Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection; all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species ( Solanum Lycopersicum L.) is one of the most important vegetable crops. The worldwide production of tomato in 2019 was more than 182 million tons . Spain is one of the world’s leading producers of tomato plants for fresh consumption, second in the European Union after Italy . Tomato cultivation in Spain is very intensive, with significant acreage devoted to greenhouse production. The major threats to tomato intensive cultivation are viral diseases, which are responsible for significant yield and fruit quality losses, causing important economic damage [Tomato  technologies enable, in a relatively short period of time, the characterization of plant viromes, allowing both the detection of known viruses and the discovery of novel ones . These t species . In addi species ,10. To d species  and both species .Lettuce ring necrosis virus, Olive latent virus 1, and Tomato fruit blotch virus.Here, we used the rRNA-depleted total RNA and dsRNA approaches to identify the viruses present in tomato samples from plants exhibiting virus-like symptoms. We compared the virus diversity and mapping reads between two replicates from total RNA extractions. In addition, we adopted an in silico approach to determine the minimum sequencing depth needed to detect the less abundant viruses in our sample pools. We identified seven known virus species, three of which are reported for the first time in tomato plants in Spain: Twenty samples of tomato leaves exhibiting symptoms suggesting viral infection were collected during the 2015–2020 period from different locations in Spain and Portugal . A portiTM One Microvolume UV-Vis Spectrophotometer  and a QubitTM 3.0 Fluorometer (Thermo Fisher Scientific), and individual samples were normalized to a final concentration of 20 ng/µL. Two identical pools denoted Tom1 and Tom2 were prepared by adding 7.5 μL from each total RNA sample to obtain a final amount of 3 μg of total RNA in 150 μL of sterile MiliQ water. The dsRNA sample (denoted as TomDS) had a final volume of 50 μL in sterile MiliQ water. The samples were sent to Macrogen  for library preparation and sequencing. The quality and quantity of the RNA in the three samples were further analyzed using a 2100 Bioanalyzer . For the three samples, cDNA libraries were synthesized using a TrueSeq Stranded Total RNA sample preparation kit  with ribosomal depletion using a Ribo-Zero plant kit (Illumina). Sequencing was performed with the Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads.For the total RNA extraction, 100 mg of leaf tissue from each individual sample was ground until attaining a fine powder with a mortar and pestle in the presence of liquid nitrogen. Total RNA was extracted using TRI Reagent  following the manufacturer’s protocol. For the dsRNA extraction, 100 mg of leaf tissue from each sample was pooled and ground. dsRNA was purified using the protocol from Valverde et al.  with Whahttp://www.bioinformatics.babraham.ac.uk/projects/fastqc; accessed on May 2020). Adapters and low-quality reads (Phred < 30) were trimmed from each data set using Trimmomatic v0.39 [https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/; accessed on May 2020). The database was built using makeblastdb, and low-complexity sequences were filtered out with dustmasker [−4, and length of alignment between the query and the hit ≥ 300 nt. Pyfasta v0.5.2  was used to retrieve the sequences of the reference viruses detected. Only one random accession for each virus was retrieved in the case that more than one accession for the same virus was found by BLASTn. These viral sequences were used as the reference to re-map the filtered reads using BWA with the mem algorithm [https://www.ncbi.nlm.nih.gov/orffinder/; accessed on May 2020) to predict ORFs of new putative viruses, and BLASTx against non-redundant proteins NCBI database to find the closest virus species. Subsets of reads used to determine the minimum number of reads needed to detect the viruses present in the datasets were obtained with Seqtk [Raw reads were analyzed using the custom bioinformatic pipeline implemented in the R language, as described in ic v0.39 . After tic v0.39 . Host reic v0.39 . Unmappeic v0.39 . For viric v0.39 . To builstmasker . After Blgorithm . From thlgorithm  and our lgorithm . In caselgorithm , using tth Seqtk , using dhttps://eu.idtdna.com; acceded on September 2020). The specificity of all the primers and probes was confirmed in silico by a BLASTn search against the NCBI database. Primers’ and probes’ sequence information and the length of the amplicons are detailed in All viruses were detected in individual samples by qRT-PCR, except southern tomato virus (STV), which was detected by conventional RT-PCR using the primer pair described in Twenty samples from leaves of tomato plants exhibiting virus-like symptoms were collected from 2015 to 2020 from different locations in the South of Spain and Portugal . The samAfter sequencing the three libraries, we obtained 86,284,538, 84,739,174 and 64,540,826 reads for Tom1, Tom2 and TomDS, respectively . The rawIn order to evaluate the reproducibility of the library construction and sequencing, the results from the two independent libraries, Tom1 and Tom2, were compared. The filtered reads were de novo assembled, and long contigs were mapped by BLASTn against our own plant virus database . After fTo calculate the average sequencing depth and the genome coverages, the filtered reads were mapped against the reference sequences of the identified viruses. In the cases where multiple accessions were found for the same virus species, the accession of the reference sequence used to map the reads was randomly selected. Viral reads constituted 7.99% and 8.57% of the clean reads for Tom1 and Tom2, respectively . The numProcedures for sample preparation and RNA extraction for dsRNA and total RNA were obviously different, but the amount of plant material used for both methods was the same, allowing some comparisons. Since Tom1 and Tom2 are almost identical replicates, only the comparison between TomDS and Tom1 is described. Fifty-five assembled contigs, with an average length of 3698 nt, derived from TomDS, mapped with our plant virus database, representing six virus species previously found in the analysis of the total RNA sample . The virIn an attempt to determine the minimum number of reads needed to detect the viruses infecting our tomato samples, we performed three different in silico simulations by decreasing the number of initial raw reads used in the bioinformatic analysis. For this, three subsets of raw data, consisting of 50% (Subset 1), 37.5% (Subset 2) and 25% (Subset 3) of the original Tom1 and TomDS reads were extracted randomly and analyzed following the same pipeline described previously . We nextAfter the analysis of the different subsets , we founPotexvirus), a (+)ssRNA virus, was the most abundant virus in our sample pools, with more than 28 M reads across the three datasets: more than 13 M reads in both total RNA samples, and more than 15 M reads in the dsRNA sample , which possesses a dsRNA genome, was the second virus for which a higher number of reads mapped along its genome in the dsRNA sample (more than 3 M), although very few viral reads were obtained from the total RNA samples (1782 in Tom1 and 1812 in Tom2)  . We werein Tom2) . These sin Tom2) .Crinivirus), with a bipartite (+)ssRNA genome, was the fourth most abundant virus among the seven viruses identified, with 457,789 reads in TomDS and around hundred times less in the total RNA samples (3842 in Tom1 and 3870 in Tom2)  . A uniquin Tom2) .Begomovirus), a (+) ssDNA virus, was detected using the two different RNA inputs but, with the exception of OLV1, it was the virus for which we obtained the lowest number of reads mapping to its genome (around 1694 and 1596 in Tom1 and Tom2 and 1344 in TomDS)  . DiffereWe also identified three virus species that, to our knowledge, were not previously reported or not frequently reported to infect tomato plants in Spain: LRNV, OLV1 and ToFBV ,28. To fOphiovirus) is a four-segmented (−)ssRNA virus, and was detected in the pooled sample using the two RNA extraction approaches. We determined the consensus sequences for the four LRNV RNA segments (RNA 1 to 4). The RNA 1 consensus sequence (MW594439) was 7604 nt in length, covering 99% of the reference segment (AY535016), sharing a 99.4% identity with it. This RNA comprises two ORFs, which are 582 nt and 6834 nt in length, respectively. The RNA 2 consensus sequence (MW594440) was 1826 nt in length, lacking only 4 nt at the 5′ end compared to the reference RNA2 sequence (AY535017). Our sequence shared 99.2% at the nt level and 99.1% at the amino acid level with its RNA2 reference. The RNA 3 consensus sequence (MW594441) was 1505 nt . Its vcRNA has one ORF of 1311 nt that encodes the CP protein. The CP showed a 99.1% amino acid identity with the CP of this reference isolate. The consensus sequence of RNA 4 (MW594442) was 1378 nt in length, covering 97% of the LRNV RNA 4 genome (AY535019) and sharing a 99.1% of nucleotide identity. The presence of LRNV was confirmed in the pooled sample as well as in four out of 20 individual samples by qRT-PCR using specific primers and probes targeting the CP  is a four-segmented (+)ssRNA virus, recently discovered to infect tomato plants in Italy and Australia [ToFBV , with a (+)ssRNA genome, was the least abundant virus in our datasets  and citrus dwarfing viroid (CDVd)  infecting grapefruit, rendering better coverage for CTV but not for the viroid [Physalis peruviana L.). They found higher sequence diversity for a specific virus species in total RNA as compared to dsRNA, but the total RNA extraction method failed to detect viruses present at low concentrations [HTS was used to identify viruses present in a pool of twenty samples of tomato leaves showing virus-like symptoms collected in areas where tomato cultivation is very important. We first assessed the reproducibility of the sequencing method by generating a replicate of the total RNA with a ribosomal depletion sample; after analysis, we did not find important differences between replicates in terms of virus species detected or the number of reads mapping to each reference virus genome. In contrast, our data suggest that the extraction method seems to have an impact on the viruses that could be identified by HTS, in agreement with previous reports. For instance, Kutnjak et al.  comparede viroid . Anothere viroid . Ma et ae viroid  comparede viroid . Gallo-Ge viroid  used tottrations . Hence, trations ,30. In otrations .Mastervirus) in two quarantined sugarcane plants, showing the importance of including this method to assess plant health status [Some previous works have discussed the possibility of the application of HTS for routine plant virus diagnostics, mentioning different parameters to take into consideration, such as sensitivity, specificity and reproducibility . Al Rwahh status . Howeverh status ,37,38,39h status  providedh status . Here, wThree viruses known to infect tomato plants, and to induce important crop losses, were detected in our samples. PepMV was detected in 19 samples by means of qRT-PCR, confirming the HTS results. Two strains, EU and CH2, were detected in mixed infections in 13 out of 19 PepMV-infected samples. This high incidence could be due to the generalization of cross-protection as a means of disease control in the South of Spain , though Lactuca sativa) crops in 1996, associated with lettuce ring necrosis disease. LRNV is transmitted by the soil-borne fungus Olpidium brassicae [In addition to viruses frequently cited in tomato plants, three viruses that are seldom if ever cited to infect these species were detected in this study. The first report of LRNV was in lettuce  but which remained unreported. The increased ability to detect new or infrequent viruses using this technique raises several questions relating to how to deal with them from a crop protection point of view, as well as the complexity of their biological characterization, particularly for the newly-identified plant viruses and viroids, and their impact at biosecurity, commercial, regulatory and scientific levels . Here, w


Pepper mottle virus (PepMoV) is a destructive pathogen that infects various solanaceous plants, including pepper, bell pepper, potato, and tomato. In this review, we summarize what is known about the molecular characteristics of PepMoV and its interactions with host plants. Comparisons of symptom variations caused by PepMoV isolates in plant hosts indicates a possible relationship between symptom development and genetic variation. Researchers have investigated the PepMoV–plant pathosystem to identify effective and durable genes that confer resistance to the pathogen. As a result, several recessive pvr or dominant Pvr resistance genes that confer resistance to PepMoV in pepper have been characterized. On the other hand, the molecular mechanisms underlying the interaction between these resistance genes and PepMoV-encoded genes remain largely unknown. Our understanding of the molecular interactions between PepMoV and host plants should be increased by reverse genetic approaches and comprehensive transcriptomic analyses of both the virus and the host genes. Pepper mottle virus (PepMoV), which is in the genus Potyvirus and the family Potyviridae [Capsicum sp.), potato, and tomato in North America, India, and Asia [yviridae , has beeand Asia ,6,7,8,9.and Asia ,11. PepMand Asia ,13. Whenand Asia ,15.PepMoV has a single-stranded positive-sense RNA genome that is about 10 kb in length ,16,17. TPotyvirus genus . Comneage 5; ,27,28.CP) genes of nine PepMoV isolates from pepper in Southern China, they detected two distinct groups and subgroups.To date, PepMoV has been reported from many regions in North America, East Asia, and India, and full genome sequences of 23 isolates are available and sequences of 45 isolates are partially released at the NCBI database. A phylogenetic analysis based on the deduced amino acid sequences indicated that all 13 Korean isolates of PepMoV formed one cluster that was distinct from American isolates ,30. HoweBased on its genetic variation and on its symptom severity and pathogenicity on different host plants, researchers divided 13 Korean isolates of PepMoV into two groups ,13. HaviLike all potyviruses, PepMoV forms a flexuous rod-shaped virion that consists of about 2000 copies of CP subunits with an Mr of 30.8 kDa . The virE. coli-based plasmids. For example, toxicity to E. coli has been reported for plasmids containing full-length clones of several viruses including citrus tristeza virus, tobacco etch virus (TEV), and influenza A virus; such toxicity makes it difficult or even impossible to use the clone for molecular manipulation [E. coli have also been reported to be unstable for plant RNA viruses including potyviruses [E. coli, insertion of introns into the viral genome has been extensively used [Agrobacterium-mediated inoculation of this modification clone showed faster symptom induction compared to non-modified clones when the same amount of Agrobacterium cell suspension was inoculated in plants [Both of the approaches described in the previous paragraph have been used to construct full-length cDNA clones of PepMoV. A Korean isolate of PepMoV (PepMoV-Vb1) was cloned downstream of a bacteriophage SP6 promoter and was tagged with green fluorescent protein (GFP), which was inserted between the coding regions for NIb and CP in the plasmid . In vitrpulation ,36,37. Dyviruses ,39,40,41yviruses ,43. To rely used ,40. For ely used . Relativn plants . In addin plants .In research on the role of virus-encoded proteins during the virus infection process, use of the infectious full-length cDNA clones has provided reliable information on viral RNA replication and movement ,45. Almoin trans to stimulate genome amplification [The P1 protein of potyviruses is a chymotrypsin-like serine proteinase that cleaves itself at C-terminus ,46. P1 ification . The funfication . The potfication .Arabidopsis 20S proteasome subunits , which is related to the antiviral response [Nicotiana tabacum and eIF(iso)4E and eIF4E of potato suggesting possible new role(s) in potyvirus infection cycle [The HC-Pro is a cysteine protease and well-established multitasking protein that is involved in many potyviral infection processes such as aphid-mediated transmission, RNA silencing suppression, genome replication, symptom expression, and long-distance movement ,52. HC-Presponse . In addion cycle .The protein P3 is also one of the well-characterized multifunctional potyviral proteins. Dual roles of TEV P3 in virus movement and replication have been reported ,57. A poThe 6K1 of plum pox virus (PPV) is required for viral replication and is a necessary viral element of the viral replication complexes (VRC) at the early infection stage .The multifunctional CI protein, as part of the VRC, participates in viral genome replication. In addition to replication, it also functions in viral cell-to-cell and long-distance systemic movement, probably by interacting with the recently reported viral P3N-PIPO protein ,58,61. TThe potyviral 6K2 protein has been found to be involved in long-distance movement and symptom development ,67,68. IPotyvirus VPg contains two nuclear localization signals (NLSs) and nucleotide triphosphate binding motifs . VPg is Ry-mediated HR in Solanum stoloniferum by sharing the same recognition/cleavage site for NIa [Arabidopsis [Potyvirus NIa-Pro is a cysteine protease and generally functions proteolytic processing of the potyviral polyprotein . NIa-Pro for NIa . Expressbidopsis . NIa-Probidopsis . This rebidopsis . Recent bidopsis .Pvr4- or Pvr9-mediated hypersensitive response (HR) [NIb of potyviruses acts as an RNA-dependent RNA polymerase (RdRp) or RNA replicase, and is therefore required for potyviral genome replication . Beyond nse (HR) ,21.122) of CP in tobacco vein banding mosaic virus, which is highly conserved residue among potyviruses, plays a role in maintaining stability of CP during viral replication and this involvement in viral cell-to-cell movement was also observed with the same residue of watermelon mosaic virus and PVY [The potyviral CP has also been reported to participate in the regulation of viral RNA replication ,89. Poty and PVY . An addi and PVY . The hig and PVY ,99.So far, extensive research has mainly focused on the defense response-related PepMoV-encoded proteins and their corresponding host genes. Although the functions of HC-Pro remain to be established in PepMoV, the protein is likely to be involved in replication and systemic movement. Likewise, PepMoV NIb might have roles in symptom development and virus multiplication, which is supported by our recent study (unpublished data).R gene-mediated resistance responses, and systemic acquired resistance [R) genes for application in plant breeding [Plants have evolved multi-layered systems to defend against viral invasion, including RNA silencing, regulation of RNA stability, ubiquitination-mediated protein degradation, autophagy, HR, sistance ,100,101.breeding ,102. ChaR genes [In incompatible interactions between a plant virus and host, resistance responses can be mediated by recessive or dominant host genes  21,80,8,106. RecR genes . RecessiR genes .Capsicum spp., pvr1 and pvr3 have been characterized as two unlinked recessive loci that confer distinct kinds of resistance to PepMoV [pvr1 gene, which was identified in C. chinense PI159236 and PI152225, confers relatively broad resistance to PepMoV, TEV, and PVY [pvr3 gene, which was identified in C. annuum ‘Avelar’, confers a different type of resistance to PepMoV than to TEV and PVY [Capsicum spp. against PepMoV differ depending on whether the response is pvr1- or pvr3-mediated [C. chinense PI 152225 and PI 159236, which contain pvr1, do not support replication of PepMoV at the cellular level, whereas C. annuum ‘Avelar’, which contains pvr3 allows for PepMoV accumulation in inoculated leaves and its movement into the vascular system but not its spread into upper leaves [pvr2 and pvr6, that are located on the pepper chromosomes 4 and 3, respectively, confer digenic recessive resistance to another pepper potyvirus, pepper veinal mottle virus [pvr2 and pvr6 in response to the infection of PepMoV remains to be determined.In o PepMoV . The pvr and PVY . In cont and PVY . The mecmediated . C. chinr leaves ,109. Howle virus . Howeverpvr1 locus encodes an eIF4E homolog, and pvr6 is expected to encode eIF(iso)4E [pvr1, researchers documented dominant resistance to several potyviruses, including PepMoV and TEV [pvr1 or eIF4E greatly affects the resistance/susceptibility to plant viruses, especially to PepMoV, although the specific mechanism is unclear. In general, previous studies have suggested that eIF4E is also required for cell-to-cell movement and viral RNA replication during potyvirus infections [The eukaryotic translation initiation factors (eIF4Es) have been identified and cloned from diverse hosts as resistance genes that are natural, recessive, and inherited A 80,111111. The F(iso)4E . RecessiF(iso)4E ,112. Usi and TEV . Moreove and TEV ,114. Thefections ,115. TheR genes, corresponding to pathogen effector-encoding or avirulence (Avr) genes, confer an active resistance resulting in the development of an HR that limits pathogen spread [R genes encode proteins consisting of a nucleotide-binding site (NBS), a leucine-rich repeat (LRR) region at the C-terminal, and Toll/Interleukin-1 receptor homology or a coiled-coil (CC)-domain at the N-terminal end -type protein, and ectopic expression of Pvr4 in N. benthamiana confers resistance against PepMoV [pvr4 in the susceptible allele from C. annuum ‘ECW’ had higher similarity with the coiled-coil nucleotide-binding domain than with the LRR domain, which might be involved in specific recognition of Avr factors [Pvr4 in pepper [Pvr4-bearing pepper [t PepMoV . Kim et  factors ,122. Resn pepper . The NIbg pepper . Kim et g pepper .R gene, Pvr9, is orthologous to Rpi-blb2 of Solanum bulbocastanum and was isolated via screening of Agrobacterium-based transient expression of candidate R genes that were able to induce an HR upon PepMoV infection in N. benthamiana [Pvr9 is expected to be located on pepper chromosome 6, and encodes 1298 amino acids that contain CNLs-type protein domains [Pvr9 gene expression in the resistant cultivar C. annuum ‘CM334′ but in a slightly reduced expression of the susceptible allele in the susceptible cultivars C. annuum ‘FloralGem’ [Pvr9-mediated HR, which is similar to the Pvr4-mediated HR [Another thamiana . Pvr9 is domains . PepMoV oralGem’ . Tran etiated HR ,21.Previous research has demonstrated that virus infection of plants affects host gene expression and metabolism, which results in altered host development and growth defects . The chaPvr9-mediated HR against PepMoV infection, researchers silenced selected genes using tobacco rattle virus-based virus-induced gene silencing and thereby assessed their functions [Pvr9-mediated HR requires the host genes HSP90, SGT1, NDR1, and NPR1 genes but not the EDS1 gene [Pvr9-mediated HR might involve the salicylic acid (SA) pathway but not the jasmonic acid (JA), ethylene (ET), reactive oxygen species (ROS), or nitric oxide (NO) pathways  between viral elicitor(s) and the host receptor(s) might contribute to the resistance responses. In this regard, it is noteworthy that the potyviral protein VPg, which is required for viral infection, interacts with eIF4E to induce infection; mutations in eIF4E. However, prevent VPg binding and thus inhibit viral infection, resulting in a resistance response ,115. At Mutation of eIF4E affects the infectivity of PepMoV in tomato , but thePvr4 in pepper plants [Pvr9 and its elicitor or so-called avirulence factor NIb of PepMoV was not detected in the model plant N. benthamiana [N. benthamiana but not in pepper.Several potyvirus proteins have also been identified as elicitors of resistance or determinants of avirulence, and these correspond to dominant resistance proteins in plants . As prevr plants . Howeverthamiana . The autPvr9 was co-expressed with NIbs from PepMoV, PVY, PVA, and turnip mosaic virus, but not with NIbs from zucchini yellow mosaic virus or soybean mosaic virus [Pvr9 and NIb in yeast or in plants, the mutational analyses suggested their possible relationship between Pvr9 and NIb [Pvr9 failed to trigger an NIb-elicited HR in plants, while internal regions of NIb (the residues 186–235 and 370–445) are essential for NIb elicitor activity [An HR was also triggered when ic virus . Althoug and NIb . The amiactivity .N. benthamiana [In many potyviruses, VPg and especially HC-Pro help block or interfere with RNA silencing . A recenthamiana . HoweverN. benthamiana [NbAGO4, NbMET1, NbDRM2, and NbCMT3 [As noted earlier, the use of a potato virus X-based NIa expressing vector indicated that PepMoV NIa might be responsible for symptom development in thamiana . In addithamiana . These rd NbCMT3 C. annuum ‘Zunla-1′ pepper plants, for example, transcript profiles of CMV-Fny infected leaves showed different expression patterns at different time points [C. annuum at different time points after infection by Phytophthora infestans, PepMoV, or tobacco mosaic virus [In the last decade, many researchers studied plant–virus interactions by focusing on genome-wide expression patterns of host and virus genes . The gene points . Kim et ic virus . FurtherPepMoV is one of the most important pathogens of solanaceous vegetables worldwide. Although the roles of each encoded PepMoV gene can be inferred by comparison with analogous genes in other potyviruses, the role(s) or function(s) of each PepMoV protein remain poorly characterized. Use of a PepMoV infectious clone will help researchers to identify the determinants of PepMoV pathogenicity/virulence and to understand the replication and movement of the virus in infected host plants.


Southern rice black-streaked dwarf virus (SRBSDV), a Fijivirus in the Reoviridae family, is transmitted by the white-backed planthopper , a long-distance migratory insect, and presents a serious threat to rice production in Asia. It was first discovered in China’s Guangdong Province in 2001 and has been endemic in the south of China and north of Vietnam for two decades, with serious outbreaks in 2009, 2010, and 2017. In this study, we evaluated the resistance of 10 dominant rice varieties from southern China, where the virus overwinters and accumulates as a source of early spring reinfection, against this virus by artificial inoculation. The results showed that in all tested varieties there was no immune resistance, but there were differences in the infection rate, with incidence rates from 21% to 90.7%, and in symptom severity, with plant weight loss from 66.71% to 91.20% and height loss from 34.1% to 65.06%. Additionally, and valuably, the virus titer and the insect vector virus acquisition potency from diseased plants were significantly different among the varieties: an over sixfold difference was determined between resistant and susceptible varieties, and there was a positive correlation between virus accumulation and insect vector virus acquisition. The results can provide a basis for the selection of rice varieties in southern China to reduce the damage of SRBSDV in this area and to minimize the reinfection source and epidemics of the virus in other rice-growing areas. Oryza sativa) is one of the main food crops around the world, and viral disease is a serious problem in rice production [Rice  has occurred in southern China, northern Vietnam, and Japan; it is transmitted by a long-distance migratory pest, the white-backed planthopper , and is the most serious rice virus in Asia [Since the beginning of the 21st century, the  in Asia ,6,7. The in Asia ,9. In re in Asia ,11.Southern China is a major area where SRBSDV overwinters and accumulates in early spring as a source of infection . At presA total of 10 dominant rice varieties—hybrid indica rice Guang8you 165, Baixiangyou 9978, Guyou 248, Yexiangyou 9, and Guiyou 117, and inbred indica rice Hainonghong 1, Meixiangzhan, Guiyu 9, Huaxiang, and Wushansimiao—were selected Provinces Guangdong, Guangxi, and Hanan, China. Japonica rice Nipponbare (NIP) and a local indica rice variety, U25, were used as high-susceptibility and high-resistance controls, respectively. SRBSDV and WBPHs were collected from fields in Luoding, Guangdong Province, China, were maintained on NIP plants in a greenhouse, and used in all experiments in this study.Seeds of the tested rice varieties were soaked in water for 12 h, germinated for 2 days at 37 °C, and then seeded in a 29 cm diameter circular flowerpot with organic peat soil (Jiffy) as the substrate. After the seedlings reached the 2-leaf stage, the weak seedlings were removed, and 20 healthy rice seedlings were retained in each pot. Then, 2 pots for each variety, 24 pots in total, were randomly arranged in a small net room. At the same time, the WBPH nymphs at the 3rd–4th instar fed on SRBSDV-infected rice plants at the tillering stage for over 12 days, by which time the hoppers had developed to the adult stage; then, an RT-PCR detection for SRBSDV was conducted following Yang et al. [The seedlings of the tested rice varieties were cultivated by the above methods. Each pot containing 20 seedlings at the 3–4-leaf stage was covered by a small plastic cup with some vents punched in the side. A total of 10 viruliferous WBPH adults were released into each cup. After 2 days of coculture, the insects were removed, and the rice seedlings were cultured to the 4–5-leaf stage and then transplanted in the field for symptom observation. The experiments were repeated three times, and more than 30 seedlings were tested for each variety in each experiment.At 45 days post-inoculation (dpi), the tested rice plants had developed to the elongation stage and were observed. SRBSDV-infected plants were diagnosed based on the symptoms of dwarfism and dark green color and the results of RT-PCR detection. The plants showing SRBSDV symptoms and RT-PCR positivity were defined as infected plants; others were defined as healthy plants. All tested plants were observed, and the SRBSDV infection rate was calculated for each variety. Meanwhile, over 10 infected, and healthy plants for each variety were measured for their height and fresh weight. The relative dwarfing rate and the relative weight loss rate of the infected plants were calculated and compared with the healthy plants.At 15 dpi, 30 dpi, and 45 dpi, tissues from the second developed upper leaf of infected and healthy plants were sampled and detected via RT-qPCR  for the To eliminate any influence of hopper resistance on virus infection, the survival rates of WBPH on different varieties were determined. In total, 15 to 20 WBPH adults were raised on 5 plants at the 3–4-leaf stage in a glass tube. Two days later, the hoppers were checked, and their survival rates on different rice varieties were calculated. The conditions of the culture were the same as above. The experiment was repeated three times.Approximately 30 WBPH adults were exposed to SRBSDV-infected plants at 15 dpi, 30 dpi, and 45 dpi in a cage for 6 h. The hoppers were then transferred to healthy rice seedlings to raise for 15 days, then detected individually by RT-PCR for SRBSDV [p < 0.05 or p < 0.01; the dwarfing rate and weight loss were analyzed by Duncan’s multiple range test at p < 0.05. Pearson correlation analysis was used to analyze the relationship between virus accumulation and WBPH virus acquisition rate.The statistical software package SPSS 19.0  was used to analyze the experimental data. The data were analyzed by one-way analysis of variance (ANOVA) to check for significant differences between infection rates in different varieties, virus accumulation in infected plants, WBPH survival rates on different rice varieties, and WBPH virus acquisition potency from infected rice plants at In order to detect dominant variety resistance against SRBSDV, we checked the infection rate via two methods: simultaneous virus inoculation for multiple varieties and separate virus inoculation for single varieties. The results obtained by the two methods were basically the same. No immune varieties were found, but the infection rates of different varieties could be divided into three categories A,B. U25,To determine whether the differences in the infection rates of varieties were related to insect resistance , we measAfter SRBSDV infection, typical symptoms of plant dwarfing, dark green leaves, short and stiff leaves, and crinkled, even twisted leaves, were observed on the infected plants of the 12 tested varieties. No significant differences in the types of symptoms were observed among varieties, but the severity of symptoms varied A.At 45 days post-infection (dpi) with SRBSDV, the plant height and weight of the infected plants of each variety were significantly reduced. The relative dwarfing rate and the relative weight loss rate of infected plants were calculated compared with the healthy plants. Based on these rates, we can divide all varieties into three categories: varieties U25 and Guiyu 9, with dwarfing rates of 30%–45% and weight loss rates less than 70%, were classified as resistant varieties; varieties Nip, Yexiangyou 9, and Hainonghong 1, with dwarfing rates of 40%–55% and weight loss rate of 70%–80%, were defined as moderately resistant varieties; varieties Guiyou 117, Wushansimiao, Huaxiang, Guyou 248, Meixiangzhan, Baixiangyou 9978, and Guang8you 165, with dwarfing rates over 55% and weight loss rates over 80%, were identified as low-resistance varieties B,C.Virus accumulation often reflects the resistance of varieties to a virus. We detected the virus accumulation in plants infected with SRBSDV by RT-qPCR and Western blot. There were significant differences in virus accumulation among the different rice varieties. According to the average virus accumulation in leaf tissues of infected plants at 15, 30, and 45 dpi, the 12 tested varieties can be divided into 3 categories: the varieties Guang8you 165, Guiyu 9, Wushansimiao, Meixiangzhan, Hainonghong 1, and U25 with low virus accumulation; the varieties Huaxiang, Guyou 248, Guiyou 117, and NIP with medium virus accumulation; the varieties Yexiangyou 9 and Baixiangyou 9978 with high virus accumulation. The difference in virus accumulation between resistant and susceptible varieties was more than sixfold. Additionally, there were significant differences in virus accumulation patterns among the different varieties. The varieties with low virus accumulation also showed slow virus titer increase with the development of the disease course, which indicated that these varieties had strong resistance to SRBSDV propagation. For example, varieties Guang8you 165, Guiyu 9, Wushansimiao, and NIP supported rapid virus accumulation that reached a peak at 30 dpi, then decreased; variety Guiyou 117 had higher virus accumulation in the early stage of virus infection, and the accumulation gradually decreased with the development of the disease course; variety Baixiangyou 9978 accumulated the virus to high levels at an early stage following a sharp decrease and increase; variety Yexiangyou 9 had high virus titers only in a short middle period of the course of the disease A,B. The p = 0.03 < 0.05). Based on the mean virus acquisition rates of WBPHs at the three disease time points , we can group the tested varieties into three categories: varieties Guang8you 165, Guiyu 9, and U25, had low virus supply ability; varieties Wushansimiao, Meixiangzhan, and Hainonghong 1 had moderate virus supply ability; varieties Huaxiang, Guiyou 117, Guyou 248, NIP, Yexiangyou 9, and Baixiangyou 9978 had high virus supply ability (Virus acquisition by the insect vector (WBPH) from infected rice plants determines how quickly SRBSDV can spread and become epidemic ,19. Ther ability B.In previous reports, infection rate and severity of symptoms were usually used to evaluate the disease resistance of crop varieties. However, crop resistance should include characteristics that slow down a disease epidemic. The concept of slow rusting for wheat rust resistance was put forward by Caldwell  and applIn this study, we evaluated the SRBSDV resistance of 10 dominant rice varieties from southern China, where the virus overwinters and accumulates as a source of early spring reinfection, by artificial inoculation. We checked the varieties for four characteristics: infection rate, symptom severity, virus accumulation in infected plants, and WBPH virus acquisition potency from infected rice plants. Among these four characteristics, only the infection rate is directly related to the yield loss of rice caused by the disease and reflects the disease resistance of varieties in the traditional sense. The virus accumulation in infected plants and WBPH virus acquisition potency from infected plants represent a typical case of slow diseasing resistance. While the symptom severity regarding dwarfing and weight loss is not associated with rice yield loss due to the infected plants being unable to ear and produce grain, the degree of dwarfing and the reduction in the fresh weight of diseased plants have a deleterious effect on the living space and food quantity of the virus-transmitting vector WBPH; therefore, symptom severity is also a characteristic of slow disease resistance.Among the 10 dominant varieties tested in this study, variety Guiyu 9 showed resistance in the above 4 characteristics; the two varieties Guang8you 165 and Hainonghong 1 showed resistance in the infection rate, virus accumulation, and WBPH virus acquisition ability; and the two varieties Meixiangzhan and Wushansimiao showed resistance in virus accumulation and WBPH virus acquisition ability. These varieties can be recommended for use in production to reduce losses due to disease and slow the spread of disease.In Asia, WBPH, the vector of SRBSDV, is a typical long-distance migratory insect. In spring, it migrates from low latitude to high latitude with the southeast monsoon , resultiThis study confirmed that the resistance of different rice varieties to SRBSDV exhibits diverse properties, including reducing virus infection and slowing disease epidemics. Although there was no immune resistance found in southern China’s dominant rice varieties, some indications of infection resistance and disease-slowing resistance were screened. Rice varieties with low virus accumulation levels and vector virus acquisition ability in infected plants can be used to minimize the source of reinfection and slow the spread of the disease.


These results indicated that the CN-INMP@TPGS were absorbed rapidly after pulmonary administration and resulted in increased systemic absorption. Therefore, the inhalable CN-INMP could significantly improve the bioavailability of CN after inhalation administration. The developed mucus-penetrating nanocrystals-in-microparticles might be regarded as a promising formulation strategy for the pulmonary administration of poorly soluble drugs.To effectively achieve the pulmonary delivery for curcumin (CN), novel inhalable mucus-penetrating nanocrystal-based microparticles (INMP) were designed. The D-Tocopherol acid polyethylene glycol 1000 succinate (TPGS) modified CN nanocrystals (CN-NS@TPGS) were prepared by high pressure homogenization and further converted into nanocrystal-based microparticles (CN-INMP@TPGS) using spray-drying. It was demonstrated that CN-NS@TPGS exhibited little interaction with the negatively charged mucin due to a strong electrostatic repulsion effect and PEG hydrophilic chain, and exhibited a much higher penetration ability across the mucus layer compared with poloxamer 407 modified CN-NS (CN-NS@P407) and tween 80 modified CN-NS (CN-NS@TW80). The aerodynamic results demonstrated that the CN-INMP with 10% TPGS acting as the stabilizer presented a high FPF value, indicating excellent deposition in the lung after inhalation administration. Additionally, in vivo bioavailability studies indicated that the AUC Unf mL/min) , as well mL/min) . Meanwhi mL/min) ,10 and m mL/min) ,12. Owin mL/min) ,14. More mL/min) ,16. The  mL/min) . So, by  mL/min) . AlthougThe curcumin (CN) was found to have strong anticancer, antioxidant, anti-inflammatory activities ,20, but Curcumin  was purchased from Chengdu Herbpurify Biotechnology Co., Ltd. . The mucin was commercially purchased from Sigma-Aldrich . D-Tocopherol acid polyethylene glycol 1000 succinate (TPGS) was commercially purchased from Up-style Biotechnology Co., Ltd. . Both tween 80 (TW80) and poloxamer 407 (P407) were commercially purchased from Beijing Fengli Jingqiu Pharmaceutical Co., Ltd. . Alexa Fluor-488-WGA was obtained from Thermo Fisher Scientific . Lactose (LA) were kindly obtained from Dingsheng Chemical Co., Ltd. . Beas-2B cell and A549 cell lines, cell counting kit-8 (CCK-8) were purchased from KeyGEN BioTECH Co., Ltd. .w/w, relative weight to CN), respectively. The CN nanocrystals suspensions (CN-NS) were then prepared using AH100D high-pressure homogenizer  at 800 bar pressure for 40 cycles. Thereafter, the different concentrations of lactose (w/w) as matrix formers was added into the CN-NS, respectively. Spray-drying was performed using a Büchi spray dryer B-90 . The gas inlet temperature and flow rate were set to 75 °C and 4 mL/min, respectively. The atomizing air flow was at 55 mmHg. The CN-INMP was deposited at the bottom of the collector. The samples were stored in a desiccator at room temperature for further evaluation.The CN-NS were prepared according to the reported method with some modification . BrieflyThe particles size of CN-NS was measured by a Mastersizer Micro Plus . The Zeta potential of the CN-NS was determined using the Zetasizer Nano-ZS . All samples were measured in triplicate at 25 °C.Transmission electron microscopy (TEM) was used to observe the morphology of CN-NSs. The samples were dropped onto a copper grid  and dried in air. The copper grid loading CN-NS was observed using the JEM-1200EX .The mucin was dissolved in PBS (1 mg/mL) and stirred for 2 h. Then, 5 mL different CN-NS solution was reacted with a 1 mL mucin solution (2 mg/mL) at 37 °C for 4 h in a shaker (150 rpm). The size and zeta potential of the mucin-NS complex at a predetermined time point were measured using the Zetasizer Nano-ZS  .appP, cm/s) of CN-NS was evaluated according to the equation below:Qd/td represents the diffusing rate of different surface modified CN-NS.Furthermore, a 24-well transwell system with 0.4 μm pores  was used to assess the mucus-penetrating ability of the CN-NS according to the previously reported method . Briefly2) of the membrane. C0 represents the initial CN-NS concentration in the donor compartment.A represents the area  for 10 min in a shaker (150 rpm) at 37 °C. After that, 1 mL of stained mucin was deposited into a confocal dish, and placed on a shaker (50 rpm) in order to generate mucus layers with equal thickness. The CN-NS (100 μL) were carefully added dropwise onto the mucus layer and incubated for 30 min at 37 °C. The 3D images were captured every 20 μm along the Z-axis using a CLSM .The redispersibility of CN-INMP in simulated lung fluid was evaluated according to the previously reported method . To do tv/v). The UV absorption wavelength was 428 nm. The flow rate and retention time was 1 mL/min and 4.5 min, respectively. The measurements were conducted in duplicates.The in vitro release of CN from different CN-INMP was investigated using the paddle method with a stirring rate of 50 rpm at 37.5 °C. Briefly, the coarse CN and CN-INMP containing an equivalent CN at a dose of 2 mg was, respectively, dispersed into 100 mL of simulated lung fluid with 0.2% of SDS . At the The particle morphology of all the coarse CN and CN-INMP were studied using a scanning electron microscope. The test powder samples were gold coated with platinum at 10 mA for 30 s using a sputter coater . The samples were then examined using the X650 scanning electron microscope (SEM)  at 5.0 kV.Thermograms of the CN-INMP were studied using a Diamond DSC . The temperature was firstly calibrated using indium and tin as reference materials. The test CN-INMP samples were heated in aluminum pans under nitrogen purge from 20 °C to 260 °C at a heating rate of 10 °C/min. The measurements were conducted in duplicates.The powder crystallinity of the coarse CN, matrix formers and CN-INMP were evaluated using D8ADVANCE X-ray diffractometer  with Cu-Kα radiation at 40 mA and 45 kV. The patterns were recorded at a scan angular speed of 2 °/min and a step length as 2θ from 5° to 90°. The measurement was repeated in triplicate.®  with a flow rate of 95 L/min and actuation time of 2.4 s. For each experiment, the perspirator was filled with 20 mL of the collection solvent, and stages were not coated. Particles deposited on the capsule shells, the inhalation device, the induction port, the mouthpiece adapter, the perspirator, and the impactor stages were collected by rinsing with the collection solvent. The in vitro aerodynamic performance was expressed in terms of the mass median aerodynamic diameter (ADe) and the fine particle fraction (FPF%). The fine particle fraction was calculated as the ratio (% w/w) of the amount of the drug in particles with an aerodynamic diameter smaller than 5.00 μm to the amount of the drug emitted from the device.The CN-INMPs were evaluated in vitro using a Next Generation Impactor . This assay was applied as in the previously described method . In each2. The Beas-2B cells were firstly seeded in 96-well plates (1 × 105 cells/mL) and cultured for 24 h before drug treatment. Then, the different concentrations of CN-NS were dropped into the cell wells. After treatment for 24 h, the cells were incubated with 10 μL of CCK8  at 37 °C for 4 h. Then, the optical density was measured at 450 nm with a microplate plate reader .The Beas-2B cell line was obtained from KeyGEN BioTECH Co., Ltd. . The cell was cultured in a BEBM medium contain 90% RPMI-1640 and 10% FBS, and incubated at 37 °C with 5% CO2. The cellular uptake evaluation of CN-NSs was determined using flow cytometer. After culturing for 2 days, the A549 cells were treated with 1 mL CN-NSs (50 μg/mL) at 37 °C for 2 h. Additionally, the culture medium was removed, the cells were washed 2–3 times with PBS, trypsinized and centrifuged. The supernatant was removed, and the centrifuged cells were gently resuspended in 0.25 mL PBS. The mean fluorescence intensity of cells was determined using the flow cytometer.The A549 cell line was obtained from KeyGEN BioTECH Co., Ltd. . The A549 cell was cultured in a medium contain 90% F-12K and 10% FBS. The cells were incubated at 37 °C with 5% COw/v). Then, the tissue homogenates were centrifuged using high-speed freezing centrifuge at 6000 rpm for 10 min at 4 °C, and the obtained supernatant was stored at −20 °C. Each tissue sample (50 μL) was vortex-mixed with 100 μL of emodin solution (100 ng/mL) as an internal standard (IS). After centrifuging for 10 min at 12,000 rpm and 4 °C, 10 μL of supernatant sample was assayed using HPLC/MS/MS. The full scan spectra of samples showed the parent ion at m/z 367.1/134.0 for CN and m/z 269.0/225.0 for IS, respectively. An elution system of aqueous formic acid and acetonitrile at a ratio of 40:60 was used. The flow rate was set as 1 mL·min−1.Animal welfare and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals and the ethics regulations of Jiangxi University of Traditional Chinese Medicine. The male rats were firstly anesthetized with an i.p. injection of ketamine/xylazine cocktail. The CN-INMP was intratracheally administered using a lavage needle at a dose of 10 mg/kg. Six rats for each time point were euthanized at predetermined time points (0.5 h and 2 h) after pulmonary administration, and the lungs were collected and washed for analysis. The obtained lungs were homogenized by adding an 80% acetonitrile solution at a ratio of 1:3  rats (180–220 g) were divided into the following three groups: (A) CN-INMP@TPGS, (B) CN-INMP@P407, and CN-INMP@TW80. Group A, B and C were subjected to pulmonary administration by dry powder insufflators. The administration dose of each group was equivalent to 10 mg/kg of CN. At least six rats were tested for each time point . Approximately 0.5 mL of blood was withdrawn from the orbital plexus at predetermined time intervals post administration. The blood samples were centrifugated at 6000 rpm for 10 min (4 °C), then the plasma was obtained and stored at −20 °C until further analysis. The CN plasma concentrations were assayed by a validated HPLC/MS/MS method as described above.Next, DAS 2.1.1 was used for the pharmacokinetic parameters analysis. Two-way or three-way analysis of variance (ANOVA) were used for statistical comparison. The significance level was set to 0.05 unless otherwise stated.Stabilizer was an important factor influencing particles size and the zeta potential of drug nanocrystals . The parThe change in particle size of the CN-NS incubated with mucin is shown in appP values of different CN-NS in the mucus. The penetrating ability and permeability coefficient (appP = 12.53) of the CN-NS@TPGS in mucus were significantly enhanced compared with the CN-NS@P407 (appP = 8.32) and CN-NS@TW80 (appP = 4.24). These further proved that TPGS-modified CN-NS exhibited a much higher penetration ability across the mucus layer compared with CN-NS@P407 and CN-NS@TW80, which could effectively block the adhesive interactions with mucus components.The ability of different CN-NSs to penetrate through the mucus layer was quantified using a transwell system. To directly observe the distribution of CN-NS in the mucus layer, the diffusion behavior of CN-NS was evaluated using confocal laser scanning microscopy (CLSM). e) and the fine particle fraction (FPF%) can generally be used as screening parameters to assess the aerodynamic performance and lung deposition ability of inhalable CN-INMPs. All the tested CN-INMPs had a relatively low tap density in the range of 0.302–0.431 g/mL , and the FPF value of tested CN-INMP increased remarkably with the increase in the lactose concentration (p < 0.05). The results also showed that the FPF of CN-INMP@P407 was not remarkably different to those of CN-INMP@TPGS and CN-INMP@TW80 using 200% of the lactose as matrix formers. This could be attributed to the suitable deposition efficiency of CN-INMP@P407 owing to a decreased adhesion among particles. However, using 100% of LA as the matrix former, the CN-INMP@TPGS and CN-INMP@TW80 exhibited similar ADe, but lower FPF values were obtained, which could be attributed to strong adhesion among particles and poor flowability owing to their hygroscopicity. Therefore, the 200% concentration of LA as matrix formers seemed to be the optimum choice for CN-INMPs with excellent lung-deposition performance.The results also showed that the concentration of lactose as matrix formers remarkably influenced the ADThe redispersibility index (RDI) could be used to evaluate the reconstitution ability of CN-INMP, indicating whether the CN-INMP could completely redisperse into CN-NS in the simulated lung fluid. The RDI of tested CN-INMPs were displayed in DSC thermograms of coarse CN, LA, CN-INMP@TPGS, CN-INMP@P407 and CN-INMP@TW80 are presented in p > 0.05), as shown in p < 0.05), respectively. Additionally, as shown in p < 0.05).In this study, the Beas-2B cell line was used as the model cell to assess the viabilities of CN-INMP. The viability of Beas-2B cells treated with different CN-INMPs was evaluated using Cell Counting Kit-8 methods. The results show that the viabilities for all the CN-INMPs did not change remarkably with the increasing concentration (p < 0.05) at 0.5 h post administration. Additionally, the drug concentration in blood for the CN-INMP@TPGS group was also significantly higher than that of the CN-INMP@P407 (p < 0.05). This could be attributed to the fast dissolution behavior of CN-INMP and the obstacles presented by the mucus. However, 2 h post administration, the retention of CN in lung was further decreased in tested groups. The drug retention amount of the CN-INMP@TPGS group was still higher than for the other two groups (p < 0.05), while the difference between CN-INMP@TW80 and CN-INMP@P407 was not obvious. It was concluded that the difference in the retention among CN-INMP@TPGS, CN-INMP@TW80 and CN-INMP@P407 could be attributed to the surface modification of nanocrystals.The amount of CN-INMP retained in the lung at different time intervals was then determined quantitatively. p < 0.05) within 2 h following pulmonary administration. Among the three groups, the Cmax (485.23 ± 18.11 µg/L) of the CN-INMP@TPGS group significantly increased (p < 0.05), compared with those of CN-INMP@TW80 (289.26 ± 12.38 µg/L) and CN-INMP@P407 (242.42 ± 29.59 µg/L). The AUC(0-t) value of CN-INMP@TPGS (2413.18 ± 432.41 µg/L h) was 1.497- and 3.32-fold greater compared with those of CN-INMP@TW80 (1612.35 ± 261.35 µg/L h) and CN-INMP@P407 (777.59 ± 196.81 µg/L h), respectively. These results indicated that the CN-INMP@TPGS was absorbed rapidly after pulmonary administration and resulted in increased systemic absorption. This might be attributed to the excellent redispersibility of CN-INMP@TPGS after lung deposition. Additionally, TPGS modified CN-NS after redispersion exhibited an excellent penetrating capability in lung mucus, owing to the negative potential surface and PEGylating modification [The plasma concentration profiles of CN-INMP@TPGS, CN-INMP@TW80 and CN-INMP@P407 after the pulmonary administration of CN-INMP are presented in fication . ConsequIn the present study, we fabricated three surface-modified inhalable nanocrystal-in-microparticles for pulmonary delivery and studied the effect of different types of surface modification on the mucus penetration in vitro and absorption in vivo. The results demonstrated that TPGS- modified nanocrystals could improve the retention time in lung, plasma concentrations and the systemic absorption of CN after pulmonary administration. The improved absorption of CN-NS@TPGS was attributed to the increase in the mucus-penetrating ability and cellular uptake in lung epithelium cells. Additionally, CN-INMP could also enhance the lung deposition of CN after pulmonary administration. Therefore, the mucus-penetrating nanocrystals-in-microparticles might be used as a promising formulation strategy for the inhalation administration of poorly soluble drugs.


The objective of the present study was to investigate whether differences in bull fertility are associated with variations of sperm quality. Differences between high- and low-fertility bulls were found mainly in parameters related to sperm acrosome integrity when using a new fluorescence method that allows clear and precise detection of the sperm plasma membrane and acrosome: the ISAS3Fun method. It was concluded that the simultaneous assessment of sperm viability and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than the more conventional in vitro sperm quality test. These results may help to predict the breeding soundness of bulls used in artificial insemination, which is important for the dairy industry.The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane–acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests. The prediction of breeding soundness of bulls and of the fertilising ability of semen used in artificial insemination (AI) is important for the dairy industry . HoweverPisum sativum (edible pea) agglutinin (PSA) and Arachis hypogaea (peanut) agglutinin (PNA) are the most commonly used lectins for this purpose [TM Green DND-189) or heterogeneous (LysoTracker® DND-99 and dapoxyl 2-aminoethyl sulphonamide) labelling [The development of acrosome-specific fluorescent probes, among which lectins are the most widely used, has facilitated the determination of acrosomal damage, even using flow cytometry.  purpose . They bi purpose . The useabelling . Althougabelling .In previous studies ,8, we deUnless otherwise indicated, all chemicals were obtained from Sigma .7 sperm per 0.25 mL semen straw (IMV Technologies). All straws were cryopreserved following standard procedures [This study analysed cryopreserved semen samples collected from commercial Holstein–Friesian bulls. Ejaculates with a total sperm motility or with morphologically normal spermatozoa lower than 70% were discarded. The ejaculates were extended in BullXcell  to a final concentration of 1.3 × 10ocedures . Before To achieve the proposed objectives, two experiments were performed.Experiment 1: Relationship between in vitro sperm quality test and field fertility of bulls.Frozen semen samples from 20 bulls, 10 of high field fertility and 10 with low field fertility, were selected for this experiment with an age range between 2 and 6 years. Field fertility was determined by a retrospective study using data from the first and second AI postpartum results of at least 100 Holstein–Friesian cows in their second lactation over a period of 5 y (2009 to 2014) in different herds. Then, 353 bulls were evaluated and, between them, the 10 bulls with the highest (>53%) and 10 bulls with the lowest (<39%) fertility were included in the study. Cow fertility (pregnancy rate) was determined through pregnancy diagnosis using ultrasonography or rectal palpation of the genital tract at 35–45 days post-AI. Two frozen samples from the same ejaculate of each bull were collected between those used for the field fertility analysis. Semen samples were thawed, pooled, and processed for sperm quality assessment as detailed below.®, version 1.32; PROISER, Paterna, Spain) was used to assess sperm motility [Within a few minutes after thawing, a diluted semen sample was placed in a pre-warmed Makler chamber , and a computer-assisted sperm analyser  was determined using an acridine orange and propidium iodide combination . The numCombined sperm plasma/acrosome membrane integrity was determined using the ISAS3Fun technique (PROISER), as described in a previous work . The labSperm head morphometry of the fluorescent sperm subpopulations was determined as described in . BrieflyExperiment 2: Validation of the use of the ISAS3Fun method for the assessment of sperm acrosome integrity in bulls.g, 10 min), and resuspended in S-TALP  medium [2. Ionomycin  was added to the sperm suspension to induce the acrosome reaction, and the incubation period was extended for 1 h in the same conditions.Frozen semen samples from 18 bulls were thawed in a water bath, centrifuged (600× ) medium  with 10 Aliquots were evaluated at 0 h, 4 h (after induced capacitation), and 5 h (an hour after the induced acrosome reaction) for sperm motility with the CASA system and for sperm plasma/acrosome membrane integrity as detailed below.® system [For each incubation time, a sample aliquot was mixed in a vial with PNA (10 μg/mL) for 10 min and PI (15 μM) for 5 min at 37 °C. Then, 3 μL of the labelled sample was placed on a prewarmed slide, covered, and pressed with the Trumorph® system . StainedAnother sample aliquot was labelled with the ISAS3Fun kit (PROISER), as detailed above.p < 0.05. Distribution normality and the homogeneity of variance of the median score for each dataset were checked using the Kolmogorov–Smirnov and Levene tests, respectively. As data were normally distributed, parametric analysis was performed throughout. In the first experiment, differences in the sperm quality results between high- and low-fertility bulls were evaluated by Wilk’s lambda ANOVA. A discriminant analysis was also performed with the linear stepwise procedure to identify the most useful parameters for the classification of high- and low-fertility bulls. Wilk’s lambda ANOVA was used to compare the fraction of the total dispersion of data not accounted for.Statistical analyses were performed using the SPSS package, version 23.0 . Statistical significance was set at In the second experiment, the effects of time  using the ISAS3Fun method vs. the PNA/PI combination on the sperm membrane and acrosome integrity , as well as on the distribution of sperm subpopulations , were analysed by General Linear Models (GLM) repeated measures analysis of variance.p < 0.05) than those of low-fertility bulls. No statistical differences were found for the other sperm quality parameters analysed.Significant differences between high- and low-fertility bulls were observed in acrosome integrity, the proportion of IADM sperm and the head width of the IAIM sperm subpopulation . Highly p < 0.001), with a globally correct assignment of 90% of the samples  and 4 h (27.86% ± 3.25) of incubation, and most spermatozoa were immotile at 5 h. The main model of the GLM repeated measures analysis of factors affecting sperm membrane and acrosome integrity is depicted in In the fluorescence sperm subpopulations, spermatozoa with damaged acrosomes and intact plasma membranes (DAIM) were barely observed at 0 h, then increased slightly at 4 h, and finally increased noticeably at 5 h d. This iMale fertility assessment based on test inseminations is expensive and time consuming , and it In porcine, it was observed that the number of spermatozoa with reacted acrosomes after capacitation was correlated with prolificacy, and the difference in the rate of reacted acrosome sperm before and after capacitation was correlated with fertility . TherefoIn a previous study in rams, we found clear differences in motility and plasma membrane integrity between high- and low-fertility males, which were not observed in this study of bulls. Unlike in rams, AI in cows is highly efficient, with the semen deposited deeply into the uterus. Consequently, spermatozoa might not have difficulties reaching the site of fertilisation; therefore, high motility and viability might not be as restrictive as in the ewe. Another possible explanation for the differences between these two studies is that the experimental design was different. In the study of rams, fresh semen samples of only eight rams with extreme fertility (four high and four low) were checked weekly during six consecutive weeks. The analysis of frozen/thawed doses of a single ejaculate per bull and the selection of more males per fertility group in the current study would probably reduce the chances of finding differences in more sperm quality parameters. In fact, Bernecic et al.  observedFor the establishment of a viable pregnancy, spermatozoa must reach the site of fertilisation, then penetrate and fertilise the oocyte, and, finally, produce a viable embryo. Acrosomal exocytosis is a crucial and irreversible process key in ovum fertilisation that occurs when the acrosomal outer membrane fuses with the plasma membrane at multiple points, releasing proteolytic enzymes ,19. UntiGiven the difficulty in clearly discerning the sperm acrosome in most animal species, its evaluation is usually limited to research or to occasional studies of sperm quality. In a recent work, we described a simple method that allows the simultaneous visualisation of sperm plasma and acrosomal membrane integrity in bulls by combining the fluorochromes Hoechst 33342, CFDA, and PI (ISAS3Fun) . To faciCFDA and PI have been combined to assess sperm membrane integrity in several species ,22,23,24During sperm capacitation, hyperpolarisation of the plasma membrane occurs, and this hyperpolarisation has been attributed to an increase in calcium during acrosomal exocytosis . CalciumThe study of the evolution of the fluorescence sperm subpopulations allowed us to clarify the effect of the treatments. Live spermatozoa showed a clear induced acrosome reaction, with extensive acrosomal damage at 5 h after calcium ionophore was added. This evolution was more clearly appreciated when using the ISAS3Fun combination, with a sudden increase in the proportion of live spermatozoa with damaged acrosomes after ionomycin treatment. The evolution of dead spermatozoa was less evident, showing a reduction in spermatozoa with intact acrosomes when using the ISAS3Fun method, while the proportion of this subpopulation even increased with time when using PNA/PI, likely because the number of spermatozoa with complete acrosomal loss was higher in dead spermatozoa.Surprisingly, the induction of the acrosome reaction with ionomycin caused an apparent increase in spermatozoa with intact plasma membranes when using the ISAS3Fun kit a. We repIn conclusion, the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method seems to have a higher ability to discriminate between high- and low-fertility bulls than a conventional in vitro sperm quality test. Moreover, the use of this technique to quantify acrosome integrity was validated in bulls, with a higher sensitivity than traditional PNA/PI staining.


Musca domestica, to act as a mechanical vector of the virus. Homogenate of crusted scabs from ORFV-positive sheep  were used to infect laboratory-reared flies. Flies walking on viral mixture and flies inoculated on their wings were individually placed in Falcon tubes and the ORFV DNA was searched by PCR on tube walls; flies were fed on the same homogenized crusts and their crop and spots (vomit and feces) molecularly examined for ORF DNA at 2, 4, and 6 h. All of the flies (100%) used in the experiments were able to pick up and transmit the viral genome to contact surfaces; 60% were found ORF virus (DNA)-positive in both spots and crop. These results suggest that M. domestica could play a role as potential mechanical vector and/or reservoir in the epidemiology of the ORF virus infection. Thus, house fly management should be considered in the measures to control the disease in ovine–caprine farms.ORF virus (Poxviridae) is the causative agent of contagious ecthyma (soremouth), a disease primarily affecting sheep and goats worldwide, but also humans exposed to disease-ridden animals. Pathogens are shed with scabs, and infection mainly occurs by direct contact. Although the disease is relatively benign and self-limiting, the morbidity rate is high in livestock with subsequent significant financial and economic impact. The aim of the study was to experimentally investigate the potential for the housefly,  Musca (M.) domestica, is a classic synanthropic species closely linked to human populations and their activities, including animal husbandry [Microsporum canis [M. domestica (Diptera: Muscidae) flies collected during an outbreak in Russia [M. domestica has long been documented in cattle, more abundantly in anatomical areas moistened by secretions  [M. domestica, carries ORFV on its body surfaces and/or internal organs and thus has a role in the spread of the virus, we investigated the potential role of this dipteran in ORFV mechanical transmission under experimental conditions.ORF virus (ORFV), the prototypical species of parapoxviruses (Poxviridae), is an epitheliotropic DNA-virus causative agent of the contagious ecthyma (EC), a worldwide pathology primarily affecting sheep and goat farming, but also affecting wild ungulates . Occasiousbandry . It can usbandry ,10,11,12um canis . Pathogeum canis ,15,16; mn Russia . In Italt, etc.) ,19. Furtt, etc.)  suggestsM. domestica, and introduced into a new cage for breeding. After eggs were laid, the emerging larvae were randomly selected and confirmed to be free of ORFV by using molecular analysis [Adult flies were caught from an ovine farm  using an insect net and rapidly transferred to the laboratory in a box. The specimens were chilled at 4 °C for 5 min, morphologically identified as analysis . At the ®Safe  and viewed with the Molecular Imager DocTM XR +  transilluminator. Amplicons were subsequently purified using the commercial GeneJet PCR purification kit and sequenced using the Sanger method  using external facilities . All sequences were compared with those present in GenBank using Blast to confirm the identification.The virus (starter) was obtained from buccal crusts of sheep from a farm in southern Italy and naturally infected with ORF virus; diagnosis was previously achieved using Electron Microscopy, cell culture, and molecular analyses, as reported in the study by Galante et al., 2019 . Subsequ9 to 1 × 10−1 copies/µL) were tested in triplicate and used as a quantitation standard to construct the curve resulting from the detection in reaction of the number of copies of the plasmid and the value of the corresponding threshold cycle (copy target). The detection limit of the reaction was determined based on the highest plasmid dilution that could be amplified  with good reproducibility. It was found to be 10 copies/µL of the ORF genome. The melting curve analysis was performed with continuous fluorescence reading between 65 °C and 95 °C. The amplification and melting curve analysis of the PCR products from ORF virus DNA showed a melting temperature in a range of 88.22 °C and 87.92 °C.The starter template was analyzed by a second PCR assay. Amplification and quantification of the ORF virus were carried out based on the principle of Syber Green technology using the assay described by Kottaridi et al., 2006 , modifieM. domestica adults were used. Experiments were conducted in a Biosafety Level 3 (BL3) laboratory.A total of three tests were conducted to determine the potential of house flies to harbor and transfer ORFV. For each test, third-generation Test 1: Efficiency of housefly legs in ORF virus acquisition and dislodgment.Flies used: 5.6,4 copy target).Positive control: ORF virus DNA from ovine scrabs .Positive control: ORF virus DNA from crusts  and crop examination for ORFV.Flies used: 15.6,4 copy target).K+ = ORF virus DNA from crusts (10K− = spots (vomit/feces) and crop from flies fed with milk/water mixture (ratio 1:1).House fly spot (vomit/feces) collection.The flies fasted for 24 h in the breeding cage were individually collected using a sterile 50 mL Falcon Conical Centrifuge tube. Each fly was chilled at −20 °C for 1–2 s and allowed 30 min of feeding time in a Petri dish where 200 µL of ovine homogenized crusts was previously distributed A. SubseqTests 1 and 2: The proportion of flies able to dislodge the virus from wings/legs to contact surfaces after contamination was also estimated at the level of confidence of 0.472 using the Clopper and Pearson procedure.Test 3: The Friedman test was used to assess whether the virus concentration in the spots (regurgitation/feces) and crops of each tested fly was statistically significant at the three different time points at the level of significance of 0.05.Test 1: Efficiency of housefly legs in ORF virus acquisition and dislodgment.Test 2: ORFV dislodgment from contaminated wings of house fly during simulated flight motion.In each test, ORF virus DNA was detected in the washing liquid collected from each fly specimen (5/5)  exhibited 100% identity homology with SY17 ORF virus strain (accession number: MG712417.1) All of the flies exposed to the virus (5/5) (100%) were able to acquire the virus on their legs and wings and also to transfer it to contact surfaces . No PCR products were generated in the negative control.Test 3: House fly spots (regurgitation/feces) and crop examination for ORFV.p = 0.554). The viral concentration in fly spots (8/9) and in the crop (1/9) was shown to be between 103 and 105 copy target (Ct) values.Out of the 15 tested house flies, 9 (9/15) (60%) fly specimens were found to be ORFV-positive and the remaining (6/15) (40%) were found to be negative. Out of nine flies that were ORFV-positive, four (4/9) flies were positive in both the crop and spots with four (4/9) flies and one only (1/9) positive in spots or crop, respectively. In relation to time, 26% (4/15), 33.3%  (5/15) and 40.0%  of the infected flies eliminated the virus in spots at the second, fourth and sixth hour from the meal; in 33%  (5/15), the virus was detected in the crop, either exclusively or in association with spots . By meanM. domestica can mechanically carry ORFV (DNA) via both its legs and wings and transfer it to contact surfaces; the dipteran is also able to excrete the ORFV genome by oro-fecal routes (vomit/feces) and to store it in crop. In particular, 100% of the used flies acquired and transferred the ORFV (DNA) to contact surfaces via their legs and wings, while in 60% (9/15) of the fed flies, it was detected in their spots (8/9) (vomit/feces) and crops (1/9) for up to 6 h following the infective food. Our tests confirm that M. domestica is structurally able to pick up and mechanically carry ORFV (DNA), such as reported for pathogens; in fact, its external surface is entirely covered by long bristles capable of carrying environmental detritus and pathogens, with over 32 million bacteria carried [M. domestica a role in the epidemiology of ORF virus infection, probably as a mechanical vector in short distances, as already shown for a number of pathogens [3 and 105, slightly lower than the starter . This result is particularly interesting because it further strengthens the importance of the potential role of the common house fly in the mechanical dispersion of contagious ecthyma; however, more studies using live virus to infect flies and subsequent viral isolations are necessary to exclude or to confirm the replication of ORFV in adult fly’s gastrointestinal tract. Frequent meals on infected substrates could be expected to accumulate pathogens in a fly’s alimentary system [The results presented herein provide the first demonstration that  carried . All sixin 60% 9/ of the f carried ,25. Evidathogens , includiathogens . ORF virin 60% 9/ of the fM. domestica. ORFV is very hardy and persists on farm material and in the environment for months to years [M. domestica population also reaches its highest density. Particular attention should be given to other viral diseases, such as lumpy skin disease in cattle, where discharges from the infected hosts [Thus, while low virus titers within nasal or other discharges are indeed likely to lower the risk of contact transmission, there is a need to re-investigate the direct mode of transmission as it pertains to spread of LSDV (lumpy skin disease virus)”. In that same paper, the authors address insect vectors as potential vectors of LSDV and even make a case that suggests house flies might play a role. As for the practical aspects of the ORF virus, we must focus not only on the economics of this virus [Furthermore, the presence of milk receptors on this insect makes it particularly attracted to some anatomical areas, such as the nipples of lactating females and the snout of lambs and kids; such locations are also those where the flies tend to concentrate and/or where ORFV-related lesions begin to develop . For theto years ; for thito years , includito years ; interesed hosts  make a red hosts , “Thus, is virus , but alsis virus ,32,33 bois virus . The per-Particularly, crusty lesions in infected sheep/goats represent the preferential site for nourishment of house flies .-Fresh ORFV-infected crusts, containing live virus, are the main source of environmental contamination.-Our study identified the presence of ORF viral genome in flies; no information is available on the presence of live virus in the flies.-More flies should be involved in future studies to better take into consideration the variability of behavior of flies.-Long-term studies based on sampling of flies in the field during ORF virus outbreaks would be needed to really understand the role of house flies in the spread of the pathogen.The study was based on observations related to the behavior of house flies during ORFV outbreaks; consequently, our results can be considered paradigmatic until “in field” studies become available:


Dengue virus is a major and rapidly growing public health concern in tropic and subtropic regions across the globe. In late 2018, Senegal experienced its largest dengue virus outbreak to date, covering several regions. However, little is known about the genetic diversity of dengue virus (DENV) in Senegal. Here we report complete viral genomes from 17 previously undetected DENV cases from the city of Thiès. In total we identified 19 cases of DENV in a cohort of 198 individuals with fever collected in October and November 2018. We detected 3 co-circulating serotypes; DENV 3 was the most frequent accounting for 11/17 sequences (65%), 4 (23%) were DENV2 and 2 (12%) were DENV1. Sequences were most similar to recent sequences from West Africa, suggesting ongoing local circulation of viral populations; however, detailed inference is limited by the scarcity of available genomic data. We did not find clear associations with reported clinical signs or symptoms, highlighting the importance of testing for diagnosing febrile diseases. Overall, these findings expand the known range of DENV in Senegal, and underscore the need for better genomic characterization of DENV in West Africa. Recent years have seen large outbreaks in endemic areas across Asia, Africa and the Americas as well as the appearance of the disease in new areas. Climatic and demographic changes, including urbanization and increasing temperatures, may be contributing to this emergence, creating more favourable conditions for the Aedes mosquitoes, the disease vector3. The majority of dengue virus infections are either mild or asymptomatic; these can be difficult to diagnose as symptoms—commonly including fever, myalgia, arthralgia, and headaches—are usually non-specific, meaning diagnostic tests are needed to confirm infection. A small fraction of cases result in a severe disease, especially following secondary infection with a different serotype of the virus via antibody-dependent enhancement4.Dengue virus is a major and growing problem across the world, with an estimated 390 million infections per year5. Dengue is caused by a virus of the Flaviviridae family and there are 4 distinct serotypes . All four serotypes have been reported in Africa, with DENV2 being associated with the greatest number of epidemics on the continent5. In Senegal, dengue virus was first isolated in 1970 in the rural Bandia area6. Aedes (Ae.) aegypti, the main mosquito vector of DENV, has been isolated in the three different ecological areas in Senegal7, as have other potential DENV vectors8. All serotypes of DENV have since been detected in Senegal, with DENV3 causing an epidemic in 2009 Dakar9 and DENV2, often confined to the forest cycle, spilling over into urban areas in Senegal in 2014–20158. Since then, recurrent urban outbreaks associated with multiple serotypes have been reported in both 2017 (138 cases) and 2018 (314 cases)10, raising the possibility that environmental changes and high mobility of people has led to a more widespread distribution of dengue virus across the country.Dengue is endemic to 34 countries across Africa, and outbreaks of this disease have been reported in all regions12. Given the risk of disease transmission at this large gathering, additional measures to enhance disease surveillance were put in place, including a mobile laboratory from the Institut Pasteur equipped with molecular tests for rapid diagnosis11. However, several factors indicate that many cases were undetected: the incubation period of the disease is typically 5–7 days, dengue fever was detected elsewhere in Senegal even before the Magal, and the similarity in symptoms and seasonality of dengue fever and malaria suggest that many more cases, especially of mild disease, remained undetected. Dengue is, therefore, likely to be an underdiagnosed cause of non-malarial fever.In late 2018, Senegal experienced the largest dengue outbreak to date, with 314 confirmed cases detected in several regions of the country. Over 60% of cases were in the region of Diourbel, concentrated in the city of Touba, where in late October the Grand Magal—a 2-day religious pilgrimage that gathers over 3 million people in this holy city—took place15. Here we used metagenomic sequencing to characterize the genetic diversity of 17 dengue virus cases from the city of Thiès; part of a cluster of 19 previously undetected cases identified in the course of surveillance for the causes of acute febrile disease in the region, and evaluated clinical metadata to identify risk factors associated with dengue virus infection. Despite the growing burden of dengue fever in Senegal and West Africa more broadly, little is known about the genetic diversity of the virus and few viral genomes are available. This information could help explain disease transmission dynamics and enable a better understanding of dengue virus circulation and transmission in Senegal.Genomics can provide important information regarding the origin, evolution and spread of a viral outbreak, such as dengue16. Individuals enrolled in this study presented to the SLAP (Service de Lutte AntiParasitaire) clinic (Thiès) with either an acute fever (temperature > 38 °C) or a recent history of fever in the last 4 days. All samples were collected between October 2nd and November 12th, a period just following the rainy season in this area of Senegal and when malaria transmission is at its annual peak17. In total, 198 individuals were enrolled into the study, of whom 36 tested positive for malaria by Rapid Diagnostic Test. Individuals were aged between 2 and 72 years of age with 121 males and 77 females.Thiès is the third largest city in Senegal and the administrative capital of the region of Thiès. It is located 72 km east of Dakar, Senegal’s capital and largest city, and 123 km west of Touba, Senegal’s second largest city. Thiès has a very low malaria endemicity with an annual entomological inoculation rate of 1–518. Here, no samples that tested positive for malaria (N = 36) also tested positive for dengue virus infection, suggesting no co-infections were present in our dataset.Following RNA extraction, all 198 samples were tested for the presence of flavivirus RNA using an in house RT-qPCR assay with previously published primers . The average coverage ranged from 28 × to 5370 × and the genome assembly length varied between 10,525 bp and 10,680 bp Table . Four saWe also performed metagenomic sequencing on 17 samples that tested negative by the pan-flavivirus RT-qPCR assay Fig. . We confWe next investigated the phylogenetic relationships of these sequences to existing dengue virus genomes. DENV3 was the most frequent serotype in our data. All 11 genomes were highly similar and fell within an African cluster of samples in genotype III that also includes 5 sequences from Gabon and 1 from Senegal collected between 2009 and 2017 Fig. .Figure 319, which is shared exclusively with all West African sequences of the sylvatic clade.For DENV2, the four genomes all belonged to the Cosmopolitan genotype (II). All the new DENV2 genomes are highly similar and are closest to other available sequences from Africa, particularly those from Burkina Faso in 2016 Fig. B. It is 20, which cluster with Asian sequences of this genotype  with an equal number of males and females Table . No clinRecent travel was significantly more frequent among individuals with presumptive dengue fever . This data enables the investigation of risk factors associated with dengue virus infection and is useful for the surveillance of repetitive outbreaks recorded in Senegal, where, as in other regions, urban outbreaks of multiple serotypes are increasingly common. These findings expand the known range of local transmission of dengue virus in late 2018 in Senegal and point to a much wider distribution of three serotypes of the virus than previously reported.During the 2018 outbreak DENV1 and DENV2 were reported respectively from the southwest (Fatick) and north of Senegal (Saint Louis and Richard Toll); however, these findings suggest that it was more widespread. Of the 2 individuals with DENV1 infection, 1 reported travel to Touba, and the other reported no recent travel. Of the 4 individuals with DENV2 infection, 1 reported travel to Saint Louis prior to developing symptoms, 1 reported travel to Touba and the other 2 reported no travel, suggesting that DENV1 and DENV2 were circulating in the Thiès region. All four DENV2 isolates were genetically very similar and fell into the cosmopolitan (II) genotype, distinct from the 5 other genomes available from Senegal that are part of the sylvatic clade. The cosmopolitan genotype is widespread across Africa and Asia; however the present sequences are most similar to recent sequences from Burkina Faso in 2016. This suggests that the lineage of DENV2 associated with this outbreak was related to viruses that have been circulating in the West African region in recent years, however, the scarcity of sequence data prevents higher resolution on this.9, Côte d’Ivoire5, Gabon21 and elsewhere. Our phylogenetic analysis suggests that this is a closely related but distinct lineage to that responsible for the 2009 outbreak in Senegal, as the samples cluster more closely to those from a 2016–17 outbreak in Gabon21 than the single sequence from Senegal in 2009, sequenced from a traveler returning to Germany22. However, we lack genomic data from this region; only 7 complete DENV3 genomes from Africa were available in GenBank prior to this study, in addition to 24 short sequences of 220–1923nt. This scarcity of data makes phylogenetic inference, especially around the recent evolutionary history of DENV3 in West Africa, challenging.The majority of cases in this study (65%) were from serotype 3. This serotype has become increasingly common in West and Central Africa in the last decade and has been responsible for several outbreaks in SenegalIn Senegal, the DENV3 serotype was most frequent in Touba (Diourbel) during the 2018 outbreak and the timing of the outbreak overlapped with the Grand Magal religious pilgrimage. 45% of DENV3 positive patients reported travel to Touba (N = 5), of which 3 developed a fever in a time frame consistent with attendance at the Magal. It should be noted that we do not know when participants travelled to Touba or whether they attended the Magal. The other 6 individuals reported no recent travel. Even if we postulate that all individuals with recent travel were infected outside of Thiès, almost half of cases are consistent with locally-acquired infection.23 for finding genetically distinct lineages that may be missed by PCR-based approaches. Where knowledge of the prevalence of different pathogens is scarce, this unbiased approach can serve to identify the species of greatest concern, providing recommendations on the priorities for implementing more rapid, low-cost, targeted diagnostic testing, and characterize the genetic diversity of circulating lineages to ensure that these tests are locally suited and sensitive. However, to enable this genomics-informed surveillance and diagnostics more widespread and ongoing sequencing is needed to provide high-resolution information on the extent of genetic variation. Developing local capacity for this work in endemic countries is central to this effort and to ensuring pathogen genomics can be translated into actionable insights.Despite using a pan-flavivirus RT-qPCR for screening of all samples prior to sequencing, the two DENV1 infections were missed, owing to 4 mismatches in the forward primer binding region that compromised the sensitivity of the test. This finding demonstrates the utility of genomic surveillance, especially with metagenomic sequencingWe found very little association between the clinical and demographic variables investigated and presumptive dengue virus infection. Individuals with presumptive dengue were equally distributed with regards to sex and age. Associations were stronger after removing patients with a confirmed malaria diagnosis, but still did not reach statistical significance. These findings are consistent with the mild symptoms of these individuals and the broader difficulty of clinically differentiating dengue fever from other febrile diseases without diagnostic testing. Although dengue fever does not have a specific therapy and the majority of infections are mild, diagnosis is still important to rule out other causes that would have different courses of treatment. The availability of rapid, sensitive and widely available diagnostic tests for common pathogens, other than malaria, is still severely limited in Senegal and many other regions and presents a major challenge for public health.In conclusion, we report the analysis of 17 complete genomes from 19 previously undiagnosed dengue cases from the 2018 outbreak in Senegal. This work expands our understanding of the source and spread of this outbreak but also of dengue virus genetic diversity in West Africa more broadly. Our findings highlight the need for enhanced diagnosis and genomic surveillance to better understand the past and track the future course of this disease.Patient samples were obtained through a study approved by the Institutional Review Boards at the Senegalese Ministry of Health and Harvard University. Patient samples were obtained from individuals presenting to the Service de Lutte AntiParasitaire in Thiès, Senegal. Study staff obtained written, informed consent from all participants enrolled in the study  following an explanation of the study objectives. All methods were performed in accordance with the relevant guidelines and regulations. A 5 mL venous blood draw was collected from each enrolled patient in a BD Vacutainer EDTA tube. Demographic information as well as clinical signs and symptoms were also recorded. Blood samples were kept on ice and transported daily to the Laboratoire Parasitologie et Mycologie at Aristide Le Dantec Hospital, where plasma was isolated on the same day and frozen prior to RNA extraction.24. Qualitative detection of Dengue virus RNA in samples with suspected flavivirus infections was performed using the RealStar Dengue RT-PCR Kit 3.0 from Altona-Diagnostics on an Applied Biosystems 7500 Fast Real-Time PCR System according to manufacturer’s instructions.Total RNA was extracted from patient plasma using the QiAmp viral RNA mini kit (Qiagen) according to the manufacturer’s instructions. Detection of Flavivirus RNA was performed by RT-qPCR using the Applied Biosystems Power SYBR RNA-to-CT 1-Step Kit on a Roche LightCycler 96 for 45 cycles. Primer sequences have been shown to capture all known flavivirus speciesSequencing libraries were prepared from all dengue virus positive samples (N = 17). We additionally sequenced 17 samples with no known diagnosis, selected as follows; negative for both the pan-flavivirus RT-qPCR and RealStar Dengue RT-PCR, and negative for malaria by Rapid Diagnostic Test.25. All samples were sequenced on an Illumina MiSeq instrument at the Laboratoire Parasitologie and Mycologie in Dakar, Senegal, with paired end 100nt sequencing.Briefly, DNA was removed by Turbo DNase treatment (Thermo Fisher Scientific), cDNA was synthesized with random primers and sequencing libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina) as previously described26, implemented on the DNAnexus cloud platform. First, samples were demultiplexed and an initial taxonomic classification of metagenomic sequencing reads was performed using KrakenUniq27. Reads were compared against a database that encompassed the known diversity of all viruses that infect humans. Next, sequences were filtered by removing reads aligning to the human genome, synthetic spike-in sequences and other known contaminants. The remaining reads were filtered against a database of dengue virus sequences, and de novo assembly was performed with Trinity28. Contigs were then scaffolded against one of four RefSeq genomes, representing the four serotypes . Short read sequence data and assembled genomes have been deposited in NCBI’s SRA and Genbank databases under BioProject PRJNA662334 and accession numbers MW288024–MW288040.Sequencing data was analysed using viral-ngs v1.25.0https://dockstore.org/organizations/BroadInstitute/collections/pgs.Code availability. Computational workflows used to analyze viral sequencing data are have been made publicly available through dockstore: https://www.genomedetective.com/app/typingtool/dengue/) was used to determine the genotype and serotype of the newly assembled dengue virus genomes, as well as the genotypes of all whole dengue virus genomes  available on GenBank on February 10th 2020 (DENV2 and DENV3) or June 24th 2020 (DENV1). We further filtered genome records to remove those where collection date or country of origin were not recorded. Due to the large number of non-African dengue genomes, a subset of these sequences was selected for phylogenetic analysis. Specifically, for each serotype, we randomly selected 50 published genomes from the same genotype as the current samples as well as up to 25 genomes from each other genotype for context. All complete sequences from Africa were also included  yielding a final dataset of 136 genomes for DENV1, 188 genomes for DENV2 and 118 for DENV3.Genotype Detective  + Gamma substitution model and ultrafast bootstrapping (N = 1000)30. This model was selected as it is the most general and makes few assumptions about substitution rates31, and was previously used in phylogenetic analyses of DENV outbreaks15. Time-scaled Bayesian phylogenetic analysis was performed using a Markov chain Monte Carlo (MCMC) algorithm implemented in the BEAST v1.10.432 on the CIPRES Science Gateway33. Briefly, BEAST was run for 50 million MCMC steps using a GTR + Gamma substitution model with 1,2,3 codon partitioning, an uncorrelated relaxed clock with log-normal distribution and a Skyline tree prior . For each serotype we used the full coding sequence and sampled tip dates. Trees were sampled every 5,000 generations. The convergence was assessed using Tracer v1.7.1 and TreeAnnotator v1.10.4 was used to generate maximum clade credibility trees with a burn-in of 10%. All trees were viewed in FigTree v1.4.434.Sequences were aligned using MAFFT v7.388 implemented in Geneious and trimmed with TrimAl. Maximum likelihood phylogenies were estimated separately for each serotype using IQTREE v1.5.5.535. Briefly, the biserial correlation coefficient and the two-tailed Fisher Exact test were used to calculate the significance of continuous and categorical variables, respectively at a p-value cut-off of 0.05. Categorical variables tested were; sex, recent travel and the presence of 12 clinical signs or symptoms reported by at least 1 participant; muscle aches, diziness, vomitting, weakness, headache, backache, chest pain, difficulty breathing, rash, sore throat, inflammation and bleeding. Continuous variables tested were; age, temperature, heart rate, respiratory rate, hemoglobin level and blood sugar level.A univariate correlation analysis was performed on 14 categorical and 6 continuous variables to determine the significance of association with a presumptive diagnosis of dengue fever (determined by the presence of dengue virus RNA in the plasma detectable by RT-qPCR or sequencing). Analyses were performed as previously describedSupplementary Figures and Tables.Supplemental File 1.Supplemental File 2.Supplemental File 3.