR-RLK genes from the same species; therefore, these studies provide a useful but limited phylogenetic framework for the classification of these genes in plants. Nevertheless, previous studies did not elucidate the origin of each subfamily due to the lack of phylogenetic analysis of LRR-RLK genes from diverse plants, including algae, bryophytes, and different lineages of vascular plants.Evolutionary studies of genes can provide insights into possible gene functions and mechanisms of gene duplication and functional divergence. With regard to the evolution of g plants , 16–23. a sativa  and 14 gchocarpa . The phyLRR-RLK intron/exon structures and protein sequences evolved accompanying the plant evolution. Protein sequences and motifs are directly related to protein function. Introns have important roles in cellular and developmental processes via alternate splicing or gene expression regulation [ERECTA LRR-RLK gene in A. thaliana [LRR-RLK subfamilies is important to understand the evolution of gene function among the subfamilies [LRR-RLK genes from flowering plants [LRR-RLK genes within the same subfamily usually have similar intron/exon structures and protein motifs, while members of different subfamilies exhibit different genomic structures and protein motifs [Second, it is not known how gulation . The prethaliana . Analysifamilies . Earlierg plants , 18. Forn motifs –22. HoweLRR-RLK subfamily? Genes accumulate mutations during evolution, and this may be due to a relaxation of purifying selection or the action of positive selection [LRR-RLK subfamilies defined in A. thaliana and O. sativa [LRR-RLK genes, of which 50% contained codons under positive selection [LRR-RLK subfamilies defined in the present phylogenetic analysis were controlled by positive selection, and evaluated the relative importance of relaxation of purifying selection and positive selection in the evolution of LRR-RLK subfamilies.Finally, what was the evolutionary force driving the evolution of each election , 28. Poselection –34. Prev. sativa , 35–38. election . In thisLRR-RLK genes in plants. Previous studies have identified LRR-RLK genes mainly from flowering plants [LRR-RLK sequences in the complete genomes of representative species of other major plant lineages, including four completely sequenced green alga species , one moss species (Physcomitrella patens), and one lycophyte species (Selaginella moellendorffii). Next, these sequences and previously identified sequences in two flowering plants  [LRR-RLK subfamilies in divergent plant species and determine the origin of each subfamily, (2) to determine the evolutionary history of gene structures and the evolutionary patterns of the protein sequences of each subfamily, and (3) to evaluate potential selection pressure that promoted the evolution of each LRR-RLK subfamily.The complete genome sequences from different major plant lineages now available allow us to examine the evolutionary history of g plants , 17–23. Arabidopsis thaliana LRR-RLK sequences reported by Shiu et al. [Arabidopsis Information Resource’  [Oryza sativa LRR-RLK sequences were obtained from a previous study [LRR-RLK subfamily of A. thaliana were used as queries to conduct Blastp searches  against the protein databases of six species available on Phytozome v11.0 [Chlamydomonas reinhardtii, Micromonas pusilla CCMP1545 and Micromonas sp.RCC299, Ostreococcus lucimarinus, and Volvox carteri), one moss species (Physcomitrella patens), one lycophyte species (Selaginella moellendorffii). The resulting hits were downloaded from Phytozome v11.0. Identical and defective sequences were identified and eliminated by manual inspection in BioEdit [http://pfam.xfam.org/) [http://smart.embl-heidelberg.de/) [http://www.cbs.dtu.dk/services/TMHMM/) [LRR-RLKs if they contained LRRs in the ECD, TMs, and a KD [LRR-RLK genes were identified in four fully sequenced green alga species. Therefore, only LRR-RLK genes identified in the genomes of P. patens and S. moellendorffii were used for further analysis. Our preliminary studies found that the LRR-RLK genes identified in the P. patens genome version 3.3 were well annotated. However, the annotations of some LRR-RLK genes in the S. moellendorffii genome version 1.0 had some problems according to the analysis of sequence homology and gene structure. To prevent the inclusion of falsely annotated data that could bias our analyses, we manually re-annotated the problematic LRR-RLK genes from S. moellendorffii using available expression data and sequence similarities with the homologous genes.The u et al.  were retis.org/) . The Oryus study . The kinme v11.0 . The six BioEdit . Potentiam.org/)  and SMARerg.de/)  to confi/TMHMM/)  to confiLRR-RLK sequences were obtained, we compared the proportions of LRR-RLK genes among all protein-coding genes for different genomes. The numbers of LRR-RLK genes contained in the genomes of angiosperm species were obtained from published papers [After d papers , 17–22. LRR-RLK sequences obtained in the present study and previously reported in A. thaliana and O. sativa [. sativa , 17 were. sativa . Multipl. sativa , after w. sativa . Sequenc. sativa . The bes. sativa . The staLRR-RLK genes were retrieved from the Phytozome v11.0. The intron/exon structures of some re-annotated sequences were determined by comparing their CDS with their corresponding genomic DNA sequences, after which these structures were displayed using the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) [http://alternate.meme-suite.org/) [To study intron evolution, the intron/exon structures for each gene were mapped to their corresponding genes. The structures of most edu.cn/) . The gente.org/) . Due to N/dS) is an effective measure to detect selection on protein-coding genes: ω = 1, neutral evolution; ω < 1, purifying selection; and ω > 1, positive selection. To evaluate the selective pressures acting on the LRR-RLK genes in each subfamily, we estimated the ω value of each subfamily using a maximum likelihood method. Previous studies demonstrated that the positive selection pressure acting on orthologs and paralogs differs in extent [The nonsynonymous/synonymous rate ratio  How did the intron/exon structures of each subfamily evolved along the major plant lineages? (2) Are gene structures conserved within subfamilies? To answer the first question, a comparison of LRR-RLK gene structures in A. thaliana and O. sativa with those of the same subfamilies in P. patens and S. moellendorffii was performed. According to the evolution of gene structures along the major plant lineages, LRR-RLK subfamilies were classified into three categories. In subfamilies of category A  has two introns, one of its introns is identical to that of the other members of this subfamily. Furthermore, another gene from P. patens has only the same one intron as other members. These findings suggest that the one intron structure of subfamily IX was established early and conserved across different plant lineages; and the extra intron in one P. Patens gene may be specific to P. patens. Similarly, the same gene structures are shared by four species in members of LRR subfamilies III, VI-1, VIII-1, IX, X, XIII-1, XIII-2, XIV, and XV   program v.4.10.2 [To further investigate the protein evolution of v.4.10.2 . LRR repv.4.10.2 . The typv.4.10.2 . We idenLRR-RLK KD contains approximately 250–280 amino acid residues. MEME analysis identified the following 20 motifs in the LRR-RLK KD from the N-terminus to the C-terminus: Q-M3, Q-M4, Q-M1, Q-M2, Q-M5, Z-M2, Z-M1, Z-M5, Z-M3, Z-M4, H-M1, H-M3, H-M10, H-M9, H-M4, H-M5, H-M6, H-M7, H-M8, and H-M2  , 5. Thesversus model M0 were significant in all cases, indicating that ω was variable among sites along the LRR-RLK sequences in all UP clusters  and SO clusters (related only by speciation) were identified as reported in Fischer et al.  using a rs Table . Models rs Table : 1, 2, 6LRR-RLK genes in the Physcomitrella patens moss genome, 67 LRR-RLK genes in the Selaginella moellendorffii lycophyte genome, and no LRR-RLK genes in five green algae genomes   plants. Differences in the copy numbers of LRR-RLK genes in moss, lycophytes and angiosperms may be due to the different expansion rates of LRR-RLK genes in different genomes, but may also be due to the difference in genome sizes. To distinguish these factors, we compared the proportions of LRR-RLK genes among all protein-coding genes in different genomes. The percentage of LRR-RLK genes in moss is 0.36%, while that in S. moellendorffii is 0.30%  also contained LRR-RLK gene sequences from P. patens. Among the 18 subfamilies that included P. patens LRR-RLK sequences, two subfamilies (I and VIII-2) lacked S. moellendorffii sequences. Using the most parsimony assumption, the ancestors of subfamilies I and VIII-2 likely evolved from the common ancestor of land plants before the divergence of specific lineages, which were subsequently lost in the lycophyte. Therefore, most LRR-RLK subfamilies  were established early in land plant evolution before the divergence of moss and other land plant lineages. In addition, in subfamilies II, III, VII-2, VIII-1, X, and XI, several clades include sequences of P. patens, S. moellendorffii, A.thaliana and O.sativa, and this is the opposite situation for other subfamilies. The result could be interpreted as contrasted ancestral copy number between subfamilies. Namely, for these subfamilies, there were probably several LRR-RLK genes before the split between P. patens, S. moellendorffii and angiosperms. The early origin of most subfamilies indicates that genes in most subfamilies may have central roles in the regulation of common developmental and defense pathways of different land plant lineages. Some subfamily members with specific developmental roles, such as the control of pollen tube development (PRK in subfamily III) [P. patens or S. moellendorffii; this subfamily is only found in A. thaliana  and O. sativa, indicating that it evolved recently in higher plants. MRH1 is required specifically for root hair elongation growth [MRH1 homologs in moss may reflect the fact that mosses possess rhizoids. The absence of MRH1 homologs in the lycophyte S. moellendorffii suggests that root hair growth may be regulated differently in lycophytes and flowering plants.According to the tree topologies and clade support values, LRR-RLK genes were classified into 19 subfamilies. The subfamily definitions were supported not only by the phylogenetic analysis, but also by the unique gene structures   and vascily III) , were esily III) . Furthern growth . The absLRR-RLK genes. For nearly half of the subfamilies , identical intron/exon gene structures in the same subfamily were found in P. patens, S. moellendorffii, A. thaliana and O. sativa; these gene structures were shared by the majority members of each subfamily  . In the 0 . In thmicrobes . Hence, When the LRR domain binds a ligand, the KD is activated to trigger the subsequent activation of downstream substrates . The conversus positive selection in the evolution of LRR-RLK subfamilies, we performed selection pressure tests. Recent studies demonstrated that orthologs and paralogs in gene families evolve under different levels of positive selection pressure [LRR-RLK genes in angiosperm, 50% of which contained codons under positive selection pressure [LRR-RLK genes from more basal plants are considered . Moreover, our results are largely consistent with the findings of Fischer et al. [LRR-RLK subfamilies defined in O. sativa.Substitutions can change the functions of duplicated genes in gene families, and may be due to a relaxation of purifying selection or the action of positive selection . To invepressure , 52. Thepressure  using a pressure , after wpressure . Hence, pressure  remain tr et al.  at a subr et al.  found thr et al. . In our r et al. . Therefor et al.  also sugLRR region of LRR-RLK genes  , which are involved in innate immunity against pathogens [O. sativa Xa 21, which is involved in resistance to bacterial pathogen Xanthomonas oryzae pv. oryzae [. Furthermore, Xa 21 was found to have evolved under positive selection in rice [FLS2 showed a signature of rapid fixation of an adaptive allele in A. thaliana [The positively selected sites were located primarily in the es Table . This reignaling , Subfamiathogens , as well. oryzae . Further in rice , 37, andthaliana . The detLRR-RLK genes have been investigated in flowering plants. However, due to the lack of phylogenetic analysis of LRR-RLK genes from diverse plants, including algae, bryophytes, and different lineages of vascular plants, the classification of LRR-RLK genes in plants, and the origin, gene structure, and protein motif evolution, and the force driving the evolution of each LRR-RLK subfamily remain to be understood. Our studies identified 119 LRR-RLK genes in the Physcomitrella patens moss genome, 67 LRR-RLK genes in the Selaginella moellendorffii lycophyte genome, and no LRR-RLK genes in five green algae genomes. Phylogenetic analyses from these sequences and sequences from two flowering plant species revealed that plant LRR-RLKs belong to 19 subfamilies, most of which were established in the common ancestors of land plants. More importantly, we found that each subfamily was characterized by unique gene structures or unique basic gene structure and protein motif compositions. Four subfamilies were found to be under positive selection. Taken together, these results provide strong evidence that functional divergence occurred among LRR-RLK subfamilies and that positive selection had only an impact on the evolution of a few subfamilies of LRR-RLK genes.The evolutionary relationships among


Bunyaviridae but not currently assigned to a specific genus. We determined and analyzed the genome sequences of five Australian viruses isolated from mosquitoes collected during routine arbovirus surveillance in Western Australia  and New South Wales (12005). Based on matching sequences of all three genome segments to prototype MRM3630 of Trubanaman virus (TRUV), NB6057 of Gan Gan virus (GGV), and MK7532 of Maprik virus (MPKV), isolates K13190 and SW27571 were identified as TRUV, 12005 as GGV, and K42904 as a Mapputta group virus from Western Australia linking GGV and MPKV. The results confirmed serum neutralization data that had linked SW27571 to TRUV. The fifth virus, K10441 from Willare, was most closely related to Batai orthobunyavirus, presumably representing an Australian variant of the virus. Phylogenetic analysis also confirmed the close relationship of our TRUV and GGV isolates to two other recently described Australian viruses, Murrumbidgee virus and Salt Ash virus, respectively. Our findings indicate that TRUV has a wide circulation throughout the Australian continent, demonstrating for the first time its presence in Western Australia. Similarly, the presence of a virus related to GGV, which had been linked to human disease and previously known only from the Australian southeast, was demonstrated in Western Australia. Finally, a Batai virus isolate was identified in Western Australia. The expanding availability of genomic sequence for novel Australian bunyavirus variants supports the identification of suitably conserved or diverse primer-binding target regions to establish group-wide as well as virus-specific nucleic acid tests in support of specific diagnostic and surveillance efforts throughout Australasia.The Mapputta group comprises antigenically related viruses indigenous to Australia and Papua New Guinea that are included in the family  Anopheles (Cellia) meraukensis mosquitoes collected at Mitchell River Mission (now Kowanyama) in northern Queensland . N-glycosylation sites were variable. SW27571 and K13190 were predicted to have a glycosylated Gn, while the Gn of 12005 and K42904 did not show a potential N-glycosylation site . The RdRp sequence encoded by the L-segment was characterized by conserved block III domains pre-A, A, B, C, D and E [The organization of the M-segment was equivalent to other orthobunyaviruses with signal peptidase cleavage motifs located at the beginning of Gn, NSm, and Gc coding sequences , obtained from An. annulipes s.l. mosquitoes collected in 1993 at Thomsons Lake in the City of Cockburn (greater Perth metropolitan area), Western Australia, and isolate K13190 from An. annulipes s.l. mosquitoes collected in 1993 at Kununurra, Western Australia, were 96%, 96% and 96% identical for their S-, M- and L-segment nt coding sequences, respectively , which identifies both as isolates of the same virus. Since SW27571 had been shown to cross-react with TRUV polyclonal antibodies in neutralization assay [S1 Table). Thus, SW27571 and K13190 represent isolates of TRUV from Western Australia.Isolate SW27571 , obtained from Ae. vigilax mosquitoes collected in 1992 at Salt Ash, Port Stephens, New South Wales, and isolate K42904, obtained from Ae. vigilax mosquitoes collected in 2000 at Derby, Western Australia, showed only limited sequence identity to other sequences at the time of analysis. However, determining partial S-, M- and L-segment sequences for GGV prototype NB6057 and comparison to a recently published NB6057 prototype sequence [S1 Table). Despite the distance to other GGV isolates, K42904 M-segment sequence was more closely related to GGVs than to any other sequence. K42904 S-, and L-segment nt sequences were equally close to GGVs and MPKV, while the aa sequences showed a closer relationship to GGVs than to MPKV.Isolate 12005 (sequence  indicateTable 2), obtained from Cx. annulirostris mosquitoes trapped in 1993 at Willare, Western Australia showed highest sequence identity (100% nt and 100% aa identity) with a single Australian S-segment sequence entry submitted to GenBank in 2000 (Acc. No. AF325122) and annotated as ‘Bunyamwera virus’. However, all three S-, M- and L-segment coding sequences of K10441 also matched closely with those of characterized Batai virus (BATV) isolates . Our data suggest that K10441 (and possibly AF325122) represents an Australian variant of Batai virus.Isolate K10441 . While MAPV clearly separates from the other viruses of the group, a separation of MPKV from GGVs was less clear due to the intermediate branching of K42904, at least in the case of S- and L-segment sequence.Phylogenetic analyses supported the relationships indicated by pairwise sequence identity analysis. K10441 clustered most closely with BATVs, and SW27671 and K13190 with TRUVs . In addition, signalase sites were more conserved with regard to GGV than MPKV . Overall, this characterizes K42904 as a divergent isolate of GGV, linking GGV to MPKV.Except for K10441, our viruses mapped consistently for all genome segments in a monophyletic clade together with recently sequenced Mapputta group viruses. The sequences of TRUV SW27571 and K13190 were similarly close to TRUV MRM3630 as those of MURBV , supportFig 2 and S1 Fig). In addition, whereas L-segment sequences of the Mapputta group formed a sister clade to all other orthobunyaviruses, their M-segment sequences branched within the orthobunyavirus clade, rooting Bunyamwera, Wyeomyia, California encephalitis, Bwamba and Wuhan louse fly clades. Again, analyses of the S-segment sequences were divergent, including low bootstrap support between 10 and 40% for deep nodes. Nevertheless, our findings for L-, and M-segments indicated differences in branching patterns compatible with early reassortment events and may indicate a divergent evolutionary history for these segments.The increasing availability of sequence information for orthobunyaviruses indicates progressively more inconsistencies between the topologies of S, M and L phylogenetic trees. In the Mapputta clade, GGV was consistently closest to MPKV for all three segments, consistent with early serological data ; K13190 Our genetic analyses confirmed the absence of NSs coding sequence among all Mapputta group viruses as recently described for MAPV, MPKV, TRUV and GGV , 12, 14.Anopheles annulipes s.l., displays opportunistic host-feeding behavior , and thiewed in .Anopheles mosquitoes, primarily An. annulipes, throughout the Australian continent, including northern Queensland (Kowanyama), the Northern Territory , Western Australia , as well as New South Wales (Griffith). Prior to this study, GGV appeared to be focused in eastern Australia, mainly New South Wales (Port Stephens), involving various Aedes species, including the salt-marsh mosquito Ae. vigilax. The distribution of the sequence-confirmed isolates is largely in agreement with earlier reports of serologically characterized isolates . HoweveCx. annulirostris mosquitoes collected in 1993 at Willare, Western Australia, was identified as closely related to BATV, a virus classified in the species Bunyamwera virus of the genus Orthobunyavirus by the International Committee on the Taxonomy of Viruses (ICTV). This is the first detailed description on the occurrence of a BATV-clade virus from the Australian continent. The discovery of BATV dates back to 1955 when the prototype isolate MM2222 was obtained from Cx. gelidus mosquitoes collected in Kuala Lumpur, Malaysia (https://wwwn.cdc.gov/Arbocat/Default.aspx). With additional isolates from a variety of Anopheles, Aedes and Culex mosquitoes, as well as mammalian species in India (Chittoor virus), China (NM/12), Ukraine (Olkya virus), Europe  and Uganda (UgMP6830)[Cx. gelidus from which BATV was initially isolated in Malaysia was recently also reported in northern regions of Australia [Isolate K10441, obtained from UgMP6830)–40, BATVUgMP6830). BATV isUgMP6830), 43–45. UgMP6830). Given tustralia , 48, as ustralia , 50, or ustralia , 52.In summary, we show the presence of Mapputta serogroup viruses throughout Australia, including Western Australia. The identification of K42904 from northern Western Australia adds a novel virus to the group that is genetically related to GGV and MPKV; thus linking eastern Australian GGV with Papua New Guinean MPKV. In addition, an orthobunyavirus from north-west Australia, closely related to BATV, was identified and comprehensively characterized for the first time. Given the serological cross-reactivity among the Mapputta group viruses , sero-diagnostic assignment of human and animal disease cases to distinct viruses should be considered tentative. Comprehensive sequence data will open the way to virus-specific nucleic acid tests for enhanced surveillance and clarifying the pathologic potential of each of these viruses in humans and animals.Isolates K10441, K13190, and SW27571 were obtained from pools of mosquitoes collected during routine mosquito and arbovirus surveillance in Western Australia by The University of Western Australia Arbovirus Surveillance and Research Laboratory (UWA ASRL) using methods described previously –56. Isolhttp://www.clcbio.com).Sequences were generated by applying a combination of consensus reverse transcriptase (RT)-polymerase chain reaction (PCR) and unbiased high-throughput sequencing (UHTS). Total RNA was extracted from culture supernatants using TRI Reagent  or RNeasy Plus Mini Kit . Aliquots of total RNA extracts (0.5 μg) were treated with DNase I  for reverse transcription (RT) by Superscript II  with random octamer primers linked to an arbitrary, defined 17-mer primer sequence. The cDNA was RNase H-treated for 454 sequencing or Klenow-treated for Ilumina sequencing and randomly amplified by PCR with AmpliTaq  and a primer mix including the octamer-linked 17-mer sequence primer and the defined 17-mer sequence primer in a 1:9 ratio . AmplifiDirect PCR amplification was performed with conserved degenerate primer sets , 59, 60,http://www.cbs.dtu.dk/services) were used for functional predictions. Phylogenetic analyses were performed by using the MEGA 6.0 software package [Sequence reads were stripped of primer sequences and highly repetitive elements, quality filtered and then clustered and assembled into contiguous fragments (contigs) for comparison by the Basic Local Alignment Search Tool (blast ) to the  package . MultiplS1 FigA), M- , and L-segment  were aligned and trees reconstructed with the Maximum Likelihood method applying the best predicted substitution model using MEGA 6 software. Bootstrap values are indicated at the respective nodes, scale bars indicate the number of substitutions per site, and GenBank accession number and isolate name (where known) are given next to the virus name. MAPV, Mapputta virus; MPKV, Maprik virus; TRUV, Trubanaman virus; GGV, Gan Gan virus.Deduced amino acid sequences of the S- Click here for additional data file.S1 Tablea), and among isolate K10441 and selected other orthobunyaviruses (b) were calculated.Percent nucleotide (NT) and amino acid (AA) sequence identity among Mapputta group viruses ((XLSX)Click here for additional data file.


Viral diseases in crop plants constitute a major obstacle to food security in the developing world. Subsistence crops, including cassava, sweetpotato, potato, banana, papaya, common bean, rice and maize are often infected with RNA and/or DNA viruses that cannot be controlled with pesticides. Hence, healthy planting materials and virus-resistant cultivars are essential for high yields of good quality. However, resistance genes are not available for all viral diseases of crop plants. Therefore, virus resistance engineered in plants using modern biotechnology methods is an important addition to the crop production toolbox. Current Opinion in Virology 2017, 26:90–97Engineering for viral resistanceThis review comes from a themed issue on John Carr and Peter PalukaitisEdited by Issue and the EditorialFor a complete overview see the Available online 8th August 2017http://dx.doi.org/10.1016/j.coviro.2017.07.022http://creativecommons.org/licenses/by/4.0/).1879-6257/© 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (Geminiviridae) [Nanoviridae) [Geminiviridae) [Ipomovirus, family Potyviridae) [Machlomovirus, family Tombusviridae) and the potyvirus sugarcane mosaic virus (a member of the family Potyviridae) [sweet potato chlorotic stunt virus  and the potyvirus sweet potato feathery mottle virus [Viral diseases constitute a major threat to crop production worldwide. The problem is exacerbated in developing countries, particularly in tropical areas where crop-free seasons are rare and plants are under continuous pressure from viruses transmitted by vectors from both cultivated and wild plants . Africa viridae) •, bananaviridae)  and maizviridae) . Cassavaviridae) , 6. In eviridae) . Sweetpole virus .Natural sources of resistance to many viral diseases of tropical crops are known and deployed in breeding. In many cases, however, resistance sources are lacking, or the genetic complexity and difficulties in introgressing resistance genes to cultivars by crossing hamper the efforts of crop improvement. Therefore, development and transfer of resistance to crops by biotechnological means offers an attractive alternative solution.et al. [Nicotiana tabacum L.) to express the coat protein of tobacco mosaic virus made the plants resistant to this virus. Similarly, transformation of plants to express non-structural viral proteins [The studies of Powell Abel et al. •• in theproteins  and trunproteins . These fproteins , which iproteins •.It was later realized that some viral proteins suppress or interfere with antiviral RNAi , 15 and Solanum tuberosum L.) [Solanum lycopersicum L.) [Capsicum annuum L.) [Arachis hypogaea L.)[Saccharum officinarum L.) [Oryza spp.) [Carica papaya L.) [Passiflora edulis Sims) [Glycine max (L.) Merr.) [Considerable efforts have been invested to develop virus resistance to many different viruses affecting crops relevant to agriculture and food production in developing countries. The main target crops, besides those mentioned above, have been potato (osum L.) , 20, 21,icum L.) , peppersnuum L.) , 24, peaogaea L.), 26, sugarum L.) , rice (Oza spp.) , 30, 31,paya L.) , 33•, pais Sims) , 35 and ) Merr.) , 37. HowPotyvirus; family Potyviridae), potato leafroll virus  and potato virus X  are the most common and devastating potato viruses worldwide. The aphid-transmitted PVY and PLRV can cause significant yield losses on their own. The contact-transmitted PVX becomes significant with PVY co-infection, which induces synergistic interactions of the viruses leading to high accumulation of PVX. To solve this problem, virus-derived resistance was engineered to both PVY and PVX in potato cv. Russet Burbank, representing only the second example (after tobacco) of genetically engineered virus resistance in crop plants [Leptinotarsa decemlineata Say) in the late 1990s and approved for marketing under the name NewLeaf™ [Potato is the third-most-cultivated food crop in the world after rice and wheat. The climate in highland areas is well suited for potato and cultivation has increased quickly in Africa. Furthermore, root crops such as potato and sweetpotato are expected to suffer less from climate change than many other subsistence crops . Potato p plants , 40. ‘RuNewLeaf™ , 42.Dioscorea spp.).Potatoes are clonally propagated and thus prone to build up viral infections over generations. In industrial countries, potato viruses are controlled by planting certified virus-free seed potatoes produced under special cultivation schemes. Combined with control of the aphid vectors using pesticides, this has led to a reduction in the prevalence of PLRV over the past 30 years. In low-income countries, however, potato viruses are common and losses are severe, because healthy seed potatoes and pesticides are not commonly available or affordable . IntrogrResistance to PLRV in ‘NewLeaf’ reached commercial production in the USA, but the engineered variety remained on the market only a couple of years before it was withdrawn due to the decision by major potato processing industries to abstain from using transgenic potatoes . The demRx, which confers extreme resistance to a wide spectrum of PVX strains, has been isolated from potato and introduced to PVX-susceptible plants by genetic engineering [Rx gene has been extended by mutation [The natural dominant PVX resistance gene ineering . Furthermutation •. Thus, Potyvirus; family Potyviridae) is a major pathogen of papaya worldwide. PRSV-resistant papaya was one of the first virus-resistant transgenic plants approved (in 1998), and was for a long time the only transgenic crop developed entirely with public funding. PRSV-resistant papaya effectively saved papaya production Hawaii [Papaya is a crop predominantly produced and widely consumed in the developing world. It is an important and rich source of essential nutrients, such as vitamins A and C. PRSV  and vegetable crops such as tomato, peppers and cucurbits (Cucurbita spp.) are damaged by whitefly-transmitted begomoviruses worldwide [Common beans  is very important as a subsistence crop in Africa. The cassava mosaic epidemic started and spread quickly in East Africa in the mid-1990s, devastating cassava crops entirely in many regions. The disease escalated when African cassava mosaic virus and virulent recombinants with other whitefly-transmitted cassava-infecting begomoviruses co-infected cassava plants, resulting in synergism, very severe symptoms, growth retardation and new virulent recombinants of the viruses [Cassava  is caused by two closely related whitefly-transmitted ipomoviruses . Both vic plants .Ipomoea batatas Lam.) originates from South and Central America, but has a particularly significant role as a subsistence crop in Africa [Sweetpotato  is transmitted by aphids and is the most destructive viral pathogen of bananas and plantains worldwide. It originates in Asia, was introduced to Africa probably from the South Pacific , 64, andet al.  were ablMusa species is unlikely. Since copy numbers and integration sites of transgenes are difficult to control, application of modern genome-editing technologies could allow targeting of specific host genes [The challenge in introducing transgenic resistance to different banana cultivars lies in the sterility of the vegetatively propagated cultivars and the need to transform each cultivar separately, which is not a trivial task. On the other hand, as banana cultivars are essentially sterile, transgene flow to other cultivars or wild st genes , for exaSobemovirus) causes a major disease in rice that is used as an example of the key role of agricultural intensification in plant virus emergence [Rice yellow mottle virus  lethal necrosis caused by co-infection of maize chlorotic mottle virus and another potyvirus, maize dwarf mosaic virus was described in USA, but is currently ravaging East-Africa and threatening to spread further across the continent. The disease can be controlled by transgenic resistance against maize dwarf mosaic virus , which cAgrobacterium spp. were found integrated in the sweetpotato genome, which raises a question about their possible role in host defense or crop domestication [Agrobacterium are also found in other plant species [Agrobacterium-mediated transformation. It is more common, however, to find viral sequences integrated in plant genomes, which is comparable to plants transformed by particle bombardment and results in random integration of DNA in the plant genome.Recently, natural transgenes corresponding to the transfer DNAs (T-DNA) of tication ••. T-DNA species , analogos — like sequences are found integrated in petunia and many solanaceous crops, respectively. Viral integrations have been observed also in woody plants such as grapevine and fig [Integration of viral sequences in a plant genome was first realized in banana carrying fragments of banana streak virus, a pararetrovirus with a double-stranded DNA genome encapsidated in bacilliform particles ••, and l and fig , 78, 79. and fig •. The in and fig  (Figure Taken together, the recently adopted methods for genome editing, such as CRISPR-Cas9, are directly adopted from living organisms and, hence, incompatible with the traditional definitions of genetic engineering and transgenic plants. It is also apparent that RNA silencing against virus-derived sequences occurs naturally in plants on a large scale based on viral sequences integrated into plant genomes. It could be time to reconsider what is ‘transgenic’ and requires regulation. Reassessment of the rules and regulations could conclude that deregulation of virus resistance mediated by homology-based RNA silencing might be reasonable.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as:


Ebola and Marburg virus diseases are said to occur at a low prevalence, but are very severe diseases with high lethalities. The fatality rates reported in different outbreaks ranged from 24–100%. In addition, sero-surveys conducted have shown different seropositivity for both Ebola and Marburg viruses. We aimed to use a meta-analysis approach to estimate the case fatality and seroprevalence rates of these filoviruses, providing vital information for epidemic response and preparedness in countries affected by these diseases.metaprop command in STATA.Published literature was retrieved through a search of databases. Articles were included if they reported number of deaths, cases, and seropositivity. We further cross-referenced with ministries of health, WHO and CDC databases. The effect size was proportion represented by case fatality rate (CFR) and seroprevalence. Analysis was done using the 2 = 97.98%] whereas that of Marburg virus disease was 53.8% . The overall seroprevalence of Ebola virus was 8.0% , whereas that for Marburg virus was 1.2% . The most severe species of ebolavirus was Zaire ebolavirus while Bundibugyo Ebolavirus was the least severe.The weighted average CFR of Ebola virus disease was estimated to be 65.0% [95% CI (54.0–76.0%), IThe pooled CFR and seroprevalence for Ebola and Marburg viruses were found to be lower than usually reported, with species differences despite high heterogeneity between studies. Countries with an improved health surveillance and epidemic response have lower CFR, thereby indicating need for improving early detection and epidemic response in filovirus outbreaks. Filoviridae and are both associated with high case fatality rates (CFR). The World Health organization (WHO) reports that the CFR of EVD ranges from 25.0 to 90.0% while that of MVD ranges from 24.0 to 88.0% [Ebola Zaire and Ebola Sudan species being most pathogenic (with a reported CFR of 100%), while Ebola Bundibugyo appears to have a lower CFR at 34% [et al. that used data from WHO database estimated the CFR of EVD to be 65.4% irrespective of the Ebola virus species [Ebola virus disease (EVD) and Marburg virus disease (MVD) are caused by filoviruses in the family to 88.0% . In the Taï Forest ebolavirus, formerly known as Côte d’Ivoire ebolavirus, has not been associated with any fatality and the only case ever reported recovered from the disease [Ebola Sudan and Ebola Zaire have shown a CFR of 100% [Although EVD is known to be very severe, there are some species of Ebola virus that cause less serious disease. For example,  disease . While t disease , 8. It s of 100% . The lar of 100% .rousettus aegyptiacus, which are believed to be reservoirs for Marburg virus, and responsible for the spill over into human populations [There is evidence that a substantial proportion of infected humans in Central Africa seem to recover without being detected by the health care system, and apparently healthy individuals have been found to be seropositive for Ebola and Marburg viruses –15. Furtulations –19. Becaulations , 5, thesProcedures for systematic reviews and meta-analysis have been developed to summarize scientific evidence from the literature. This work was done following the guidelines published in the PRISMA statement  and MOOSth October 2015. In cases where there was no peer-reviewed publication for a known outbreak, data was retrieved from websites of WHO and CDC. The following key words were used; “ebola”, “ebolavirus”, “viral haemorrhagic fevers”, “marburg virus disease”, “marburg haemorrhagic fever”, “marburg virus outbreak”, “ebola virus disease outbreak”, “marburg virus”, “ebola outbreak”, “seroprevalence of ebola virus”, “seroprevalence of marburg virus” and “risk factors of viral haemorrhagic fevers”. The search included all articles and outbreak reports about EVD and MVD and cross-referencing of primary articles was done to obtain the original articles. Since the number of outbreaks of EVD and MVD are known and few, efforts were made to obtain all information about these outbreaks from WHO and CDC websites and Ministries of health of respective countries.A detailed literature search was conducted by the authors in PubMed (as well as Medline), Web of Science and Google Scholar until 5Studies were included in the meta-analysis if they reported the total number of cases and total number of deaths from the outbreak of EVD or MVD. Also studies that were reporting CFR and sero-prevalence in percentages were included. Studies or reports that did not include total number of deaths or cases were excluded as well as studies that did not report original data Fig. . We alsoLN compiled a list of articles and discrepancies were discussed and resolved by consensus between FM, CK and JL. We used a standardized data extraction form and the following information was extracted for each qualifying study and outbreak report: i) author; ii) Country; iii) number of cases; iv) number of deaths; v) CFR (if reported); vi) month and year of outbreak; vii) year of publication viii) and species involved. For population-based sero-prevalence studies, the following additional information was retrieved: i) sample size and ii) number of seropositive samples.metaprop command [metaprop command was preferred to metan command because it implements procedures that are specific to binomial data and is appropriate for dealing with proportions close to or at the margins and also uses the Freeman-Tukey double arcsine transformations to stabilize the variances [Data were collected in a Microsoft Excel® spreadsheet and outcome measures were calculated. CFR was calculated as number of deaths divided by reported cases whereas seroprevalence was calculated as number of individuals seropositive divided by total sample size in each study. Our effect size (ES), the principal summary measure, was the proportion represented by CFR and seroprevalence. We used the newly developed  command  for perfariances . The met2) and Higgins I2 which is an estimate of what proportion of the observed variance that reflects real differences in ES. If I2 is close to 0, then almost all the observed variation is spurious, and there is nothing to explain. If I2 is large, then reasons for the observed variance should be evaluated [The following parameters were estimated: Cochran’s Q indicating differences in true ESs, an estimate of the true variance of ESs between studies  [Sudan ebolavirus with 30.3% (7/23) outbreaks [Bundibugyo ebolavirus 8.7% (2/23) [Two more outbreaks have occurred without human mortalities namely Ebola Reston , 75 and st virus . Zaire e (60.9%) , 37, 76 utbreaks , 77 and % (2/23) . Most ar% (2/23) , 42, 76 % (2/23) , 7, 42 a% (2/23) , 36, 78,% (2/23) , 37, 41,% (2/23) , 29, 38 % (2/23) –82. Inte2 = 97.98%. On stratification by Ebola virus species, the CFR for Sudan ebolavirus was 53%, Bundibugyo ebolavirus was 34%, whereas that of Zaire ebolavirus was 75%. From the meta-regression, the CFR for Zaire ebolavirus was higher compared to other Ebola species . In sub-analysis analysis by country, the highest CFR for EVD was observed in Republic of Congo  whereas the lowest was found in Uganda    . The pooled seroprevalence of Marburg virus was lower than that of Ebola virus at 1.2% (0.5–2%)  Fig. . There w1% Fig. .P = 0.001, P < 0.001, p = 0.032, and 0.046 respectively). However, the Begg’s bias test was not significant for studies reporting CFR of EVD and MVD (p = 0.091 and p = 0.293 respectively), seroprevalence of MVD (p = 0.95), but was significant for studies reporting seroprevalence of EVD (p = 0.007).In the funnel plots, asymmetry was evident which gives rise to suspected publication bias Fig. . Egger’set al. [Our findings show that the overall pooled CFR of EVD of 65% was lower than the previously reported CFR of 90% . This inet al. , who repet al. , 7, theset al. , 37, 41,et al.  where thSudan ebolavirus and Bundibugyo ebolavirus) as compared to DRC and West African countries that have experienced Zaire ebolavirus Also, it is important to look at the denominators and numerators when interpreting the CFR. In this analysis, we see that CFR of EVD in a large outbreak in West Africa that affected multiple countries is at CFR of 40% using WHO data, but this alone would be misleading if the real numbers of deaths and cases were not looked at. As of 30th March 2016, there were 11323 deaths and 28646 cases due to EVD from all countries affected by that outbreak.The high CFR of EVD in Republic of Congo (89%) compared to Uganda (43%) may be due to partly, differences in health care system and response mechanisms to outbreaks, but also the severity of the species of Ebola virus involved. For example, Uganda has developed a robust surveillance system for detecting these viral haemorrhagic fevers and epidemic response is started within hours of a positive diagnosis at a CDC supported laboratory in the country . The welebolavirus  was found to be the most severe followed by Sudan ebolavirus , while Bundibugyo ebolavirius  was the least severe species. This finding is supported by McCormick et al., who described differences in severity and filovirus dynamics [Zaire ebolavirus are unclear, thus there is a need for further research to determine whether genetic differences are responsible for the variation in pathogenesis of these species. There was also heterogeneity within Zaire ebolavirus outbreaks (P < 0.001) meaning that these outbreaks, although caused by the same species are not always similar. The heterogeneity could further be explained by differences in outbreak investigation designs or approaches, location of the outbreak and data collection methods. This is further supported by the strains that have been found within Ebola Zaire species [Bundibugyo ebolavirus and Sudan ebolavirus probably due to few outbreaks that have been caused by these species. However, the meta-regression did not show any influence on CFR of EVD by country of outbreak (p = 0.249). This is probably due to low power given the few number of outbreaks that we have had globally.Another significant finding of our study was the variation in the severity and CFR among the pathogenic species of Ebola virus. Zaire dynamics , 87. The species . There wMetaprop command for meta-analysis of marginal proportions [p = 0.913).With the portions , it was We found that apparently healthy individuals in central African countries, that are endemic for viral haemorrhagic fevers, had a 5 and 1% chance of having antibodies against Ebola and Marburg viruses, respectively. This finding suggests that some individuals who get infected with filoviruses make a full recovery without severe complications and being documented by healthcare systems. Although the sero-prevalence is low, it is important that these seropositive individuals are detected early enough because of greater mortality and socio-economic implications associated with these infections. Because serological tests have been reported to have low specificity and there is a lot of cross-reactivity of filoviruses with other viral haemorrhagic fevers , this fiThe limitation of our ES estimates was the heterogeneity that was observed between studies. Efforts to identify sources of heterogeneity were made, and many unmeasured factors could have influenced CFR during outbreaks. These reports had data that were collected using different methods and hence combining them to produce one effect was likely to produce high heterogeneity. Sensitivity analysis by dropping single cases with 100% mortality did not have substantial impact on the result. Funnel plots and Beggs tests suggested that publication bias might have been present, meaning that studies with negative results about Ebola and Marburg viruses are less likely to be published hence affecting the estimate of seroprevalence and CFR for EVD and MVD.The fact that laboratory tests for Ebola and Marburg viruses are expensive, used only in specific laboratories and that serological tests are not specific might influence the publication of studies done with these tests.The CFR for Ebola and Marburg viruses is still moderately high but not as high as has been reported in the media and other publications. The CFR of EVD and MVD is higher in countries with poor disease surveillance systems. This calls for an improved surveillance system that will enhance early detection and response to these filovirus outbreaks to avoid a pandemic. The presence of seropositive individuals in apparently health populations indicate that cases go undetected by the health care system in affected countries; further calling for robust surveillance for Ebola and Marburg viruses.


Tomato mosaic virus and Turnip yellow mosaic virus, put in contact with a highly hydrophobic plasma textured silicon surface. Results show a high sensitivity of the system towards the virus particles with an interestingly low detection limit, from tens to hundreds of attomolar corresponding to pg/mL of sap, which refers, in the infection time-scale, to a concentration of virus particles in still-symptomless plants. Such a threshold limit, together with an envisaged engineering of an easily manageable device, compared to more sophisticated apparatuses, may contribute in simplifying the in-field plant virus diagnostics.Early diagnosis of plant virus infections before the disease symptoms appearance may represent a significant benefit in limiting disease spread by a prompt application of appropriate containment steps. We propose a label-free procedure applied on a device structure where the electrical signal transduction is evaluated via impedance spectroscopy techniques. The device consists of a droplet suspension embedding two representative purified plant viruses i.e.,  The development of modern multifunctional biologically-oriented platforms for a sensitive, reliable, and rapid detection of plant viruses plays a critical role in virus disease management. Although a single diagnostic test may provide adequate information on the identity of a virus, a combination of methods is generally needed for unequivocal diagnosis. Thus, methods for detection of plant viruses should be sensitive, specific, and should be completed within a relatively short period of time at a low cost.Generally, diagnostic techniques in virus detection fall into two categories due to the intrinsic properties of the virus itself. Methods are based on the detection of coat proteins and genomic nucleic acids as enzyme-linked immunosorbent assays (ELISA) and immunoblotting (DIBA), whose virus protein limit of detection (LOD) is from 1 to 10 ng/mL of sap, and on the reverse transcription polymerase chain reaction (RT-PCR), the latter being more sensitive than other methods reaching a LOD of 1 fg/mL of virus RNA ,3,4. NowTobacco mosaic virus (TMV) [Asian rust virus on the soybean leaf with a declared LOD of μg/mL [In recent years, biosensor-based techniques were developed for the detection of intact plant viruses. Quartz crystal microbalance (QCM) immunosensor demonstrated the potential to detect as low as 1.0 ng each of two orchid viruses . Surfaceus (TMV) , and Asiof μg/mL .Nevertheless, all the sensors developed until now are quite expensive, highly complex, require many steps, as well as expert operators and furthermore, and are not suitable for rapid in-field testing.Prunus necrotic ringspot virus in plant extracts was obtained by electrical impedance spectroscopy (EIS)-based immunosensing technique via the virus antigen-antibody interaction, with the latter immobilized on a modified glassy carbon electrode [Nowadays, the impedance bio-sensing technique represents a valuable alternative as it can shed light on the changes in the electrochemistry during the analyte detection. In spite of all of the other techniques, the impedance-based methods have the advantages to be, in most cases, label-free and easily miniaturized with a simple design at low cost. Recently, label-free identification of human and animals virus-based suspension via capacitance based measurements and using electrical parameters was recently performed ,11,12. Slectrode . In thisThe innovative droplet-on-a-surface configuration recently opened new perspectives in the design of highly sensitive label-free lab-on-a-chip EIS based devices for virus detection. Droplets were displaced on microfluidic devices and acted as chemical and biological reactors where changes in droplet impedance allowed the analysis of processes occurring inside ,16. A hiTurnip yellow mosaic virus (TYMV) [Tomato mosaic virus (ToMV) [Two well-known viruses, s (TYMV)  and Tomas (ToMV)  were choTomato mosaic virus [L = 300 nm long and d = 18 nm in diameter, with a 4 nm wide inner channel  of 3.7 and 4.5, respectively ,21. ConsNicotiana tabacum and Chinese cabbage were mechanically inoculated by ToMV and TYMV, respectively, by gently rubbing the sap from infected tissues macerated in 100 mM tris(hydroxymethyl)aminomethane chlorohydrate (TRIS-HCl) buffer, pH = 8.0. Both viruses were purified as in Foster and Taylor [Leaves of young seedlings of d Taylor .dpi) were crushed in a blender with ice-cold buffer, filtered and clarified with 0.4 vol chloroform/n-butanol (1:1). The mixture was stirred on ice for 15 min and centrifuged at 6000 rpm for 30 min. The supernatant was stratified on a 3 mL cushion of 30% sucrose in TRIS-HCl solution and centrifuged at 30,000 rpm for 2.5 h. The resulting pellets were re-suspended in the same buffer and subjected to a further similar round of centrifugation. The partially-purified viruses were finally re-suspended and stored indefinitely at 4 °C.Infected leaves at 15 days post inoculation  observation by using a Morgagni 282D . The virus samples were prepared by depositing 20 μL of the resuspended particles on carbon-coated copper grids, stained for 4 min with 2% uranyl acetate water solution .ρ underwent to several ten-fold dilutions starting from the stock suspension in a range from ρ = 1.0 mg/mL, corresponding to the molar concentration ρM of 25 nM for ToMV and 177 nM for TYMV, down to 1.0 pg/mL, corresponding to ρM of 25 aM in ToMV and 177 aM in TYMV.Both suspensions of partially-purified viruses were scanned for optical density reading (320 to 220 nm) on a Nanodrop 2000 . The different extinction coefficients of the viruses, which depend on their relative nucleic acid contents , were us2, polished wafers, with resistivity 1–50 Ω·cm, 750 μm thick, provided by MEMC  were plasma textured in a radiofrequency )-powered parallel plate reactor [P-doped crystalline silicon slabs (pSi)  slabs was required in order to make these supports suitable as platforms for viral sample spotting. To this aim a fast washing with acetone and ethanol followed by rinsing with bi-distilled water and sonication for 1 min at maximum power kept micro-pollutants away. Finally, the clean silicon surfaces were put in a vacuum chamber for 6 h.The surface water contact angle, WCA, was evaluated using the sessile drop method, from triplicate measurements onto different points of the sample, returning an average advancing WCA of 152° ± 6° and a receding one of 120° ± 4°, indicating a quite uniform hydrophobic surface.The T-pSi slices were highly hydrophobic respect to the suspensions droplet on the overall region constituting the sensing area, i.e., the droplets displayed a spherical shape with a similar contact angle of the droplets see c. Simila2) slices exposed to a set of droplets (7 μL each) of ToMV or TYMV in buffered TRIS-HCl in water suspension at pH 8.0  droplets . The latter have been subsequently analyzed by modeling them with suitable equivalent circuits using a free downloadable EIS spectrum analyzer software [The lets see . From thsoftware ,27.tr, onto the surface tr = 20 min were collected both at VDC = 0 and by adding a continuous negative bias (VDC = −200 mV) to the AC voltage. The reasoning behind the choice of a residence time was due to the ascertained droplet volume and impedance stability after consecutively collecting the droplet impedance spectra for a similar residence time. The choice of collecting the impedance spectra under a negative DC voltage is aiming to induce a repulsive electric field drifting the negatively surface charged virus (pI (virus) < (pH(TRIS-HCl) = 8.0) from the gold electrode towards the textured silicon surface.The time evolution of NPs for a droplet residence time, R), purely capacitive (C), constant-phase (ZCPE), and diffusive (ZW) circuital elements each representing a specific phenomenological contribution to the impedance [The equivalent circuits representing the NPs were designed by properly combining resistive  readout, and the very low evaporation rate of suspension buffer ruled out any significant droplet volume reduction and, consequently, any relevant changes in virus concentration. Rather, after 20 min and at lets see . More splets see , furtherVDC = 0 V of the virus-based suspension, displayed a significant variation respect to the blank one in the whole frequency range  was observed at the lowest concentration (corresponding to 1 pg/mL) in the case of ToMV, while in TYMV the steeper diffusion occurs at the same frequency but at higher ReZ values. Moreover, the higher the virus concentration, the more the ToMV (TYMV) curve shifts to the right (to the left).The NPs inspection in the range 10 MHz < 9.0 kHz, c,d, evidUnder the application of the continuous voltage, the NPs of virus suspensions displayed a more faradic behavior that was, furthermore, different on that of the biased blank one, the latter still retained a non-faradic trend . MoreoveReZ values at 0.1 Hz vs. ρM , relates to infection stages where the plants are no longer symptomless with the disease already at an advanced stage.The correlation between the NPs behavior with the molar concentration suggested to use them for the extraction of parameters suitable for virus quantification. By representing the z vs. ρM  a linearReZ and −ImZ values are selective with respect to the charging and polarization of the insulator in the lower frequency ranges meaning, in this case charging and polarization of the droplet [The “water gated” MIS-like configuration confer to the droplet the role of the insulating part of the structure. Following the MIS device theory, the  droplet .ReZ and ImZ values with the concentration implies higher droplet ion charge-mediated conductivity, as could be expected by the increase of the virus charge density.As a consequence, the returned lowering at 0.1 Hz of the ImZ vs. the virus concentration, relates to the droplet/T-Si interface charging and polarization capabilities under the AC field [C. For a better evidence, the capacitance contribution C was extracted, in the overall measurement range, as a function of the frequency directly from NPs data following general calculation rules as [δ, the phase angle, given by:On the other hand, the behavior of the imaginary part −AC field  that canrules as :(1)Z=ReInterestingly, the virus-based suspensions displayed a remarkable increase (up to three order of magnitude) of the capacitance at all frequencies (except at those >1 MHz) and a loss factor  spectra different with respect to those of blank TRIS-HCl together with both a concentration and a virus-related behavior .C of the virus-based suspension, CTRIS-HCl, was higher than that of the buffer, we considered the elicited capacitance CTRIS-HCl and that of the viruses, CVi, acting as two parallel capacitors [Since the measured capacitance pacitors , likewisΓ was defined as:A proper frequency was selected where this effect was maximized, and the parameter Γ parameters were calculated by choosing the frequency of 9.0 kHz, i.e., lying in the plateau region of the C vs. f curves and maximizing the capacitance variation respect to that of the suspending medium. Interestingly, the substantially different behavior and values of the Γ parameters vs. the molar concentration on the virus type (AC polarization field. These findings are particularly relevant since the highlight the definition of a possible marker (Γ) for the recognition of AC-responding viruses even at very low concentrations and, finally, directly extracted from rough experimental data [The rus type  suggestetal data .ReZ at 9 kHz respect to the TYMV, at the onset of diffusion, and on the reversed shift when increasing the molar concentration.The data in The stepped shift in ToMV at the lowest concentration  can be explained not only as being due to a different electrical conductivity of the virus suspension, dictated by the number of the dielectric virus particles  but also to a specific and peculiar virus behavior that will be better detailed in the next section.L/d~17(ToMV) > 1(TYMV)) and corresponding surface area higher than TYMV, both implying a higher electronic surface charge density available for conduction. In fact, both can be explained by examining the phase angle values at 9 kHz of each virus suspension at similarly low concentrations . ConsideReZ and ImZ at 9 kHz) vs. the concentration can also be a hint of a specific charge balance of the peculiar virus surface charges when interacting with the suspending aqueous medium, whose effect is strictly dependent on the intrinsic virus structure.Within this framework, it can be argument that the virus-depending behavior of the impedance (pH (isoelectric point), while keeping inside the folded particle a highly hydrophobic status that helps in stabilizing the structure and the protein-protein and low electrostatic nucleic acid-protein interactions.We recall that main and essential virus structure is made of a shell of folded protein sub-units that assemble with each other to maintain an outside hydrophilic surface, the latter particularly sensitive to any charge unbalance in the medium. The resulting surface electric charge is, therefore, very specific and dependent on the chemical composition in the primary amino-acid chain, and eventually remarks a peculiar neutrality AC electric field-mediated mechanisms affecting the virus particle dynamics in the droplets, providing that the viruses are sensitive to the AC polarizing field. As for the latter requirement, this has already been assessed in a previous section. The NP simulation is accomplished by designing a suitable electrical circuit whose impedance is able to replicate the experimental ones, whereas a specific mechanism is assigned to each electrical component. As underlined, the viruses have a negative charge on their outer surface, being that the ToMV and TYMV pI are lower than the buffer pH. Moreover, the nanometer-sized virus dimensions allow the assumption that the droplet suspension is, likewise, a ‘charged’ nanoparticle-based system.As illustrated in previous sections, the NP behaviors of the plant virus droplet-based system were found to depend on the virus type and concentrations. In this section more insights will be reported on the NP behavior as representative of HF) impedance (corresponding to the first NPs’ semicircle). of the experimentally virus-based droplets consisted of a component made up by the series of the Au-droplet contact resistance Rc with the parallel of a resistor and a capacitance labeled as RHF and CHF (RHF//CHF). The latter consist of the indistinguishable contribution of both the geometrical droplet suspension capacitance and resistance and that of the silicon , ZW = Warburg element). The virus-embedding droplet impedance Zdroplet contributed via an in-homogenous double layer capacitive term , while the part of the droplet interacting with, or adsorbed onto, the wet textured surface was represented by the series of a non-ideal capacitive contribution  and a resistance (Rads) . At tal data ,30. The ZCPE,ads) with a general decrease of the adsorption resistance, Rads ·cm−2 compared to 1.5 × 10−8 nF·s(n−1)·cm−2 in TRIS-HCl).The second component of the equivalent circuit showed increasing values of the charge surface adsorption term . Conversely, the TYMV particles are probably strongly agglomerating during their drift towards the textured surface , and remain adsorbed onto the textured surfaces without diffusing . The SEM pictures partially support the data interpretation and provide evidence of ToMV particles found inside the textured surface while no TYMV ones were observed  moving more or less freely in the suspending medium depending on their aspect ratio, the suspending medium viscosity, and the virus polarizability under an ZW values soon after the bias application and during the droplet residence time suggested, again, a progressive reduction of the virus charge mobility [D being the diffusion coefficient, R is the gas constant, T is the temperature in Kelvin (K), A is the electrode area, n is the valence of the species (n = 2 in this case), F is the Faraday constant, and ρM is the molar concentration. Findings on nanoparticle dynamics in liquid suspension showed that the diffusion coefficient relevantly differs depending on the nanoparticle aspect ratios [ηs, and in a highly-diluted regime, the particles are able both to rotate freely without interacting each other due to the large distance and to diffuse due to the Brownian forces only (~KBT). The Brownian rotational and translation diffusion coefficients are defined as Dr,B and Dt,B; for nearly spherical particles of diameter d, given by:L and diameter d, and if the aspect ratio L/d is in the range 2 < L/d < 30, they are given by [kB being the Boltzmann constant, and T the absolute temperature). The Equations  and  indicate the dependence of the diffusion on the geometrical radius and aspect ratio. Considering the ToMV and TYMV geometrical shapes and dimensions, we estimate the corresponding rotational and translational diffusion coefficients. Due to the high dilution of TRIS-HCl in water (100 mM), we considered water as the suspending liquid and placed ηs = 10−3 Pa, i.e., the water viscosity. The Dt,B values were found to be 1.45 × 10−11 m2·s−1 and 0.88 × 10−11 m2·s−1, while the Dr,B was found to be 4.8 × 104 s−1 and 292 s−1 in TYMV and ToMV, respectively. Following these results, TYMV translational and rotational diffusivity were found to be 1.6 and 160 times higher than in ToMV, respectively. Furthermore, under Brownian forces, the diffusivity does not depend on the particle concentration. The increase of the diffusion impedance term mobility ,31,34. It ratios . In partgiven by ,37:(8a)D values obtained from Equation (6) . Afterwards, the diffusivity progressively reduces up to ten orders of magnitude with respect to D0 with the molar concentration. This effect is commonly found in suspension embedding nanoparticles and is generally referred to as “diffusion hindering” [By representing the experimental  (6) see , it can ndering” , whose oAC voltage (0.1 Hz) while the virus recognition was made via the definition of a parameter specifically related to the viruses’ polarizability.The present work demonstrates a feasible and practical route for label-free and fast detection of plant viruses in aqueous medium suspension through an impedance method. The EIS technique was applied to a water-gated MIS-like device built on a hybrid droplet on a highly-hydrophobic surface, where the droplet was constituted by a suspension of virus in a TRIS-HCl solution at different virus concentrations. The virus quantification was derived by the extraction of a calibration curves in the low-frequency region of the DC bias. We also demonstrated that the impedance behavior, and specifically the diffusivity in the suspension, were related to the virus aspect ratio.Two well-known plant viruses (ToMV and TYMV), were considered as model nanostructured systems with different substructures, shapes, and controlled surface net charge. Specifically, the potential to detect very low molar virus concentration and to extract a specific parameter allowing the label-free virus identification via a single real-time measurement was demonstrated. The method showed a peculiar sensitivity toward lowest virus concentration and selectivity to charged virus when applying a AC field, this method could be extended also to other droplet-shaped suspensions embedding one purified virus, since it would be an easy task to relate the parameter variations to the corresponding concentration and virus type, therefore leading to a set of calibrated curves for virus recognition. However, for a reliable applicability of the elicited approach, further validations are required as that via quantification/recognition of a much wider class of plant viruses with different shapes. Nonetheless, the presented results, although not exhaustive due to the elementary and few virus model systems examined up to now, , pave a possible route for increasing the device sensitivity and test speed by using droplet-based devices engineered for label-free plant virus detection, quantification, and recognition.In principle, provided that viruses are responding to an An interesting approach could also be the testing of a single droplet of sap extracted from plant tissues, which is necessary to prime the test, while reducing the cost, as it does not requires any antiserum or labelling and uses an inexpensive active electrode surface. A potential application of the EIS on our hybrid droplet-on-surface device can be envisaged even to samples with multiple virus infections or at different stages of the infection and, in the crude plant sap, assured that baseline reference measures of the expected targets have been recorded.Considering that standard DAS-ELISA detects virus protein from 1 to10 ng/mL of sap , while t