Ixodes ricinus; it is rarely found in other tick species and in Dermacentor reticulatus it has, so far, only been reported in Poland.Tick-borne encephalitis (TBE) virus is transmitted to humans and animals through tick bites and is thought to circulate in very strictly defined natural environments called natural foci. The most common tick serving as a vector for the TBE virus in central Europe is Between autumn 2016 and spring 2018 ticks were collected by the flagging method in a new TBE focus in the district of northern Saxony, Germany, outside the known risk areas as defined by the national Robert Koch Institute. Ticks were morphologically identified and tested in pools for the presence of TBE virus using a real-time RT-PCR. TBE virus from positive pools was isolated in A549 cells, and the E gene sequences were determined after conventional RT-PCR, followed by a phylogenetic comparison.D. reticulatus  and only twice in I. ricinus nymphs . All other ticks, I. ricinus males (n = 33), females (n = 30) and larvae (n = 58), as well as 5 I. inopinatus  and 14 Haemaphysalis concinna , tested negative for TBE virus. TBE virus was not detected in I. ricinus during the summer, when D. reticulatus is not active. Sequence comparison of the entire E gene of the isolated virus strains resembled each other with only 3 nucleotide differences. The most closely related viral sequences belong to TBE virus strains from Poland and Neustadt an der Waldnaab , approximately 200 km east and 200 km south-west of the new focus, respectively.TBE virus was detected in 11 pools, 9 times in flagged adults D. reticulatus and I. ricinus, indicating that D. reticulatus plays an equal role to I. ricinus in virus circulation when both tick species are sympatric.TBE virus was found in northern Saxony, Germany, with similar MIRs in  Flavivirus, family Flaviviridae) is the etiological agent of TBE, the medically most important member of the tick-borne serocomplex [Tick-borne encephalitis (TBE) virus  includes 35 species and has a worldwide distribution, except for Australia [Dermacentor species are found mostly in Europe, Asia and North America [D. reticulatus (‘the ornate dog tick’) and D. marginatus (‘the ornate sheep tick’), as well as one in Asia, D. nuttalli, are associated with TBE virus. Both D. marginatus and D. reticulatus are competent vectors of this virus [Dermacentor ticks in the circulation of TBE virus in the environment is, however, unclear and poorly studied [Dermacentor reticulatus appears to be spreading and its distribution area and its population density have been increasing during recent decades [D. reticulatus was 10.8%, which is considerably higher than the prevalence found in I. ricinus (1.6%) [D. reticulatus ticks from Białowieża Primeval Forest was similar (1.58%) [I. ricinus (1.3%) [I. ricinus, 3.8%; D. reticulatus, 3.9%; but Haemaphysalis punctata, 8.8%) [D. reticulatus tick population in TBE natural foci has not been studied so far and no data on TBE virus circulation in D. reticulatus in central Europe are available.The genus ustralia . Dermace America . In Eurois virus –24. The  studied , 26. Der decades –29. In es (1.6%) . The pre (1.58%)  to that s (1.3%) , which ia, 8.8%) . HoweverD. reticulatus are nidicolous and adults exophilic (non-nidicolous), only the female D. reticulatus ticks are considered to play a major role in the virus transmission to larger mammals including humans, while in case of I. ricinus, the nymph is the key stage in viral transmission. Tick males, which either do not feed at all or parasitize only for a short time, might also be involved in virus transmission. Investigations of the viral load of either nymphs, female or male ticks from German natural foci by means of quantitative real-time RT-PCR did not show any significant differences in virus load between the respective stages and sexes . However, in a Russian study the researchers stated that male ticks appear to be responsible mainly for asymptomatic infections because of their lower virus load and short time of sucking, and thus can contribute to host and population immunity [via co-feeding (direct uptake of the virus from one tick feeding on a non-viremic host in close proximity to a tick harboring the virus); (iii) transstadial, (from one life stage to the next life stage after molting); (iv) transovarial ; (v) transsexual ; and (vi) infection of the next host via saliva .D. reticulatus for a period of three seasons.In this study we describe a new natural TBE focus in Germany and the phenology of TBE virus in In July 2016, the first ever human TBE case was reported from the district Northern Saxony in the German Federal State of Saxony, so far classified as a TBE non-endemic area  programme in Battaune, Federal State of Saxony, Germany, at the end of September 2016, in February, April, July and September 2017, and April 2018. The ticks were kept alive in 50 ml plastic tubes until identification and further testing in the laboratory. Ticks were identified to species level using the morphological characters according to Feider , FilippoTicks were processed in pools of 10 nymphs or 5 adults  per pool. Ticks were homogenized using 1 ml minimum essential medium  containing an antibiotic-antimycotic solution  using a Fast Prep Savant FP120 tissue lyser , with three rounds at speed 6.5 for 30 s each. Total nucleic acid was extracted using the MagNA Pure LC RNA/DNA Kit  in a MagNA Pure LC instrument  according to the manufacturer’s instructions using 200 µl of the tick homogenate. The total nucleic acid was extracted in 50 µl and a 5 µl aliquot was tested for TBE virus RNA using real-time RT-PCR .A 100 μl-aliquot of the supernatants of each crushed RT-qPCR-positive tick pool was added to an 80% confluent cell culture of A549 cells . The supernatants were kept at -80 °C until they were used undiluted and in a dilution of 1:5 and 1:10 for virus isolation. After 1 h of incubation at 37 °C, the supernatant was pipetted off and the cells were washed 5 times with MEM containing ABAM. Five milliliters of MEM containing 5-fold concentrated ABAM and 3% fetal calf serum were added. Cells were incubated for up to 7 days at 37 °C and observed daily for the occurrence of cytopathogenic effect (cpe). In case of more than 50% cpe, the supernatant was taken and tested by RT-qPCR for TBEV as described. In case of no cpe, culture supernatant was taken after 7 days of incubation and also tested for growth of TBEV by RT-qPCR. No subcultures were conducted. From the isolated TBEV strains E genes were sequenced for confirmation (see below).de novo assembly was performed using the three chromatograms obtained from GATC  for each positive sample. Nucleotides with an estimated error higher than 1% were trimmed. Subsequently the sequences were cut to 1488 bp, the exact length of the envelope gene sequence. A ClustalW alignment with several other E genes from selected isolates was performed and a phylogenetic tree was generated using the PhyML algorithm [The E-gene was amplified with a conventional PCR directly from positive tick nucleic acid extractions . This RTlgorithm .P = 0.05. Confidence intervals (95% CI) for the prevalence of pathogens were determined by the modified Wald method.The minimum infection rate (MIR) was calculated for pools of 10 nymphs or 5 adults. It was assumed that only one tick specimen in the pool was infected if the pool tested positive. Fisher’s test was used to compare the prevalence levels for significant independence using GraphPad Prism v.4 . The significance threshold was set at Dermacentor reticulatus, Ixodes ricinus, I. inopinatus and Haemaphysalis concinna) were collected by flagging ; 173 I. ricinus ; 4 I. inopinatus  and 3 H. concinna nymphs. Of the 174 pools, TBE viral RNA was detected in 3 pools of D. reticulatus  and 1 I. ricinus nymph pool.Overall, 2023 ticks belonging to four species , 231 I. ricinus , one I. inopinatus , and 11 H. concinna  were identified. Details regarding tick species activity are presented in Table D. reticulatus female pools were positive. From ticks sampled in April, 16 pools were tested: 12 D. reticulatus pools and 4 I. ricinus pools, with 1 D. reticulatus female positive pool. From ticks from July, 27 pools were analysed: 22 I. ricinus pools and 2 H. concinna nymph pools and 3 individual females, none of which were positive. From individuals collected in September, 38 pools were investigated and 2 pools were positive, 1 D. reticulatus female pool and 1 I. ricinus nymph pool.In 2017, 863 ticks were collected during the year, from which 620 D. reticulatus  and 66 I. ricinus . Of 33 pools, one male D. reticulatus pool was positive.In April 2018, 164 ticks were collected: 98 D. reticulatus pools and two pools of I. ricinus nymphs.In total, the detection of viral RNA in 11 tick pools was confirmed by the isolation of virus strains from 3 male and 6 female D. reticulatus was 0.59% (CI: 0.29–11.3%) and 0.42% (CI: 0.01–1.64%) for I. ricinus ticks and 0.57% (CI: 0.02–2.2%) for I. ricinus nymphs. There were no statistical differences in the TBE prevalence between D. reticulatus ticks and I. ricinus nymphs (P = 1.000) or ticks (P = 1.000). The tested I. ricinus ticks were all negative in July 2017 when D. reticulatus ticks were entirely absent.In general, the minimum infection rate (MIR) for D. reticulatus ticks showed no difference to virus strains from I. ricinus ticks from the focus and from other foci . The actual sequences of the E genes were also closely related to sequences from Germany (2011) and from Poland (1971).A total of 11 E-genes sequences from the positive tick pools were generated and analysed. The phylogenetic tree is shown in Fig. D. reticulatus during three sampling seasons in an area of sympatric occurrence together with I. ricinus and, to a lesser extent, I. inopinatus and H. concinna. Although TBE viral RNA has previously been detected in D. reticulatus ticks [D. reticulatus in natural foci in the absence of Ixodes ticks was considered highly unlikely [D. reticulatus was considered to support TBE virus circulation in the presence of I. ricinus populations. Our results suggest the opposite, i.e. that D. reticulatus is at least of equal importance in the maintenance of TBE virus in this natural focus as (i) we found it throughout our sampling period and (ii) the virus was absent in the I. ricinus population during the summer when adult D. reticulatus cannot be flagged (see below).TBE virus was detected and isolated repeatedly from us ticks –32 and ius ticks , this waunlikely ; howeverD. reticulatus sympatrically occurs with I. persulcatus, a genetically closely related species to I. ricinus, the virus was also found in both tick species [D. reticulatus and I. persulcatus throughout the year while in Germany, as in central Europe, D. reticulatus adults do not quest and thus cannot be collected by flagging in summer [D. reticulatus and in only 1.6% of 875 I. ricinus [D. reticulatus [Apodemus and the bank vole (Myodes glareolus), known to serve as reservoir hosts for TBE virus [Dermacentor tick species is not very common and the reasons for this are neither known nor understood. The repeated findings of TBE virus positive D. reticulatus ticks in three consecutive years, 2016–2018, are notable. It may be speculated that because TBE virus has been recently introduced into this area (as no human TBE case has been reported in this region since 2001 when TBE became a notifiable disease in Germany), it seems very likely that the most abundant tick species became the main carrier of the virus.In European parts of Russia where  species . In contn summer , 46, 47. ricinus . As imma ricinus . As traniculatus , the mosBE virus , 36. FutI. ricinus nymphs were found only in September, when both I. ricinus and D. reticulatus activity was high. To explain if this was an accidental occurrence, more studies should be conducted. However, the positive testing of TBE virus might possibly support the theory that the tick species D. reticulatus plays a major part in the conservation of this TBE natural focus. This would be consistent with the findings of the Polish research team of Biernat et al. [D. reticulatus and I. ricinus nymphs with 0.59% and 0.57%, respectively. Interestingly the total (nymph and adults) I. ricinus MIR of 0.42% was very similar to 0.47% found in a recent study investigating the prevalence of TBE virus in the upper Rhine region in 2016–2017, more than 500 km away from the TBE focus investigated here [It is interesting to note that positive t et al.  in Polanted here .D. reticulatus or I. ricinus ticks. The branching of our sequences together with sequences from Poland (approximately 200 km east) and Neustadt an der Waldnaab (approximately 200 km south-west) are in line with a general east to west evolution of TBE viruses [D. reticulatus seems to be the key vector in TBE virus maintenance in this natural focus. The phylogenetic analyses of the E genes show a marked high homology to virus strains from Poland isolated in 1971 and from Germany isolated in 2011. It is surprising that the virus did not show mutations during this long time period. It might be an indication of a rather recent introduction from Poland or South Germany. The data also show that there is so far no adaptation of the TBE strain to different tick species belonging to different tick genera. Dermacentor and Ixodes separated more than 200 million years ago and therefore are genetically very distant. One would expect that an RNA virus would adapt to such different vectors and reservoirs. The TBE virus detections in D. reticulatus in Poland showed a sequence in the C gene, which the Polish group interpreted as specific for the tick species D. reticulatus. We did not investigate the C gene as the E gene is thought to be more informative with regards to virus strain separation [D. reticulatus. It will be interesting to follow the genetic evolution of this virus strain during the next years to show whether changes will be introduced in the genome of the virus specific for D. reticulatus ticks.In contrast to this study, where only two E gene sequences were identical , our phy viruses , but theparation . The lacDermacentor reticulatus ticks throughout their activity season and only occasionally in Ixodes ricinus sharing the same area in sympatry. In the warm summer months when adult D. reticulatus are not questing and thus cannot be flagged, the virus was not found in active I. ricinus of any life stage, further supporting the conclusion that D. reticulatus may play an important role in virus circulation in this newly established TBE focus.TBE virus was detected in


For validation of the method, total RNA and double strand RNA (dsRNA) from naturally infected trees were used. In a first trial, dsRNAs from trees of cv. “Galega vulgar” from a Portuguese orchard, were subjected to qPCR and from the 30 samples tested, 26 were TNV-D and/or OMMV-positive and 25 were OLV-1 positive. In a second trial, total RNA from trees of different cultivars from Tunisian orchards, were here tested by qPCR and all viruses were detected. From the 33 samples studied, the most prevalent virus detected in Tunisia orchards was OLV-1 (31 samples diagnosed), followed by OLYaV (20 samples diagnosed), and finally the combination in last TNV-D and/or OMMV (12 samples diagnosed). In both trials, qPCR demonstrated to be effective and sensitive, even when using total RNA as template. qPCR through the use of a SYBR® Green methodology enabled, for the first time, a reliable, sensitive, and reproducible estimation of virus accumulation in infected olive trees, in which viruses are usually in low titres, that will allow gaining new insights in virus biology essential for disease control and give an important contribution for establishment of sanitary certification of olive propagative material.Sensitive detection of viruses in olive orchards is actually of main importance since these pathogenic agents cannot be treated, their dissemination is quite easy, and they can have eventual negative effects on olive oil quality. The work presented here describes the development and application of a new SYBR Colletotrichum spp. and Spilocaea oleagina)  was not found to have a negative interference in oil yield and quality  , and morsyndrome . Regardisyndrome . Olive tsyndrome . Viral ssyndrome . These ssyndrome . A decresyndrome . Neverth quality .Strawberry latent ringspot virus; ArMV, Arabis mosaic virus; CLRV, Cherry leafroll virus; OLRSV, Olive latent ringspot virus; CMV, Cucumber mosaic virus; OLYaV, Olive leaf yellowing-associated virus; OLV-1, Olive latent virus 1; OLV-2, Olive latent virus 2), have shown high levels of infection, such as 75% in Tunisia, 51% in Syria, 33% in Italy, 31% in Lebanon, 25% in Croatia, and 31% of necroviruses infection in Portugal  category, applicable to olive plant material, demands that plants must be free of all viruses (Annex to the 93/48/EEC). The Italian certification of propagative material (DM 20/11/2006), followed by most of the Mediterranean countries, including the countries from the North of Africa, imposes the absence of several viruses such as ArMV, CLRV, SLRSV, CMV, OLV-1, OLV-2, OLYaV, and Tobacco necrosis virus senso lato, in which Tobacco necrosis virus D (TNV-D) and Olive mild mosaic virus (OMMV) are included  have been used for the detection of viruses . More re® Green qPCR assays were developed for reliable and sensitive detection of OLV-1, TNV-D and/or OMMV, and OLYaV. The work involved the use of specific primers for each target virus, using different templates . The main performance criteria were tested such as the sensitivity of the technique and the specificity of the primers. qPCR was validated through application to plant material from different olive orchards, from Portugal and Tunisia.In the work described here, three new SYBRVirus reference isolates of OLV-1, TNV-D, and OMMV were obtained according to For validation and comparative analysis of virus infection, two different experiments were conducted. In the first one, samples were collected from 30 symptomatic or asymptomatic olive trees from an orchard under traditional management. The cultivar studied was the native cultivar “Galega vulgar,” and the orchard was located in Alentejo region .The second experiment was performed in samples collected from a total of 33 symptomatic or asymptomatic olive trees. These samples were randomly selected from the 280 collected by In the first experiment, double stranded RNAs (dsRNAs) were extracted from 300 mg of cortical scrapings according to In the second experiment, total RNA was extracted from 25 mg of cortical scrapings from cuttings that were mixed together and homogenized using liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen), following the manufacturer’s instructions. The quantification of RNA and the evaluation of its purity were determined in a NanoDrop-2000C spectrophotometer (Thermo Scientific). RNA integrity was evaluated by denaturing gel electrophoresis. Total RNA (500 ng) was reverse transcribed with the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific), according to manufacturer’s instructions.For conventional PCR assays, primers used for OLV-1 and OLYaV were the same as in in silico using basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (NCBI)1. All qPCR primers are listed in These assays were performed in both experiments. For TNV-D and/or OMMV it was used the already referred primer set . For OLV®-T Easy vector (Promega) and used to transform Escherichiacoli JM109 (Promega) competent cells, by standard methodologies. Each amplicon, sequenced through Sanger procedure, was identified by comparison with corresponding virus sequences available from GenBank.qPCRs were carried out on a 7500 Real Time PCR System (Applied Biosystems) with SYBR Green q-PCR Master Mix (Nzytech). 5 μL of first-strand cDNA (previously diluted 1:10) and 560 nM of each specific primer were used, performing in a total 18 μl reaction volume. The quantification cycle (Cq) values were acquired for each sample with the following cycling conditions: 10 min at 95°C for initial denaturation, an amplification program of 40 cycles at 95°C for 15 s and 60°C for 1 min. Three technical replicates were considered for each sample. Virus reference isolates and no template controls were included in all plates. The fluorescence threshold was manually set above the background level. The specificity of qPCR reactions was evaluated by melting curve analysis. The identity of each amplicon was confirmed by Sanger sequencing. Specific amplification of target viral cDNA was confirmed by cloning PCR amplicons into pGem® Green assay against the cDNA of the other virus reference isolates was also tested by qPCR.The specificity of each SYBR(-1/slope) - 1) × 100, as well as slope and linearity . Detection sensitivities of the assays were determined from the Cq of the lowest plasmid dilution that fell within the linear standard curve. The method performed for absolute DNA quantification was based on the determination of the absolute number of target copies (TCN) previously described by For each specific viral fragment, to determine the amplification efficiencies of the primers, standard curves were generated from 10-fold dilution series of plasmid DNA , used to draw a nine-point calibration curve to validate each assay in the dynamic range chosen (0.8 to 8E7 target copies). Amplification efficiencies were calculated through the equation E =  above 0.99 . Also, the specificity of the primers and their combinations were verified via cloning and sequencing of amplified products.The specificity of the assays was confirmed To evaluate the practical robustness and accuracy of the qPCR assays, two experiments were conducted to test the presence of the viruses using field samples. Additionally, conventional PCR assays were also performed. It was also studied if total RNA template as viral target was suitable for virus detection through qPCR.In the first experiment, concerning the use of dsRNA from 30 field-collected samples from a Portuguese olive orchard, OLYaV was not detected with neither qPCR nor conventional PCR. For the other studied viruses, qPCR revealed 26 positive samples for TNV-D and/or OMMV and 25 positive samples for OLV-1 . Using cTo demonstrate the suitability of the qPCR methodology in different olive material and corroborate qPCR sensitivity, a second experiment was performed in 33 field-collected samples from 11 olive orchards, in several regions of Tunisia. For the qPCR experiment it was used as template total RNA from cortical scrapings from cuttings, instead of the highly laborious protocol for extraction of dsRNA used for conventional PCR. The results revealed that through qPCR and using total RNA as template, it was possible to detect all the studied viruses . qPCR reBoth experiments performed here demonstrate qPCR tests as suitable for detection of virus infecting olive.Since there is no known source of genetic resistance to olive viruses, a key component of viral disease management is the propagation of virus-free plant materials. The main approach used to obtain, propagate and commercialize plants free from harmful pathogens is through phytosanitary selection and certification programs. The obtention of pathogen-free material from infected trees is possible to achieve through sanitation treatments such as heat therapy, meristem tip culture and micrografting, although the use of these methodologies for virus elimination in olive is still limited . Thus, s® Green-based qPCR methodology for specific and reliable quantitative detection of highly spread olive viruses. Moreover, despite the low viral titres of the olive viruses (2 and slope consistent with the acceptance criteria (The work performed here, describes for the first time the use of a SYBR viruses , qPCR tu viruses . The pri viruses , and newcriteria , confirmcriteria . In Tunicriteria , althoug® Green methodology enabled, for the first time, a high level of reliability, sensitivity and reproducibility regarding the estimation of virus accumulation in infected olive trees, being an efficient tool for the early diagnosis of the diseases, and for the improvement of the viral diagnosis. The results achieved will allow gaining new insights in virus biology, essential for disease control. Furthermore, this robust diagnostic procedure will give an important contribute for the establishment of phytosanitary certification programs of olive and high-quality productions.qPCR combined with the SYBRThe datasets generated for this study are available on request to the corresponding author.MDC and MRF: conceptualization. MDC: formal analysis. MDC and AP: funding acquisition. MDC, MZ, and CV: methodology. MDC, MZ, CV, and PM: resources. MRF and MC: supervision. MDC: writing, original draft preparation. MDC, MZ, CV, PM, AP, MC, and MRF: writing, review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is no definitive strategy for gastrointestinal bleeding due to left-sided portal hypertension after pancreaticoduodenectomy (PD) for pancreatic cancer (PC) with concomitant portal vein resection (PVR).Case 1: A 70-year-old woman underwent a PD for PC with PVR. Seven years after her surgery, she suffered severe anemia with suspected gastrointestinal bleeding. Computed tomography scan (CT) revealed varices at a portion of the pancreaticojejunostomy (PJ). Angiography revealed that splenic venous flow drained into the varices and then into the portal vein. A diagnosis of bleeding varices of the PJ due to left-sided portal hypertension was made. Following a partial splenic artery embolization, her anemia improved.Case 2: An 80-year-old male underwent a PD for pancreatic head cancer combined with resection of the confluence of the portal and splenic veins with a reconstruction between the portal and superior mesenteric veins. Eighteen months after his surgery, he developed melena with negative endoscopy findings in his large and small bowel. CT revealed varices at the site of the PJ that communicated with the jejunal and portal veins. He underwent obliteration of the varices via a trans-portal-venous approach. As a result, he remained without melena until he died of PC 17 months after the embolization.Left-sided portal hypertension following a PD with bleeding varices can be treated by interventional radiology with minimal invasiveness. Left-sided portal hypertension (LSPH) is a localized form of portal hypertension that occurs as a result of an isolated thrombosis or obstruction of the splenic vein (SV) . LSPH isA 70-year-old woman underwent PD for pancreatic cancer (PC) with a concomitant PVR. She also underwent an SV and inferior mesenteric vein (IMV) resection. The side-to-end anastomosis was performed only between the SV and IMV Fig. . She disWe selected partial splenic artery embolization (PSE) for hemostasis. At the first stage, we embolized 70% of the splenic arterial flow. Ten months after the first stage PSE, she again complained of melena and anemia, for which we performed a second stage PSE (embolized 90% of splenic artery flow). Although follow-up CT at 3 years after first stage PSE showed the varices around the PJ  Fig. . EighteePreviously, some authors have shown that PD without SV reconstruction could cause LSPH , 4. HoweFor intestinal bleeding from a PJ due to LSPH, several types of procedures can be considered: total pancreatectomy, a splenectomy, obliteration of the varices via a trans-portal-venous approach, and a PSE. Previously, several authors have reported that PSE is an effective treatment for acute gastric bleeding due to LSPH , 13. OurIn our second case, we selected obliteration of the varices at the PJ via a trans-portal-venous approach because CT scans showed a route from the PV to the varices at a portion of the PJ. This procedure was previously reported by Sakamoto et al.  in 2014.Our cases suggested that IVR could be the first choice for intestinal bleeding at a portion of the PJ due to LSPH. Furthermore, it is assumed that trans-portal-venous approach is first-choice procedure.We presented two types of IVR procedures for intestinal bleeding at a portion of the PJ following PD. The obliteration of the varices in PJ could be first choice procedure.


Cucurbit yellow stunting disorder virus  is transmitted in a semipersistent manner by the whitefly, Bemisia tabaci, and is efficiently transmitted by the widely prevalent B. tabaci cryptic species, MEAM1. In this study, we compared transcriptome profiles of B. tabaci MEAM1, after 24 h, 72 h and 7 days of acquisition feeding on melon plants infected with CYSDV (CYSDV-whiteflies) with those fed on virus-free melon, using RNA-Seq technology. We also compared transcriptome profiles with whiteflies fed on tomato plants separately infected with Tomato chlorosis virus (ToCV), a crinivirus closely related to CYSDV, and Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, which has a distinctly different mode of transmission and their respective virus-free controls, to find common gene expression changes among viruliferous whiteflies feeding on different host plants infected with distinct (TYLCV) and related (CYSDV and ToCV) viruses.A total of 275 differentially expressed genes (DEGs) were identified in CYSDV-whiteflies, with 3 DEGs at 24 h, 221 DEGs at 72 h, and 51 DEGs at 7 days of virus acquisition. Changes in genes encoding orphan genes (54 genes), phosphatidylethanolamine-binding proteins (PEBP) (20 genes), and AAA-ATPase domain containing proteins (10 genes) were associated with the 72 h time point. Several more orphan genes (20 genes) were differentially expressed at 7 days. A total of 59 common DEGs were found between CYSDV-whiteflies and ToCV-whiteflies, which included 20 orphan genes and 6 lysosomal genes. A comparison of DEGs across the three different virus-host systems revealed 14 common DEGs, among which, eight showed similar and significant up-regulation in CYSDV-whiteflies at 72 h and TYLCV-whiteflies at 24 h, while down-regulation of the same genes was observed in ToCV-whiteflies at 72 h.Dynamic gene expression changes occurred in CYSDV-whiteflies after 72 h feeding, with decreased gene expression changes associated with 7 days of CYSDV acquisition. Similarities in gene expression changes among CYSDV-whiteflies, ToCV-whiteflies and TYLCV-whiteflies suggest the possible involvement of common genes or pathways for virus acquisition and transmission by whiteflies, even for viruses with distinctly different modes of transmission.The online version of this article (10.1186/s12864-019-5999-0) contains supplementary material, which is available to authorized users. Cucurbit yellow stunting disorder virus , is widely distributed among cucurbit production regions. The virus was initially discovered in the United Arab Emirates  whiteflies’) for acquisition access periods (AAP) of 24 h, 72 h, and 7 d. A total of 11.5–14.9 M raw reads were generated per library. These were processed to remove adapters, low quality reads, and reads from the endosymbionts (i MEAM1)   in CYSDV whiteflies compared with VF whiteflies at three different AAPs. Three DEGs  were found after the 24 h AAP, 221 DEGs (82 up-regulated and 139 down-regulated) after the 72 h AAP, and 51 DEGs (49 up-regulated and 2 down-regulated) were present after the 7 d AAP  than was found after the 72 h feeding period (5.2 × 103 copies/ug) suggesting that virus binding in the whitefly may not saturate all sites even after several days of feeding. However, it is equally likely that differences in virus levels within whiteflies between the two virus acquisition access periods could be related to greater ingestion of virus into whiteflies after the 7 d acquisition period rather than the amount of virus available for transmission from binding sites in the anterior foregut.As noted previously, feeding periods of 18 to 24 h provide sufficient virus acquisition to maximize transmission efficiency and retention of initially acquired virus will decline gradually over a period of seven days ; howeverB. tabaci MEAM1 like ToCV; however, its retention time in the whitefly vector can last up to 7 days following acquisition  that plays a primary role in binding double-stranded DNA during transcription initiation [B. tabaci in association with feeding on crinivirus-infected plants, but their association with other virus infectious diseases warrants notation and potentially further study.KEGG Pathways Analysis identified four up-regulated genes associated with the viral infectious disease category in CYSDV whiteflies. No DEGs related to virus infectious diseases were found to be down-regulated in CYSDV whiteflies compared to VF whiteflies for any of the three feeding periods , also found that MAPK and TGF-β pathways were down-regulated [KEGG pathway analysis identified five DEGs associated with signal transduction pathways at 72 h. Among the up-regulated signal transduction genes at 72 h, us study . In thatus study . A similegulated . This hiDrosophila, and they have also been implicated with species-specific adaptive processes, host-parasite interactions, and interactions with the environment [A large number of differentially regulated orphan genes with high FPKM values were found in both CYSDV and ToCV whiteflies in comparison to their respective VF whiteflies, indicating these might be associated with virus-vector interactions. As discussed before, orphan genes comprised a significant portion of the sequenced genome of different species including humans, mosquito, and ironment , 60, 61.Crinivirus, family Closteroviridae) or TYLCV . Furthermore, such genes might encode functions critical for virus acquisition and transmission by the whitefly.It appears viruliferous whiteflies shut down their carbohydrate metabolism during acquisition of criniviruses by knocking down the expression of alpha-glucosidase and sucrase genes while increasing carbohydrate transportation activity by maintaining the higher expression of facilitated glucose transporter protein 1 gene in both ToCV and CYSDV systems. In addition, four genes related to carbohydrate metabolism, which included two α-glucosidases, one glycogen branching enzyme, and one sucrase, were also downregulated, with the exception of one α-glucosidase in TYLCV whiteflies compared to VF whiteflies . Three gWhiteflies that fed on CYSDV infected melon plants compared with those fed on virus free melon plants exhibited patterns of differential expression overall that appeared to be delayed relative to patterns found in the ToCV-tomato study . As noteB. tabaci MEAM1 to two viruses with very different modes of transmission as described by Hasegawa et al. [Crinivirus genus [Interestingly, whiteflies that fed on tomato infected with either ToCV or TYLCV showed similar trends in terms of the relative number of DEGs found at three different feeding time points  Fig. a & b 1313. Higheus genus , 63, thrus genus , 63, wheus genus , 64–67.Bta03880, cathepsins B; Bta07602, UDP-glucuronosyltransferase 1–1; Bta10829, acid phosphatese-1; Bta14312, sucrase; Bta14422, alpha-glucosidase) exhibited down-regulation in whiteflies fed on each of the three virus infected host combinations compared to those fed on their respective virus-free source plants  showed similar trends of up-regulation in both CYSDV-whiteflies and TYLCV-whiteflies compared with whiteflies fed on respective virus-free plants; however, an opposite trend of down-regulation was observed in ToCV-whiteflies , genes associated with the lysosome including cathepsins, genes encoding phosphatidylethanolamine-binding proteins (PEBP), and AAA-ATPase domain containing proteins.A total of 275 differentially expressed genes (DEGs) were identified in replicated experiments over three feeding periods between whiteflies fed on melon leaves with and without infection by the semipersistently transmitted crinivirus, CYSDV. Only 3 DEGs were identified following a 24 h AAP, sharply contrasting with results of experiments involving another crinivirus, ToCV on tomato, which exhibited 989 DEGs after a 24 h AAP using the same methodology. In contrast, the number of DEGs were comparable between whiteflies fed on each of the two virus-host systems at 72 h . Following a 7 d AAP, 51 genes were found to be differentially expressed between CYSDV- and VF-whiteflies. Over the three sampling periods a total of 59 genes were found to be differentially expressed in common between Bemisia tabaci MEAM1 from the Salinas USDA-ARS colony, originally collected from Imperial County, CA in 2013, were reared on broccoli (Brassica oleracea) plants. Whiteflies were provided acquisition access periods (AAPs) on CYSDV-infected or virus-free melon plants (cv. Topmark) for 24 h, 72 h, and 7 d. The presence of CYSDV in host plants and whiteflies was confirmed by RT-PCR from each feeding time point, and similarly virus-free plants and whiteflies were confirmed virus free by negative RT-PCR results as described in [ribed in . Followiribed in .p values < 005),: genes with FC ratio ≥ 1.5 for up-regulated genes and FC ratio ≤ 0.67 for down-regulated genes. More stringent criteria were used to identify individual genes in depth with FPKM > 10 and ratio ≥ 2 (up-regulated genes) and ratio ≤ 0.5 (down-regulated genes) [RNA extracted from whiteflies was used to construct RNA-Seq libraries using the protocol described in . A HiSeqd genes) . KEGG and genes) . Venn did genes) .Additional file 1:Bemisia tabaci MEAM1, and whiteflies fed on cucurbit yellow stunting disorder virus (CYSDV) infected melon plants (“CYSDV”) for 24 h, 72 h, and 7 d. b. Correlation matrix analysis for multiple biological replicates obtained from RNA-Seq libraries prepared from virus free (VF) whiteflies fed on uninfected melon plants and whiteflies fed on cucurbit yellow stunting disorder virus (CYSDV) infected melon plants for periods of for 24 h, 72 h, and 7 d. c. Differentially expressed genes between virus free (VF) whiteflies fed on uninfected melon and whiteflies fed on cucurbit yellow stunting disorder virus (CYSDV) infected melon plants for 24 h, 72 h, and 7 d. (XLSX 33 kb)a. The number of Illumina raw and processed reads produced per RNA-Seq library from virus free (“VF”) whiteflies, Additional file 2:a. KEGG annotation, KO, and score values of the 28 genes out of a total of 82 up-regulated in CYSDV whiteflies versus virus-free whiteflies at 72 h. b. Pathway reconstruction results from the 28 genes out of a total of 82 up-regulated in CYSDV whiteflies versus virus-free whiteflies at 72 h. c. KEGG annotation, KO, and score values of the 40 genes out of a total of 139 down-regulated in CYSDV whiteflies versus virus-free whiteflies at 72 h. d. Pathway reconstruction results from the 40 genes out of a total of 139 down-regulated in CYSDV whiteflies versus virus-free whiteflies at 72 h. e. KEGG annotation, KO, and score values of the 12 genes out of a total of 49 up-regulated in CYSDV whiteflies versus virus-free whiteflies at 7 d. f. Pathway reconstruction results from the 12 genes out of a total of 49 up-regulated in CYSDV whiteflies versus virus-free whiteflies at 7 d. (XLSX 49 kb)Additional file 3:A list of 59 common differentially expressed genes between ToCV whiteflies and CYSDV whiteflies compared to virus free whiteflies at all three different feeding time points. (XLSX 33144 kb)Additional file 4:A list of 69 differentially regulated orphan genes found in CYSDV whiteflies compared to VF whiteflies following feeding for 72 h and 7 days. (XLSX 21 kb)


Live-attenuated viral vaccines are highly safe and efficacious but represent complex and often multigenic attenuation mechanisms. Most of these vaccines have been generated empirically by serial passaging of a wild-type (WT) virus in cell culture. One of the safest and most effective live-attenuated vaccines is yellow fever (YF) virus strain 17D, which has been used for over 80 years to control YF disease. The availability of the WT parental strain of 17D, Asibi virus, and large quantities of clinical data showing the effectiveness of the 17D vaccine make this WT parent/vaccine pair an excellent model for investigating RNA virus attenuation. Here, we investigate a mechanism of 17D attenuation and show that the vaccine virus is resistant to the antiviral compound ribavirin. The findings suggest that attenuation is in part due to a low probability of reversion or mutation of the vaccine virus genome to WT, thus maintaining a stable genotype despite external pressures. The molecular basis of attenuation for live-attenuated vaccines is poorly understood. The yellow fever (YF) 17D vaccine virus was derived from the wild-type, parental strain Asibi virus by serial passage in chicken tissue and has proven to be a very safe and efficacious vaccine. We have previously shown that wild-type Asibi is a typical RNA virus with high genetic diversity, while the 17D vaccine virus has very little genetic diversity. To investigate this further, we treated Asibi and 17D viruses with ribavirin, a GTP analog with strong antiviral activity that increases levels of mutations in the viral genome. As expected, ribavirin treatment introduced mutations into the Asibi virus genome at a very high frequency and decreased viral infectivity while, in contrast, the 17D vaccine virus was resistant to ribavirin, as treatment with the antiviral introduced very few mutations into the genome, and viral infectivity was not lost. The results were confirmed for another YF wild-type parental and vaccine pair, a wild-type French viscerotropic virus and French neurotropic vaccine. Using recombinant Asibi and 17D viruses, ribavirin sensitivity was located to viral nonstructural genes. Thus, two live-attenuated YF vaccine viruses are genetically stable even under intense mutagenic pressure, suggesting that attenuation of live-attenuated YF vaccines is due, at least in part, to fidelity of the replication complex resulting in high genetic stability. Flavivirus contains approximately 70 viruses, most of which are arthropod-borne (arboviruses) and major public health problems, including dengue, Japanese encephalitis, yellow fever (YF), and Zika viruses. These enveloped, positive-sense RNA viruses contain the following 10 genes: three structural proteins  and seven nonstructural (NS) proteins . The functions of the NS proteins, which constitute the host cell membrane-bound replication complex, are incompletely understood. The viral protease and helicase are contained within NS3, and methyltransferase and polymerase reside in NS5  strain Asibi virus in various cell types, including chicken embryos lacking nervous tissue, wherein it lost viscerotropic properties as well as the ability to be transmitted by mosquitoes . AlthougConcurrent to the development of the 17D vaccine virus, another live-attenuated YF vaccine, French neurotropic vaccine (FNV) virus, was developed by 128 passages of the WT strain French viscerotropic virus (FVV) in the mouse brain. Asibi virus and FVV were isolated in the same YF outbreak in West Africa in 1927. As such, these viruses are genetically very similar, with only two amino acid mutations differentiating the WT strains (E-200 and NS3-280)  and a high mutation rate with considerable nucleotide mismatching . Though 10–15–RNA virus diversity in attenuated mutants has been investigated. Studies with poliovirus have shown that increasing the fidelity of the RdRp attenuates the virus by reducing the ability of the virus to enter the brain and cause disease , 18. Whe20–Lethal mutagenesis is an antiviral principle that utilizes the concept of error threshold, a theoretical maximum rate of genetic diversity. Antivirals utilizing this mechanism of action are typically nucleoside analogs which, when incorporated into the genome, increase the viral mutation rate beyond the error threshold. At this point, the benefits of a robust mutant spectrum are outweighed by the accumulation of deleterious mutations and the generation of defective genomes that inhibit viral replication and reduce infectivity . Ribavir26–in vitro, supporting the hypothesis that the incorporation of a GTP analog is the primary mechanism of action of ribavirin against WT YFV. We propose that YFV vaccine strains are resistant to ribavirin due to an acquired high-fidelity replication mechanism and that this contributes to the attenuated phenotype.Due to the differences in genetic diversity between WT Asibi virus and its 17D vaccine virus derivative, we investigated the changes in genetic diversity of 17D and Asibi viruses following treatment with ribavirin. It was found that 17D virus was resistant to antiviral effects, whereas the drug was active against WT Asibi. Furthermore, ribavirin resistance was observed in a second live-attenuated YFV vaccine strain (FNV) and susceptibility in a second WT YFV strain (FVV). Experiments with recombinant-derived mutant viruses indicated that resistance was associated with the NS genes of the vaccine virus, suggesting a role for the replication complex in susceptibility. The contribution of anti-IMPDH activity was tested using mycophenolic acid (MPA), a nonnucleoside triphosphate antiviral that inhibits IMPDH and has been shown to be effective against 17D , 32. WT P < 0.0001). The 50% inhibitory concentration (IC50) values of Asibi  and 17D  viruses differed by 57-fold, showing that ribavirin is much more active against Asibi virus than against 17D virus (P < 0.037). The IC50 values of FVV  and FNV  viruses differed by 14-fold showing that, again, WT YFV was more susceptible to ribavirin than was a live-attenuated vaccine virus. It was noted that YFV vaccine strains 17D and FNV were not statistically different from each other (P > 0.999). The same was also true in a comparison of the two WT YFV strains, where no difference can be detected between the curves (P > 0.999).The effect of ribavirin treatment on the viral infectivity of low-passage-number WT Asibi virus and commercial 17D vaccine virus strain was investigated in Vero cells. The viral infectivity reduction curves for WT Asibi virus and vaccine YFV were significantly different (7D virus . The mulP = 0.68), and the IC50 values of Asibi  and 17D  viruses were statistically indistinguishable and within the range considered safe and effective for MPA in vitro drug screens (R2 = 0.94) and FNV  viruses, with reduction curves and IC50 values which appeared to be statistically similar (P > 0.999)  and vaccine strains (17D and FNV viruses) was investigated. MPA is an anti-IMPDH antiviral with no nucleoside triphosphate (NTP) analog activity. Viral infectivity reduction curves of MPA-treated Asibi and 17D viruses were not significantly different P = 0.68)(i) WT Asibi versus 17D vaccine virus. Four ribavirin treatment concentrations  were incubated with Asibi and 17D viruses, sampled at 48 h postinfection, and sequenced by massively parallel sequencing. Shannon’s entropy  Effect of ribavirin on single nucleotide variant populations. Genetic diversity was also evaluated in terms of single nucleotide variants (SNVs), defined as a nucleotide that differs from the consensus sequence by at least 1% of the population at that position.P = 0.033) (Treatment of Asibi virus with ribavirin resulted in a dose-dependent increase in both the number of SNVs and the number of nonsynonymous variants compared to synonymous variants. The 0.5 μM concentration caused the largest increase in genome variation . Twenty = 0.033) . With 1.= 0.033) . Interes= 0.033) . The E-410.1128/mBio.02294-19.1TABLE S1Table S1, DOCX file, 0.1 MB.17D-204 treated with ribavirin generates less SNV than does ribavirin-treated Asibi. Variants were detected using V-Phaser2, and variants below 1% frequency and those that did not pass the strand bias test were discarded. Variants highlighted in green are common throughout the samples. Variants in red text are residues which distinguish 17D-204 and Asibi. Download Copyright © 2019 Davis et al.2019Davis et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P > 0.999).Although the number of SNVs in the 17D virus samples increased with increasing concentration of ribavirin, the frequency of variants detected remained low. The number of nonsynonymous SNVs was consistently lower than synonymous SNVs, suggesting that the virus was not responding to the selection pressure posed by ribavirin. In total, only seven nonsynonymous SNVs were detected across all ribavirin treatments sequenced, with each SNV being present in less than 5% of the population . Structu(iii) NGS analysis of WT FVV versus FNV virus. In order to confirm the resistance of vaccine strains to ribavirin treatment, untreated and 0.5 μM ribavirin FVV- and FNV virus-treated samples were sequenced by next-generation methods. As with Asibi virus, WT FVV experienced large increases in viral diversity by entropy (P < 0.0001). Consistent with the 17D findings, the number of SNVs detected in ribavirin-treated  and untreated  FNV virus samples were consistent in both number and identity  Sensitivity to ribavirin is controlled by YFV nonstructural proteins. To exclude virus passage history in the antiviral activity of ribavirin and determine if the YFV replication complex was involved in the resistance of 17D to ribavirin, infectious clone-derived viruses and structural chimeras  were generated using an infectious clone system (P > 0.999). Thus, the results of infectious-clone-derived viruses show that the effects of ribavirin were not due to the passage history of the viruses. The IC50 of the ribavirin-treated Asibi/17D prME virus  was similar to that of the Asibi virus infectious clone  (R2 = 0.68) was resistant to ribavirin, similar to the 17D virus   . The 17D = 0.88) , indicatLive-attenuated virus vaccines have been successfully used to combat infectious disease for decades, but due to their empirical derivation, little is known about their mechanisms of attenuation. This lack of understanding makes the development of next-generation live-attenuated vaccines difficult. The live-attenuated YF 17D vaccine virus has been used for 80 years to successfully reduce YF disease, and over 850 million doses of the vaccine have been distributed with exceptionally low levels of vaccine-associated adverse events . The suc50 values, 0.37 μM and 0.51 μM for Asibi and FVV, respectively) is consistent with findings from studies of other RNA viruses encoding a low-fidelity RdRp, such as Lassa and Crimean-Congo hemorrhagic fever viruses   , 40, 41. strains . It has  strains . In orde strains , 32. The and FNV . Togethe and FNV .50 similar to that of Asibi virus and had increases in diversity and SNVs, of which 66% were detected in the replication complex. Our studies indicate that ribavirin is being incorporated into the genome of WT YFV and should be tested as a treatment for YFV infection. This warrants further investigation, as most studies to date have utilized 17D vaccine virus as a model system, which is not representative of WT YFV strains  of hepatitis C virus, dengue virus NS5 methyltransferase, poliovirus RdRp, and chikungunya virus nsP4, which contains the RdRp , 44, 45. strains , 49.Little variation was detected in the genotype of ribavirin-treated YFV vaccine strains, and only a negligible decrease in infectivity titer was observed, suggesting a resistance to the lethal mutagenesis of ribavirin by YFV vaccine strains. This confirms previous reports examining the activity of ribavirin against 17D virus . In compIt is possible that the extensive serial passaging of WT Asibi virus and FVV  caused the WT virus to adapt to an unchanging environment where an unwavering consensus sequence was the most fit. This adaptation caused the virus to be less fit in a biological environment where the vaccines could no longer respond to selection pressure  . ResistaIn this study, the effects of ribavirin on two WT YFV strains and the two LAV strains derived from them were compared under a hypothesis that an acquired fidelity mechanism contributes to the attenuation of an empirical flavivirus vaccine. It had been previously shown that 17D was resistant to the drug; however, the WT Asibi strain was not tested in a similar context, which was performed herein . The WT Ribavirin  was reconstituted in high-performance liquid chromatography (HPLC)-grade water and sterilized using a 0.2-μm filter. A 10 μM working solution was then obtained by diluting the stock in minimal essential medium (MEM) supplemented with 2% fetal bovine serum (FBS). This working stock was used to make ribavirin concentrations of 6 μM, 2 μM, 1 μM, 0.7 μM, 0.5 μM, 0.2 μM, 0.15 μM, 0.1 μM, 0.05 μM, and 0.02 μM for the antiviral assays.MPA  was reconstituted at 25 mM in HPLC-grade water. A 1 mM working solution was obtained by diluting the stock in MEM supplemented with 2% FBS. This working stock was used to make a dose-response curve of 2-fold dilutions from MPA concentrations of 1 mM to 0.24 μM.KF769016) and is the lowest passage number available, namely, six passages in rhesus macaques and 3 passages in C6/36 cells. FVV (GenBank accession no. U21056) and FNV-Yale (GenBank accession no. U21055) viruses used were received from the WRCEVA and passaged once in Vero cells . The Asibi virus used was obtained from the World Reference Center for Emerging Viruses and Arboviruses . Virus was harvested at 80% cytopathic effect (CPE) and sequenced by Illumina methods to confirm sequence identity .Chimeric 17D-204 and Asibi viruses with prM and E (prME) genes swapped were constructed using infectious clone systems , 55. BriAll viral infectivity assays were undertaken in biosafety level 3 (BSL3) facilities at the University of Texas Medical Branch.As described by Leyssen et al. , viral iAY640589) using Bowtie2 with paired-end, very sensitive local parameters. PCR duplicates were removed with MarkDuplicates . All samples were downsampled to the lowest mean coverage of the data set, 9,984 reads per base. Shannon’s entropy was calculated using custom R scripts over possible nucleotide variables  using the method described by Nishijima et al  and sequenced on an Illumina HiSeq 1500 platform. Reads were trimmed of adapter sequences and quality controlled for quality scores greater than 30 (Trimmomatic). The reads were aligned to a reference genome  to determine antiviral 50% inhibitory concentrations (IC50) values and significant differences between four-parameter nonlinear fits of sensitivity curves. A one-way ANOVA was performed with Friedman’s post hoc multiple-comparison test (P < 0.05) to determine significant differences in average entropy across viruses and treatments. A one-way ANOVA was performed with Kruskal-Wallis post hoc multiple-comparison test (P < 0.05) to determine significant differences in the frequencies of variants.All statistical analyses were completed in the GraphPad Prism (v7.2.1). A one-way analysis of variance (ANOVA) was performed with Friedman’s


The ferret transmission model is extensively used to assess the pandemic potential of emerging influenza viruses, yet experimental conditions and reported results vary among laboratories. Such variation can be a critical consideration when contextualizing results from independent risk-assessment studies of novel and emerging influenza viruses. To streamline interpretation of data generated in different laboratories, we provide a consensus on experimental parameters that define risk-assessment experiments of influenza virus transmissibility, including disclosure of variables known or suspected to contribute to experimental variability in this model, and advocate adoption of more standardized practices. We also discuss current limitations of the ferret transmission model and highlight continued refinements and advances to this model ongoing in laboratories. Understanding, disclosing, and standardizing the critical parameters of ferret transmission studies will improve the comparability and reproducibility of pandemic influenza risk assessment and increase the statistical power and, perhaps, accuracy of this model. The susceptibility of ferrets to influenza virus infection has been known for nearly a century. Ferrets and humans share similarities in lung physiology, cellular receptor distribution, and clinical signs of infection, making the ferret an attractive small mammalian model for laboratory study of influenza viruses . BeyAlthough some factors are considered to be controllable, much of the variability between groups cannot be easily overcome. For example, the absence of commercially available uniform caging for ferret transmission experiments (a reflection of country- and institution-specific size regulations and facilities constraints) represents a parameter that would be difficult to standardize. However, a greater understanding of the relative role and contribution of different variables can improve our ability to better contextualize and interpret results among laboratories. For example, does the virus dose affect transmissibility or otherwise influence detection of transmissible quasispecies in contact ferrets? Gustin et al. previously demonstrated the potential effects of the dose and the route of inoculation  on transmission potential  would beAs discussed at the workshop, the participating laboratories analyzed the protocols used for evaluation of influenza virus transmissibility via airborne particles in the ferret model, which highlighted the breadth of experimental designs. It was also clear that all variables that are probable contributors to differential results are not routinely disclosed in peer-reviewed publications or other platforms where results are discussed. Lack of disclosure of all variables can complicate the ease of comparing findings between laboratories, warranting a closer look at the feasibility of using a more comparable study design between multiple laboratory groups for risk-assessment studies. As such, a push toward comprehensive description of specific experimental conditions would aid this effort and would probably be valuable when risk assessments performed in different laboratories are compared.Beyond the experiment- and facility-based variability we describe, the lack of a standard protocol for the number of experimentally infected animals and the number of recipient ferrets (donor:recipient ratios) included in ferret transmission studies can affect the ability to interpret results among groups and represents a substantial controllable parameter. The ideal standard for risk assessment activities seems to be a 1:1 donor:recipient experimental setup to assess virus transmissibility via the airborne route, where each virus-infected ferret is exposed to only 1 recipient ferret. This design facilitates ease of interpretation of results and provides added rigor from a statistical perspective. This design also restricts exposure to virus-laden particles expelled from each donor ferret to its respective recipient, ensuring that any detected transmission event would have originated from exposure to separate donors. However, because of space limitations, these experiments are often conducted in replicates inside a single physical containment area with a shared ventilation system  while still maintaining a 1:1 donor:recipient ratio. If transmission is mediated by virus-laden particles expelled by donors, increasing the number of donors within a single containment area is likely to increase the concentration of virus-laden particles in the air, thereby increasing the observed transmissibility. Specifically, it is not known whether transmission kinetics would be comparable if 3 independent experiments were performed with 1 donor to 1 recipient (each ferret housed singly) versus 1 experiment with 3 donors and 3 recipients per containment area. Air sampling devices that allow monitoring of the quantities of virus-laden particles in the air throughout the experiment would help refine the experiment outcomes and are likely to become part of these experimental designs in the future.Further impeding efforts to compare results among laboratories, many experiments include an additional contact ferret co-housed with an experimentally infected ferret to evaluate virus transmissibility in a direct contact setting while still assessing the respiratory droplet transmission to a recipient ferret housed in an adjacent cage. In this design, several ferrets may serve as donors . Moreover, the donor and direct contact–infected ferrets are likely to shed virus-laden particles at different times, further complicating the results of the transmission experiments. Ideally, the effects of different experimental designs should be investigated in systematic experiments, and researchers should strive to disclose this information as comprehensively as possible.During workshop discussions, most researchers agreed that it would be helpful for the field to coalesce around a fixed 1:1 donor:recipient ratio  for risk-assessment transmission experiments. Introduction of direct contact ferrets into the experimental setup would probably extend the amount of time that virus-laden particles can be released in the air. Virus amplification by direct contact ferrets may also lead to virus adaptation and emergence of variants with increased respiratory droplet transmission potential. Applying a 1:1 donor:recipient ratio would increase the consistency of the experimental design under which risk-assessment experiments are conducted across multiple laboratories, differentiating them from broader, more heterogeneous research-based assessments that would include more experimental designs and variables. However, individual laboratories have built up datasets and experience over the years while performing risk-assessment studies with different strains of influenza viruses; thus, adopting a common protocol may be difficult to achieve within a short time.Although viruses that are readily transmitted by the airborne route will exhibit robust transmission in a direct contact setting, some influenza viruses that are not transmitted efficiently via respiratory droplets are nonetheless transmitted between ferrets placed in direct contact, which facilitates transmission via multiple modes. Studies evaluating virus transmissibility between ferrets placed in direct contact may be influenced by many of the experimental drivers discussed here; when using this model, further contributions to variability are introduced by animal behavior and housing practices. Although scoring for the Centers for Disease Control and Prevention Influenza Risk Assessment Tool includes data derived from the direct contact transmission model in risk assessment, it is not fully clear how to interpret the relative pandemic risk resulting from viruses that transmit in a direct contact setting but not via respiratory droplets. As discussed above, a greater understanding of what confers virus transmissibility in both models will improve our ability to interpret results from more permissive direct contact models with the more stringent respiratory droplet transmissibility. This knowledge will improve our ability to appropriately include and aggregate results from both types of transmission studies in influenza virus risk assessments.Although the ferret transmission model has greatly improved our understanding of influenza virus transmissibility, there are limits to what this model can contribute to risk assessment and how results are interpreted. In particular, inefficient virus transmission  remains a difficult outcome to understand. It is often unclear whether this event results from genetic changes in the virus during the transmission event, reflects the transmitted infectious dose, or results from other contributing factors; concurrent contextualization of these results with other laboratory paramaters  can often provide additional insight. Similarly, interpretation of seroconversion in the absence of detectable virus in respiratory secretions or detectable virus in the absence of seroconversion can be difficult. Moving toward a consensus on the implications of inefficient transmission events would be helpful because, currently, efficient and inefficient transmission are not well defined.Another major limitation of current ferret transmission studies is the small group size, which is driven by cost, size of the animals and their associated housing requirements, and ethical and practical constraints. For this reason, statistical analyses of data from transmission experiments are infrequently performed  or deep-sequencing approaches have provided, and probably will continue to provide, additional information to aid in the interpretation of inefficient virus transmission events, elucidate transmission bottlenecks, and differentiate between within-group variability and larger differences in experimental setup and design (An additional avenue for improved understanding of virus transmissibility via respiratory droplets are aerobiology-based approaches. These approaches include analysis of the exhaled breath of infected ferrets and the amount and size distribution of virus-containing aerosols released by infected ferrets (In vivo ferret transmission studies are not performed in isolation. The incorporation of these data into larger research efforts has greatly expanded our understanding of the complex determinants of influenza virus transmission in mammals. For example, hemagglutinin acid stability and the hemagglutinin–neuraminidase balance have been linked with virus transmissibility via the airborne route in ferrets, as have receptor binding preference, gene constellation, neuraminidase stalk length, and other parameters (The plasticity of the ferret model permits a wide range of experimental approaches to assess influenza virus transmissibility. This plasticity represents a great advantage when designing research experiments to evaluate viral, host, environmental, and other factors that contribute to transmission between mammals. However, it might be beneficial for studies conducted primarily for risk-assessment purposes to be performed under conditions as uniform as possible. For example, moving toward a standardized 1:1 donor:recipient ratio in risk-assessment studies would probably enhance the comparability of results found by different research groups and would enable inclusion in meta-analyses .Current knowledge regarding the viral, host, and environmental parameters that drive transmission outcomes is limited. Understanding these parameters would be beneficial for infection control, and future studies should aim to validate these factors empirically. As data regarding the exact role of each of the potential parameters discussed in this article are developed, improved documentation of variables  associatMany unanswered questions are relevant for understanding the pandemic risk posed by novel and emerging influenza viruses that lie outside a standardized risk-assessment experimental setup. Some studies that would provide additional valuable information include modulation of distance between cages when assessing virus transmissibility via the airborne route, shortening the duration of exposure between inoculated donors and contact ferrets (A useful exercise might be for several research groups to test and compare results of a transmission experiment by using 1 selected virus strain prepared by 1 laboratory. Many practical considerations would need to be discussed, including the particular strain to test and the anticipated transmission phenotype of this virus. Complete standardization of ferret transmission studies conducted worldwide is not possible; however, exercises that seek to examine the relative contributions of laboratory-specific drivers of experimental variability may identify critical parameters or conditions. Similarly, meta-analyses of published data, with included disclosure of the parameters listed in Influenza viruses will continue to jump the species barrier and cause human infection and disease. Virus-transmissibility assessments in the ferret model represent one of numerous activities conducted to aid pandemic preparedness efforts in the event that a pandemic virus does emerge in the future. Continued refinement of the ferret model, concurrent with advances in identification of viral, host, and environmental factors that influence transmission risk (


Solanum lycopersicum infected by a nuclear-replicating virus, we highlighted miRNA-mediated cleavage events that could not be revealed in virus-free systems. Among them we confirmed miR6024 targeting and cleavage of RX-coiled-coil (RX-CC), nucleotide binding site (NBS), leucine-rich (LRR) mRNA. Cleavage of paralogs was associated with short indels close to the target sites, indicating a general functional significance of indels in fine-tuning gene expression in plant–virus interaction. miR6024-mediated cleavage, uniquely in virus-infected tissues, triggers the production of several 21–22 nt secondary siRNAs. These secondary siRNAs, rather than being involved in the cascade regulation of other NBS–LRR paralogs, explained cleavages of several mRNAs annotated as defence-related proteins and components of the photosynthetic machinery. Outputs of these data explain part of the phenotype plasticity in plants, including the appearance of yellowing symptoms in the viral pathosystem.Plant viruses modify gene expression in infected tissues by altering the micro (mi)RNA-mediated regulation of genes. Among conserved miRNA targets there are transcripts coding for transcription factors, RNA silencing core, and disease-resistance proteins. Paralogs in these gene families are widely present in plant genomes and are known to respond differently to miRNA-mediated regulation during plant virus infections. Using genome-wide approaches applied to  RNA silencing refers to conserved pathways affecting gene expression through negative regulation mediated by non-coding RNAs, such as short interfering (si)RNAs. In plants, micro (mi)RNA comprises one of the most abundant classes of 21- to 24-nucleotide (nt)-long small RNAs that mediate post-transcriptional regulation of endogenous messenger (m)RNAs . The biomiRNAs may also initiate regulatory cascades that have a massive effect on the regulation of gene expression, reinforcing miRNA activity and ensuring gene expression homeostasis of large gene families. These cascades involve secondary siRNAs that associate with AGO proteins, similarly to miRNAs. Secondary siRNAs arise predominantly from RNAs that are targeted and cleaved by 22-nt-  or, if cBegomovirus of the family Geminiviridae. Geminiviruses are circular single-stranded (ss)DNA viruses forming mini-chromosomes associated with cellular histones and are transcribed and replicated in the nucleus through dsDNA intermediates  the direct activity of viral silencing suppressors on endogenous sRNAs or on silencing related effectors ; (ii) thmediates . Geminivmediates  or with mediates . Geminivf plants . TYLCSV Nicotiana benthamiana possesses two functional NbAGO1-like paralogs. One 18-nt-long insertion/deletion (indel), which is located in the immediate vicinity of the miR168 target site, considerably affects the miR168-guided, post-transcriptional regulation of NbAGO1 mRNAs. The indel effect is underpinned under conditions of viral infection. Indeed, NbAGO1 homeologs are redundantly involved in their susceptibility to viral infection but are divergently involved in the phenotype associated with the infection , we found that geminivirus infection in tomato parallels previous observations in tombusvirus-N. benthamiana  that were exclusively revealed in virus-infected plants and identified their closest evolutionary paralogs. Concerning the capacity to reveal the miR168-mediated cleavage of a longer thamiana . SimilarS. lycopersicum L. cultivar “Moneymaker” were inoculated, as previously described, either by TYLCSV or by mock  were detected using a mixture of P32-labeled DNA antisense oligoprobes. Radiolabeled signals were observed by Storm 860 Molecular Imager.For Northern blot analysis, 10 μg of total RNA from either mock-inoculated or virus-infected leaf tissue were resolved on 12% PAGE gels. Once transferred to the positively charged nylon membrane Hybond N+ (Roche Diagnostics), miR171, and miR168 were detected as internal controls with oligoprobes, as previously described , whereas32-labeled probe generated by random priming on a CP-amplicon, as previously reported  and sequenced with standard sequencing oligos on the Illumina HiSeq 2500 platform. Differential expression of selected transcripts was confirmed by qRT-PCR .The poly-A fraction of total RNA extracted from leaf tissue was analyzed for the identification of target transcripts of conserved miRNAs. To generate miRNA-cleaved target libraries (RNA-end-dataset) from leaf tissue of either mock-inoculated or virus-infected, symptomatic tomato plants we applied the previously described high-throughput experimental approach that identifies mRNAs targeted by miRNAs, also known as “PARE” . PARE li1. mRNAs were aligned to the selected paralogous genes using PatMaN aligner with 0 mismatch (NC_003828.1) was performed as above and SAM output files were used as input in Geneious® software 8.1.7 (Biomatters Ltd.) in order to obtain a graphical representation of viral siRNAs along the viral genome.A quality check of the transcriptome was performed with FASTX toolkitmismatch . A raw cmismatch . sRNA-semismatch ] and virmismatch . miRNAs mismatch  with permismatch ]. AlignmRNA-end libraries underwent the same bioinformatics flowchart indicated above for sRNAs, with the only difference being the 20- to 21-nt filter size that was used before performing the alignment.sly-AGO1a and sly-AGO1b were discriminated based on the indels and SNPs revealed by Sanger sequence downstream of the cleavage target site. PARE datasets were aligned with the NCBI seq IDs of sly-AGO1a and sly-AGO1b using Bowtie with 0 mismatches. The SAM file output was used in Geneious® software 8.1.7 (Biomatters Ltd.) to obtain the graphical representation.5′-RACE was performed as previously described , except 2.cDNA targeted by sRNA were identified using PAREsnp software . miRNAs The selected targeted cDNAs from PAREsnp analysis were searched in the Solgenomics platform  and the The gene orthology and paralogy predictions were downloaded from Ensembl (Ensembl Plants release 34—December 2016) . Both PProtein domain predictions were obtained through the NCBI CD-Search  searchincleavage and translation types of regulation were studied.psRNATarget  was usedCentroid plain RNA secondary structure drawings and positional entropy were obtained using RNAfold with default parameters on the Vienna RNA website .p-value of 0.01. A subtraction step of common sRNAs of mock-inoculated from TYLCSV-inoculated plant tissue dataset was run on Galaxy website3. The TYLCSV-infected plant tissue dataset filtered from the previous step was used in a PaTmaN alignment with 0 mismatches using sequences selected from mRNAs as reference . Total RNA (500 ng) was retro-transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). All quantitative reverse-transcription PCR (qRT-PCR) assays were carried out using iTaq Universal SYBR Green Supermix  in an apparatus CFX Connect Real-Time PCR Detection System . The comparative threshold cycle method was employed to calculate relative expression levels using tomato UBC  as the reference gene . The lisFigure 1A and Supplementary Table S1). sRNAs not matching the tomato genome were used in order to retrieve v-siRNAs, i.e., 0.7 million redundant reads in the case of TYLCSV-infected plants. Traces of v-siRNAs found in mock-inoculated plants, i.e., 71 in total, were not considered reliable marks of viral infection: they are rather siRNAs in datasets that likely align randomly with 0 mismatches with the all viral reference sequence (Ref_Seq) dataset. In this case, we almost found a 0 level of redundancy , whereas v-siRNAs from full replicative viruses in infected tissues were characterized by a high level of redundancy due at least to the replication process and the production of secondary v-siRNAs . Southern blot hybridization on the total DNA extracted from plant tissues confirmed the infection and the active replication of the virus. Indeed, all TYLCSV replicative forms were detected only in virus-infected plant tissues , indicating an active TYLCSV infection in the tissues analyzed.Small (s)RNA datasets were obtained from the leaf tissue of mock-inoculated and TYLCSV-infected plants. Primary quality checks yielded a total of 17.8 million reads in the range of 18–34-nt . In addition, 64.6 and 71.9% of the reads aligning to the S. lycopersicum genomic sequence  also aligned to the S. lycopersicum mRNA dataset .PARE datasets were obtained from the same RNA preparations used for the sRNA libraries in line with previous reports . RNA remFigure 1A). Some of the miRNAs analyzed were up-regulated in the presence of viral infection, while others were down-regulated in line with previous observations in Geminivirus-infected plants (Supplementary Tables S1–S3)  .Table 1). We also interrogated psRNATarget .The PARE datasets allowed us to assess the miRNA-mediated regulation of genes belonging to gene families coding for transcriptional factors and disease-resistance proteins, which are known to be altered under biotic stresses . TherefoNATarget  and annoSolyc05g008070 (Table 1). Worthy of note, Solyc05g008070 has been previously described as targeted by miR482 (Supplementary Table S3), although PARE analysis never found 5′ remnants explained by miR482 for this gene family.Regarding NBS-LRRs targeted by miR6024, one gene belonging to this large family was specifically cleaved only in the virus-infected tissues, i.e., y miR482  and, indsly-AGO1a and sly-AGO1b and found that both transcripts can be cleaved at the same position . Cleaved sly-AGO1b could only be found in the case of TYLCSV-infected plants  and not in mock-inoculated plants. It is noteworthy that, the “A” at the 3′-end of sly-miR168a-5p v2 ensures pairing with the target site in both sly-AGO1a and sly-AGO1b transcripts .As a proof of concept, we extended the analysis also to mRNAs coding for AGO1. We performed a 5′-RACE analysis of miR168-mediated cleavage of transcripts from sly-AGO1a and sly-AGO1b by PARE analysis confirmed what was found with 5′-RACE. Figure 2B shows the distributions of 5′-remnants found in mock-inoculated and in virus-infected plants along transcripts of either sly-AGO1a or sly-AGO1b. The degradation patterns of the two transcripts revealed a peak at the cleavage position, which was absent in the case of sly-AGO1b in mock-inoculated plants . The two sly-AGO1 mRNAs differ in size: sly-AGO1a is 3,546 bp vs. 3,930 bp of sly-AGO1b; while the main difference is located at the 5′-terminal part of the coding sequence .The high-throughput analysis of Sly-AGO1a and sly-AGO1b, which differ by one 21-nt-long indel immediately downstream of the miR168 target site, have been previously highlighted in S. lycopersicum , and aligned their transcript sequences in pairs. This was carried out for all the gene families in Table 1, except for the HAP genes since the closest tomato paralog to HAP Solyc03g121940  is not annotated in the region of the miR169 recognition site .miRNA/target RNA recognition is a critical feature for miRNA functionality, target cleavage occurrence, and regulation of gene expression. persicum  and herely-AGO1a and sly-AGO1b confirmed the presence of a 21-nt-long indel, 9-nt downstream of the miR168 target site ; (ii) a 39-nt-long indel 48 nt upstream the miR160 cleavage site for ARFs ; (iii) a 3-nt-long indel, 113 nt upstream the miR166 recognition site in the case of HD-ZIPIIIs ; and (iv) a 12-nt-long indel, 24 nt downstream the miR172 recognition site in AP/RAP .SSolyc05g008070  to Solyc07g049700  disclosed a 15-nt-long indel, 123 nucleotides upstream of the miR6024 recognition site .Finally, alignment of Figure 4). We, therefore, set up transcriptome quantification via mRNA-seq, of selected target mRNAs, to confirm the presence/absence of miRNA targeting in the mock and infected plant tissues. All paralogs with no indels were significantly more expressed than those having indels in either mock or virus-infected tissues . When comparing the expression of each couple of paralogs in virus-infected vs. mock-inoculated plant tissues we found significant variations only in the case of sly-AGO1b and Solyc05g008070; the latter was down-regulated in virus-infected plant tissues . Thus, we highlighted a possible correlation between the cleavage event (Table 1), the indel , and the down-regulation  in the case of the NBS–LRR transcript Solyc05g008070.PARE analysis can verify target cleavage events, although it is not sufficient to exclude miRNA-mediated regulation of paralogs that were not found cleaved , and the artificial insertion of the indel in the latter strongly affected the structure . We also annotated the location of miRNA target sites in the gene structures with respect to the functional protein domains . Therefore, the functional indels described could impact secondary structures of the transcript rather than protein domains (see later in “Discussion”).When predicting the secondary structure of the stretches of sequence in Supplementary Table S2) that explained cleavage events in our PARE analysis (Table 1). We thus restricted the analysis to HD-ZIPIII, HAP, AP/RAP, and NBS–LRR transcripts cleaved by miR166, miR169, miR172, and miR6024, respectively. Given that AGO1 mRNA is also known in Arabidopsis and tomato to have secondary siRNAs initiated by miR168 . None or trifling reads mapped to Solyc02g024070 and Solyc11g069470 transcripts of the HD-ZIPIII clade, respectively. A modest number of siRNAs mapped to either sly-AGO1a or sly-AGO1b transcripts in agreement with a previous report . Within the selected mRNAs in Table 2, we identified only two cases of phased register with respect to the miRNA target site: miR482/Solyc02g036270  and miR6024/Solyc05g008070 . Surprisingly, phased siRNAs from Solyc02g036270 were only present in mock-inoculated plant tissue datasets and not in the case of virus-infected tissues . Phased siRNAs from Solyc05g008070 were detected only in TYLCSV-infected plant tissues where miR6024 was active , despite the fact that the majority of secondary siRNAs from the two NBS–LRR transcripts were not in phase . Interestingly, we found that the miR6024 target site overlapped to some extent with that of miR482 , which is not functional according to PARE analysis.We aligned the siRNA datasets uniquely from virus-infected plant tissues against selected mRNAs; the output is reported in 2g036270 , i.e., 1s report . A similTable 2. Functional siRNAs of 21 and 22 nt are detailed in Supplementary Tables S4, S5, respectively, and are summarized in Table 2. Among functional secondary siRNAs in TYLCS-infected plant tissues deriving from Solyc02g036270, we could find 22 and 15 unique functional siRNAs of 21 and 22 nt in length, respectively. Instead, one phased, seventeen 21-nt-long un-phased and thirteen 22-nt-long non-redundant functional siRNAs were associated with Solyc05g008070 .Secondary siRNAs including those in a phased register are known to be functional upon incorporation into RISC complex . Being PFigure 6C  derived from a highly statistically significant  phased locus starting at position 555 of Solyc05g008070, which corresponds to the miR6024 cleavage site . siRNA 8070_742 explained the cleavage of Solyc03g083250 coding for a putative ATP-dependent CIp protease.The 21-nt-long siRNA indicated as siRNA 8070_742 in Supplementary Tables S4, S5).At least one secondary siRNA deriving from NBS–LRR genes has been shown to target other mRNAs of defense-related proteins ; this waFigure 7A, we report eight secondary siRNAs from Solyc05g008070 originated downstream of the miR6024 cleavage site uniquely present in virus-infected plant tissues that target and cleave core members of photosystem I and II, chlorophyll-binding proteins, and ribulose bisphosphate carboxylases (RuBiSCO). Conventional and specific Sanger sequenced 5′-RACE were also performed on the library besides the high-throughput sequencing. Validation by Northern blot analysis revealed the expression of the secondary functional siRNAs only in the case of RNAs from TYLCSV-inoculated plant tissues showing typical yellowing symptoms . No secondary functional siRNAs were detected in either mock-inoculated  or TYLCSV-infected plant tissues not-showing yellowing symptoms . miR168 and miR171, used as a control of the Northern analysis, were detected in all the samples under investigation . Finally, DESeq analysis resulted in down-regulation of all targeted transcripts in TYLCSV-infected plant tissues. For those transcripts that could be investigated by qRT-PCR, a significant 1.5- to 2-fold down-regulation in virus-infected vs. mock-inoculated plant tissues, thus confirming the functionality of these siRNA cleaving targets solely associated with virus infection .Worthy of note, 11 out of 42 targeted mRNAs were key players of plant photosynthetic machinery. In Plant viruses are infectious entities that alter gene expression and are therefore recognized as “phenotype extenders.” For instance, it has been shown that virus infection can enhance plants’ attractiveness to pollinators  as well S. lycopersicum possesses two AGO1 homologs named sly-AGO1a and sly-AGO1b, differing for 21-nt-long indels just downstream of the miR168 target site. Sly-AGO1b (the transcript containing the insertion) is cleaved in TYLCSV-infected plants, whereas it is not in mock-inoculated plants. Consistent data came from mRNA-seq analysis since the sly-AGO1b appeared significantly up-regulated in virus-infected tissues, which is in agreement with our previous observations (sly-SPB6b and sly-SPB13), one ARF gene cleaved by miR160 (Sl-ARF16A), two HD-ZIPIII genes cleaved by miR166 (Solyc02g024070 and Solyc11g069470), one AP transcription factor cleaved by miR172 (Solyc09g007260), and one NBS–LRR disease-resistance protein-coding gene cleaved by miR6024 (Solyc05g008070). We show that 12-, 15-, 3-, and 39-nt-long single indels are in close vicinity to the target sites of miR172, miR6024, miR166, and miR160, respectively. Nevertheless, in all these cases, the indels discriminate between cleavages of close paralogs: the version containing the insertion was never cleaved, whereas the other was found cleaved in TYLCSV-infected plant tissues. All paralogs with no indels were significantly more expressed than those having indels in either virus-infected or mock plant tissue. A significant differential expression of paralogs in mock-inoculated tissues suggests fine and independent gene regulation that renders unclear the interpretation of the role of indels in miRNA-dependent regulation. However, Solyc05g008070 lacking one 15-nt-long indel, 123 nucleotides upstream of the miR6024 recognition site, is cleaved and is down-regulated solely in virus-infected plant tissues. All the analyzed genes are homologs, thus they derive from a whole genome duplication and belong to different subgenomes  and the mRNA decay causes the production of secondary siRNAs that in turn down-regulate other mRNAs of disease-resistance genes: this has been proposed as a novel pathogen-inducible layer of plant defense  genes could be directly implicated in symptom development through secondary cascade pathways. Indeed, one NBS–LRR transcript releases secondary siRNAs that target transcripts coding for RuBISCO, chlorophyll a/b binding protein 6A, and photosystem II reaction center W protein. These are significantly down-expressed in infected tissues at least 1.5-fold. In addition, Northern blot analysis unveiled the expression of these secondary sRNAs exclusively in plant virus-infected tissues showing yellowing. Thus, here we reveal a novel mechanism that could be added to earlier findings in virus–host interaction. Leaf chlorosis is often seen in plants infected with viruses and viroids and is known to be associated with a reduced content of chlorophyll in leaf cells . As chloThe understanding of miRNA biology is still limited by our poor appreciation of their cell-type specific and spatio-temporal regulation. Many miRNAs exhibit discrete expression patterns and their regulation depends largely on the availability of particular AGO proteins and distinct target mRNAs within a given cell/tissue type. Next-generation sequence approaches to facilitate the simultaneous analysis of large gene sets but they typically provide a very limited spatio-temporal resolution of gene expression changes. Either in plants or animals , miRNA eThe sRNA and PARE datasets have been deposited in GEO Omnibus under the entry code GSE93648. The DESeq analysis of selected paralogs was carried out using alignments deposited in SRA under the entry code SRP133920.(To review GEO accession GSE93648:https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93648Go to Enter token onsrwgugbxmpnwd into the box)https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP133920).(To Access to metadata about SRA submission VP designed the experiments and prepared PARE and sRNA datasets. LM prepared total RNA from tomato plants and performed quantification analysis. MC, CC, WP, and VP analyzed the data. MC, CC, and VP prepared the manuscript. All authors discussed the results and contributed to editing the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.