Cotton leaf curl Kokhran virus that lacks a full complement of a gene encoding a transcription activator protein (TrAP). In order to understand the ongoing changes in CLCuD complex in Pakistan, CLCuD affected plants from cotton fields at Vehari were collected. Illumina sequencing was used to assess the diversity of CLCuD complex. At least three distinct begomoviruses characterized from the first epidemic; Cotton leaf curl Multan virus, Cotton leaf curl Kokhran virus and Cotton leaf curl Alabad virus, several distinct species of alphasatellites and cotton leaf curl Multan betasatellite were found associated with CLCuD. These viruses were also cloned and sequenced through Sanger sequencing to confirm the identity of the begomoviruses and that all clones possessed a full complement of the TrAP gene. A new strain of betasatellite was identified here and named CLCuMuBVeh. The implications of these findings in efforts to control CLCuD are discussed.The first epidemic of cotton leaf curl disease (CLCuD) in early 1990’s in the Indian subcontinent was associated with several distinct begomoviruses along with a disease-specific betasatellite. Resistant cotton varieties were introduced in late 1990’s but soon resistance was broken and was associated with a single recombinant begomovirus named Burewala strain of  Cotton leaf curl disease (CLCuD) is the major biotic constraint of cotton, transmitted by the insect vector Bemisia tabaci (whitefly)3. CLCuD affects cotton crops across Pakistan and northwestern India causing severe yield losses4. Cotton plants affected by CLCuD exhibit specific symptoms such as upward and downward leaf curling, vein thickening and swelling, stunted growth and development of leaf-like enations. In Pakistan, CLCuD was observed for the first time on cotton near Multan in 1967. The disease spread widely and some more cotton varieties were affected by late 1970, but were not considered significant5. In 1989, the disease was noted on a newly released variety S12 grown at Kokhran near Multan and became epidemic, spreading to all the growing areas of Pakistan. The first epidemic of the disease was associated with the “Multan strain” of begomoviruses6. By introduction of resistant varieties developed through conventional breeding, cotton production in Pakistan was restored to pre-epidemic levels in the late 1990s7. Unfortunately, in 2001 it became evident that resistance was broken and symptoms of CLCuD were observed in all previously resistant varieties at Burewala, Pakistan8. This signaled a second epidemic of CLCuD which spread to all the cotton growing areas of Pakistan9.Cotton is the largest fiber producing and important cash crop in Pakistan and IndiaBegomovirus, family Geminiviridae) transmitted by B. tabaci10. Begomoviruses are of two types, bipartite and monopartite; bipartite consists of two genomic components DNA-A and DNA-B equal in size (~2.7 kb). Monopartite begomoviruses have a single genomic component similar to DNA-A of bipartite begomoviruses and are often associated with satellite molecules called betasatellite and alphasatellite. Betasatellite (~1.4 kb in size) is a pathogenicity determinant, encodes a single βC1 protein and depends on the helper virus for movement, replication and encapsidation11. Alphasatellites (~1.4 kb in size) encode their own replication associated (Rep) protein, replicate independently from their helper virus and are usually not required for pathogenicity11.The CLCuD complex is caused by monopartite begomoviruses (genus Cotton Leaf curl Multan virus (CLCuMuV), Cotton leaf curl Kokhran virus (CLCuKoV), Cotton leaf curl Alabad virus (CLCuAV) and Papaya leaf curl virus (PaLCuV)12, and a single betasatellite (Cotton leaf curl Multan betasatellite [CLCuMuB])14. Following the appearance of the second CLCuD epidemic in cotton, to present time, a predominant single recombinant begomovirus named as Cotton leaf curl Kokhran virus- Burewala (CLCuKoV-Bu), previously known as Cotton leaf curl Burewala virus (CLCuBuV), was associated with CLCuD in Pakistan and India9. However, the occasional occurrence of four other virus species and strains  was also observed on cotton in Pakistan  and a mutated C2 protein of only 35 amino acids (aa)9. Recently CLCuKoV-Bu, with a full complement TrAP, has also been identified showing severe symptoms of CLCuD16. The betasatellite associated with CLCuKoV-Bu was also found to be recombinant with most of its sequence from CLCuMuB, but also containing a small fragment of SCR region derived from tomato leaf curl betasatellite16.During the first epidemic, CLCuD was caused by begomovirus-betasatellite complex. CLCuD was associated with several distinct monopartite begomoviruses – an Table . CLCuKoV17. Recently a bipartite begomovirus, Tomato leaf curl New Delhi virus (ToLCNDV) was identified associated with CLCuD in Pakistan19. Interestingly, ToLCNDV isolated from cotton maintained a full complement of TrAP. Another study identified Chickpea chlorotic dwarf virus (CpCDV), a Mastrevirus in cotton showing leaf curl virus disease symptoms20. These results suggest that CLCuD complex has captured viruses that may have contributed to complete breakdown of resistance.The CLCuD complex is in a state of continuous change, evolving rapidly to overcome resistance by component capture, recombination and mutationHere we have characterized begomoviruses and associated satellites from symptomatic samples of cotton collected in Vehari from lines that were being screened for virus resistance. In this study, we tried to understand the evolving nature and recent changes in begomovirus disease complexes by using rolling circle amplification (RCA) followed by next generation sequencing (NGS) and Sanger sequencing. Based on NGS and Sanger sequencing data, three distinct begomoviruses  characterized from the first epidemic and several distinct alphasatellites and a single betasatellite species were identified associated with CLCuD. We also performed Southern blot hybridization for semi-quantification of begomoviruses and betasatellites. An important feature of the complex found in recent samples from Vehari is the absence of CLCuKoV-Bu in recent samples. Implications of these findings on begomovirus disease complexes are discussed.21, followed by ethanol precipitation and DNA quantification. To amplify circular molecules RCA22 was performed using phi 29 DNA polymerase . RCA product was purified, enriched and processed for NGS.Cotton leaf samples from a total of six lines showing the typical CLCuD disease symptoms were collected from the Cotton Research Station (CRS) Vehari  in July 2015  was used with library kit, Illumina #NP-101-1001, “TruSeq Nano DNA Library Kit for NeoPrep”, which includes the adapter set “TruSeq LT”. The target insert size was 350 bp, with size selection performed by the NeoPrep instrument. Actual lower size limit of the libraries was ~300 bp as measured by the Agilent 2200 TapeStation. Sequencing was performed on the Illumina MiSeq, v2 chemistry, 2 × 150 bp.De novo as well as reference-guided assemblies were made. Reference-guided mapping was performed using begomovirus and satellite sequences present in Genbank. Based on good quality of data, twelve sequences from six plant samples  were selected as shown in Table The MiSeq Reporter software was set to automatically trim the adaptors. These short sequences were processed using CLC Genomics Work Bench 7.5. The paired-end reads obtained from the Illumina MiSeq Sequencer pipeline were subjected to quality filtering using quality score 0.001 and Phred quality score of 30. 23, Beta01/Beta0224 and DNA 101/DNA 10225 were used. For amplification of NGS-assembled CLCuAlV new sets of primers were designed  v1.228. RDP using 9 recombination analysis methods, RDP29, GENECONV30, BOOTSCAN31, MAXIMUM CHI SQUARE32, CHIMAERA33, SISCAN34, 3SEQ35, PHYLPRO36, LARD37 and VisRD38 for possible recombination breakpoints within query sequences. Recombination events identified by multiple recombination methods with high p-values and good phylogeny were considered potential targets for recombination.Recombination among viruses and satellites were detected using RDP4 Beta 4.74+ membrane  and UV cross-linked. The blot was pre-hybridized at 42 °C for 2–3 h in DIG Easy Hyb solution  and hybridized overnight at 42 °C in DIG-labelled probe. For begomoviruses, primers CLCV1/CLCV2. (5′-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3′/5′-CTGCCACAACCATGGATTCACGCACAGGG-3′) and for betasatellites Beta01/Beta02 (5′-GTACCGGCTGCTGCGTAGCGTAGT-3′/5′-GGTACCTACCCTCCCAGGGGTACAC-3′) primers were used for synthesis of probes. Genomic DNA of infected cotton plants was used as insert to amplify DIG labeled probes. Hybridization signals were detected on blot after treatment with Nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate .For Southern blot hybridization an agarose gel (1.2% [w/v]) stained in ethidium bromide was cast in a tray and an equal amount of DNA (~10 µg) per sample was resolved on the gel. After washing in depurination (0.25 M HCl), denaturation (1.5 M NaCl and 0.5 M NaOH) and neutralization buffer [1 M Tris (pH 7.4) & 1.5 M NaOH] the gel was blotted on Hybond-Nde novo assembly of the high-throughput Illumina sequencing data was carried out on CLC Genomics Work bench 7.5 (CLC bio) software. Resulting contigs of size ~2.8 kb and 1.4 kb, based on putative genome size of begomoviruses and alpha/betasatellites respectively, were selected from the data. The selected contigs were searched by using the BLASTn search tool for closely related begomoviruses and satellites molecules available at NCBI-GenBank database. From the NGS data, BLASTn indicated three kinds of begomoviruses, Cotton leaf curl Multan virus-Pakistan (CLCuMuV-PK), Cotton leaf curl Alabad virus-Multan (CLCuAlV-Mu) and cotton leaf curl Kokhran virus-Kokhran (CLCuKoV-Ko) were present. However, CLCuKoV-Ko sequences identified here with NGS have low percent (50–60%) coverage as shown in Table The Gossypium darwinii symptomless alphasatellite (GDarSLA), Gossypium davidsonii symptomless alphasatellite (GDavSLA) and cotton leaf curl Burewala alphasatellite (CLCuBuA) and one type of betasatellite, cotton leaf curl Multan betasatellite (CLCuMuB) in the collected infected samples . The ORF analysis shows that 2.8 kb clones resembled and had an arrangement of genes typical of the DNA-A component of begomoviruses (Table Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Raj) and one strain of CLCuKoV, Cotton leaf curl Kokhran virus-Shadadpur (CLCuKoV-Sha) were identified (Table The 2.8 kb and 1.4 kb clones obtained here were checked for the presence of putative open reading frames (ORF) using ORF finder at , Gossypium davidsonii symptomless alphasatellite (GDavSLA) and cotton leaf curl Burewala alphasatellite (CLCuBuA) and one type betasatellite, cotton leaf curl Multan betasatellite (CLCuMuB) in the collected infected samples  in the coat protein and Tomato leaf curl Palampur virus  in Rep in the complementary sense.Recombination analysis of CLCuMuV-Raj isolates show that CLCuMuV-Raj is a recombinant of two viruses CLCuMuV and CLCuKoV-Ko as reported previouslyA phylogenetic dendrogram of begomoviruses was constructed on the basis of pairwise multiple MUSCLE alignment (MEGA6) of CLCuMuV, CLCuMuV-Raj and CLCuKoV-Sha and their similar isolates available in the database. The phylogenetic tree of CLCuMuV, CLCuMuV-Raj and CLCuKoV-Sha isolates shows that they are separated with closely related begomoviruses interspersed with the three begomoviruses as shown in Fig. Three types of alphasatellites GDarSLA, CLCuBuA and AConSLA were identified from Sanger sequencing data. The phylogenetic tree of alphasatellites indicated that they clustered with closely related GDarSLA, CLCuBuA and AConSLA as shown in Fig. et al.41, and known as Multan betasatellite (CLCuMuBMul). The second group consisted of a single clone (MZ-35) and is closely related to recombinant CLCuMuB associated with CLCuBuV known as (CLCuMuBBur). The clone (MZ-35) contains approximately 95 nt derived from Tomato leaf curl betasatellite (ToLCuB) within the SCR region42. The third group consisted of two clones  and their sequence in SCR does not match either CLCuMuBMul or ToLCuB. Instead they contain approximately 90 nt within the SCR derived from cotton leaf curl virus betasatellite defective interfering DNA (KT228331), recently identified from India, and here we designate it as Vehari betasatellite (CLCuMuBVeh). Phylogenetic trees of full length betasatellites and their SCR regions is shown in Fig. A single type of betasatellite, CLCuMuB was obtained from Sanger sequencing data. Analysis of the SCR region of these betasatellites showed that three types of betasatellites separated into three groups as shown in Fig. lone MZ-3 and is cTo determine relative titer of begomoviruses and betasatellites, Southern blot hybridization of the infected cultivated cotton samples was performed. Southern blot analysis of DNA samples extracted from symptomatic leaves showed weak hybridization signals for virus probe in sample 1 and 2 indicating that virus titer was low in these samples, while virus titer was high in samples 3, 4, 5 and 6 showing good hybridization signals for virus probe as shown in Fig. . The tit43. We have found that some recent isolates of CLCuKoV-Bu encode a full length TrAP . Our current study shows that the CLCuD complex can occur as result of infection of multiple begomoviruses. The begomovirus disease complex can easily evolve due to component capture, recombination and mutation in order to overcome disease resistance and to expand its host range44.The begomovirus disease complex can severely affect yields of cultivated cotton in Pakistan and India, and previously often occurred as an infection of multiple begomoviruses. The CLCuKoV-Bu strain, with a truncated TrAP, is the only begomovirus strain associated with the resistance breaking of CLCuD currently found throughout Pakistan, with the occasional identification of other strains Table 9, 43. WeTo understand ongoing changes in the CLCuD complex, symptomatic leaves of previously resistant cotton varieties were collected from Vehari. Vehari is a major cotton growing area of Pakistan and exhibits a high diversity of begomoviruses. To evaluate the samples collected, a highly sensitive method of RCA coupled with NGS was used followed by confirmation with Sanger sequencing. Our NGS data showed that the three distinct begomoviruses, CLCuMuV, CLCuAlV and CLCuKoV-Ko associated with the first epidemic of CLCuD in early 1990s, a betasatellite, CLCuMuB and 3 alphasatellites GDarSLA, GDavSLA and CLCuBuA were identified in infected samples from Vehari. The reason for the low coverage (50–60%) of CLCuKoV-Ko in NGS data is not known. To confirm our NGS data results, genomic DNA from the collected samples were amplified with universal begomo, alpha and beta primers, cloned and dideoxy nucleotide chain termination sequencing was performed. From the Sanger sequencing data, two strains of Multan, CLCuMuV-Mu, CLCuMuV-Raj and one strain of Kokhran, CLCuKoV-Sha associated with the first epidemic of CLCuD, were identified. Our results from both NGS and Sanger sequencing data shows that CLCuKoV-Bu was not identified in infected cotton samples from Vehari. The presence of previously identified begomoviruses and the absence of CLCuKoV-Bu which was the only begomovirus found in infected cotton plants, indicate a potential major change in the begomovirus complex associated with CLCuD in Pakistan. A single species of betasatellite (CLCuMuB) and three species of alphasatellites  were identified in these samples. Sanger sequencing data complementing our NGS data, identified and confirmed the presence of CLCuMuV, CLCuMuV-Raj and CLCuKoV-Sha. However, CLCuAlV identified with NGS was not amplified with universal begomo primers. The CLCuAlV was amplified from infected samples with CLCuAlV specific primers designed according to the assembled sequences of NGS Table .14. TrAP encoded by begomoviruses is a multi-functional protein, functioning as a pathogenicity determinant44 and also a strong suppressor of post transcriptional gene silencing activity (PTGS) of the host45. Full length and truncated TrAP both exhibited PTGS activity but truncated ones have lower PTGS activity as compared to full length TrAP46.A closer detailed analysis of the TrAP gene of CLCuMuV, CLCuMuV-Raj and CLCuKoV-Sha isolates identified here encode a full length TrAP protein of 150 aa. The CLCuKoV-Bu which is the dominant strain associated with CLCuD from 2000 onward in cultivated cotton, encodes a truncated C2 protein of 35 amino acids (aa)48. The recombination analysis shows that diversification of begomoviruses identified here occurs as a result of recombination between CLCuMuV and CLCuKoV. Most of begomoviruses identified here are recombinant between CLCuMuV and CLCuKoV exchanged as either virion-sense or complementary-sense genes but some isolates of CLCuMuV contain fragments of PeLCV and ToLCPalV in virion and complementary sense. Three types of betasatellites, replicating with begomoviruses, were identified from infected samples. A new type of recombinant betasatellite named as CLCuMuBVeh, has a recombinant region within the SCR region different from the previously identified CLCuMuBMul and recombinant CLCuMuBBur.Recombination is a major process of evolution in begomoviruses51.The emergence of viruses associated with the first epidemic of CLCuD in cultivated cotton indicate that the begomovirus complex may be changing to resemble the original CLCuD complex found during the first epidemic of the 1990s before the introduction of resistant varieties derived from LRA5166 and CP15/2 as a source of resistance. The arrival CLCuMuV, CLCuAlV, and CLCuKoV back into cultivated cotton Table  is a sigSupplementary Dataset 1


Bombus terrestris, and whether the presence of possible functional viral suppressors could alter the potency of the host’s immune response. For this, a two-fold approach was used. Through a functional RNAi assay, we observed an enhancement of the RNAi system after IAPV infection instead of its suppression, despite only minimal upregulation of the genes involved in RNAi. Besides, the presence of the proposed suppressor 1A and the predicted OrfX protein in IAPV could not be confirmed using high definition mass spectrometry. In parallel, when bumblebees were infected with cricket paralysis virus (CrPV), known to encode a suppressor of RNAi, no increase in RNAi efficiency was seen. For both viruses, pre-infection with the one virus lead to a decreased replication of the other virus, indicating a major effect of competition. These results are compelling in the context of Dicistroviridae in multi-virus/multi-host networks as the effect of a viral infection on the RNAi machinery may influence subsequent virus infections.RNA interference (RNAi) is the primary antiviral defense system in insects and its importance for pollinator health is indisputable. In this work, we examined the effect of Israeli acute paralysis virus (IAPV) infection on the RNAi process in the bumblebee,  No domain of life is exempt from the threat of viruses , which hDicistroviridae, a family of positive single-stranded RNA viruses infecting arthropods, a 166 amino acids (AA) long protein, called 1A, has been proven to be a functional VSR in both the drosophila C virus [Dicistroviridae, Israeli acute paralysis virus (IAPV). The proof of its functionality was based on reduced virus titers after silencing the 1A region, compared to targeting the non-coding 5’ internal ribosome entry site (IRES) region [orfX (or pog), has been predicted in IAPV and its close relatives but not in CrPV [Solenopsis invicta virus 1 (SINV-1) [During evolution, viruses have not stood by idly while their hosts developed this RNAi defense mechanism. Viruses known to encode viral suppressors of RNAi (VSRs) have been found in plants  and inse C virus  and the  C virus . Recentl) region , indicat in CrPV ,21, and  in CrPV  but not (SINV-1) . As of yBombus terrestris. This virus is known to pose an important health danger to pollinators such as honeybees and bumblebees [Drosophila, was included. CrPV has not been reported as a problematic infection of the pollinator community and has a broad host range as it was reported to infect species within the insect orders of Heteroptera, Diptera, Lepidoptera and Hymenoptera, at least in experimental conditions [The aim of this study was to investigate to what extent IAPV can influence RNAi efficiency, and to which direction this balance will sway, in the bumblebee mblebees ,25 and imblebees . As an enditions ,28,29. Wnditions , the prenditions , an imponditions ,33.B. terrestris, we used a dual approach. First, a proteomic analysis was performed to confirm the translation of the VSR 1A and predicted OrfX proteins using the data-independent acquisition method with high definition mass spectrometry (HDMSE). Second, the RNAi efficiency after IAPV and CrPV infection was determined. An assay was developed in which bumblebees were infected with a fixed amount of viral particles and after an incubation period injected with dsRNA targeting peptidylprolyl isomerase A (ppia), a gene known to remain stable during virus infection [dcr-2 and ago-2, and the systemic RNAi genes, ninaC, egghead and sid-1 [In order to evaluate the effect of IAPV infection on RNAi efficiency in nfection . Silencind sid-1 .Bombus terrestris colonies . Several workers were collected from the colonies and verified to be virus-free by PCR [w/v, BIOGLUC®; Biobest NV) and gamma-irradiated pollen . Prior to injections with virus- or dsRNA-containing solutions, the bumblebees were anaesthetized by placing them on ice in a plastic container for 5 min. The bumblebees were then injected through the abdominal intersegment membrane between the second and third segment using an Femtojet Microinjector . A volume of 5 µL was used for all virus solutions (and the appropriate controls), while dsRNA was injected in a volume of 20 µL.All experiments were performed using 5-to-10 day old workers, age fixed within each experiment, collected from e by PCR . One or g and 4 h at 100,000× g (4 °C). The resulting pellet was resuspended in 6 mL demineralized water. Subsequent dilutions were in phosphate buffered saline (PBS). Contamination of other common bumblebee viruses such as acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), slow bee paralysis virus (SBPV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Varroa destructor virus-1, Sacbrood virus and black queen cell virus, was determined to be less than 0.1% of the IAPV level by RT-qPCR [IAPV was produced by injecting 160 virus-free bumblebees with 500 IAPV particles and waiting three days for the virus to amplify within the body. Then the bumblebees were crushed in 10 mM phosphate buffer (pH 7.0) supplemented with 0.02% diethyldithiocarbamate. The resulting suspension was centrifuged for 15′ at 800×  RT-qPCR  and the Drosophila melanogaster X virus, Drosophila melanogaster American nodavirus, Drosophila melanogaster totivirus and Drosophila melanogaster birnavirus [6 particles/µL) was used to infect 40.106 S2 cells and 15 h later the resulting viral particles were obtained by repeated freezing and thawing in a ultrafreezer (−70 °C) and subsequently applied to 300.106 cells. 15 h later the resulting viral suspension was cleared of cell debris by centrifuging twice: 15′ at 800× g, followed by 30′ at 20,000× g. The viral suspension was cleaned further by ultracentrifuging 2 h, at 4 °C, at 100,000× g in a 15% sucrose gradient. The pellet was collected, resuspended in PBS and tested negative for IAPV, ABPV, KBV, DWV and SBPV using PCR [CrPV was produced within the Schneider-2 (S2) cell line, which was tested to be negative for flock house virus, rnavirus . 40 µL osing PCR .For both viruses, the concentration of viral particles was estimated by transmission electron microscopy . Briefly, Alcian blue-treated grids were deposited on a 15 μL drop of solution for 10 min and rinsed two times with water. Afterwards, the grids were stained for 10 s on a drop of 2% uranyl acetate , blotted and air-dried. The samples were imaged in bright field (BF) mode using a Tecnai Spirit TEM  with Biotwin lens configuration operating at 120 kV. Five micrographs were recorded per sample using a 4K x 4K CCD camera  at a magnification of 30,000 times.The dsRNA was prepared using the MEGAscript RNAi Kit  according to the manufacturer’s specifications. The T7-DNA products used as dsRNA template were generated during a PCR reaction  using cDNA of bumblebee workers and the appropriate T7-primers . For pre60S ribosomal protein L23 (rpl23) and polyubiquitin B (ubi) were used for normalization of the data [+ software . Reference gene stability was evaluated using the geNorm M value and the coefficient of variation on the normalized relative quantities (CV) values generated by the software. The thresholds for the M and CV values were set at 0.5 and 0.2 respectively for within-tissue comparisons and 1 and 0.5 for between-tissues comparisons [RNA was isolated using the RNeasy Mini Kit  according to the manufacturer’s instructions and treated with the Turbo DNA-free kit (Life Technologies). For cDNA synthesis, 500 ng RNA was used in each reaction, performed with the SuperScript II Reverse Transcriptase Kit (Life Technologies). The RT-qPCR reactions were performed in duplicate in a C1000 Touch Thermal Cycler  using the GoTaq qPCR Master Mix . Each reaction was composed of 1 μL of each primer (10 μM), 10 μL of GoTaq Master Mix and 8 μL of cDNA (diluted 1:40 or 1:100). The amplification conditions were 95 °C for 5 min followed by 40 cycles of 95 °C for 30 s, and 60 °C for 60 s. As reference genes the data . Amplifiparisons . An overparisons  or desigparisons .ppia was chosen as it remains stable in the presence of IAPV [ppia level, were injected with 5 µL PBS and 24 h later with 20 µg dsGFP (baseline control group) [6 CrPV particles, followed by an injection of 20 µg dsPPIA (virus treatment group). Because of a slower replication of CrPV the time between virus infection and dsRNA treatment was extended to 48 h and not longer to limit the effect of a bumblebee age difference. This way, there is an equal time between the end of the experiment and the onset of death for both viruses. Each group consisted of 7–9 individuals (n). Within one group, all bumblebees were placed in the same microcolony. This institutes a potential problem of pseudoreplication, however, the stable climatic conditions and ad libitum feeding limit the risk of microcolony effects. Moreover, the chosen reporter gene ppia is very stable , thus, any large effects are very unlikely to be the result of pseudoreplication. Brain, fat body, midgut and ovaries were dissected 48 h after dsRNA treatment and stored separately in 350 µL RLT buffer. In order to assess the expression levels of the RNAi genes 24 h after IAPV treatment, a separate experiment was set up (n = 8–10). For the pre-infection experiments, similar timelines were used as in the RNAi efficiency assays. In a first experiment, injection with 500 particles of IAPV was followed by injection of 106 particles of CrPV 24 h later (n = 9–10), in the other 106 particles of CrPV were injected and 48 h later, 500 particles of IAPV (n = 11–15). Viral titers were analyzed using RT-qPCR 48 h after the second virus infection.In order to test the effect of viral (IAPV or CrPV) presence on the RNAi efficiency, an assay was developed, containing three treatments. As a reporter gene, the endogenous  of IAPV . Bumblebl group) ,40. A se2 transformed as advised in [t-test testing.All statistical analyses of RT-qPCR data were performed within SPSS . For experiments that evaluated viral titers, non-parametric methods were used . In the functional RNAi assay and the evaluation of the RNAi gene levels, the data was logvised in  after whv/v) and acetonitrile , in the presence of Halt Protease and Phosphatase Inhibitor Cocktail  and 100 U of benzonase nuclease . The sample was sonicated on ice and centrifuged. The protein concentration was determined using the Bradford assay and 2.5 µg of protein was reduced in 0.5 M TEABC by adding 1 µL reducing agent and incubating for 30 min at 60 °C, followed by alkylation using a 10 mM methyl methanethiosulfonate for 10 min at room temperature (RT). The samples were digested in 1 mM CaCl2 by adding trypsin/lysC . They were placed overnight at 37 °C and evaporated, after which the sample was resuspended in 0.1% formic acid (FA). 100 ng of peptide sample, spiked with 25 fmol Hi3 standard and 25 fmol MSPD standard, was injected of each sample.Fifteen bumblebees were injected with 500 IAPV particles and the ovaries were dissected after three days at standard rearing conditions. However, the workers were not given pollen as to limit the development of the ovaries. The ovaries were pooled per three individuals and crushed in liquid nitrogen. Two third of the resulting powder was stored at −80 °C for mass spectrometry, while one third was dissolved in RLT buffer  awaiting RNA extraction (for confirmation of infection). The powdered sample was resuspended in an Eppendorf protein LoBind tube in 45 µL 0.5 M triethylammonium bicarbonate , supplemented with sodium dodecyl sulfate  with time of flight (TOF) mass analysis. The spectral acquisition time of a low and elevated energy scans was 0.6 s over an m/z range of 50–2000.Peptides were analyzed using a nanoscale UPLC system  coupled to a Synapt G2-Si mass spectrometer (Waters Corporation). Peptides were first trapped on a 180 µm × 20 mm C18 Trap column in 0.1% FA and separation was performed on a HSS C18 1.8 m, 100 m × 250 mm analytical column at a flow rate of 300 nL/min and a temperature of 45 °C. A 0.1% formic acid with 4% dimethyl sulfoxide (DMSO) in water was used as mobile phase A and 80% ACN containing 0.1% formic acid as mobile phase B. Peptides were separated for 60 min at 1%–40% solvent B, for 1 min 40%–85% solvent B. After 7 min of rinsing using 85% solvent B the column was re-equilibrated to the initial conditions. Eluted peptides were analyzed in positive mode ESI-MS using HDMSribed in  and the Data analysis of the raw files obtained from the Synapt G2-Si was performed in Progenesis QI . The PLGS Threshold Inspector was used to iterate through a range of apex low energy and high energy settings to determine the optimal threshold for protein identification. Peptides with charges from two to five were retained following the data normalization in the Progenesis. For protein identification, the LC-MS data were processed and searched by Protein Lynx Global SERVER  with peptide tolerance and fragment tolerance set to auto. Protein identifications were obtained by searching a compiled database of UniProtKB/Swiss-Prot entries belonging to IAPV supplemented with the cRAP database (laboratory proteins and dust/contact proteins) and sequences of spiked standard proteins, which was concatenated to a randomized decoy database. For peptide identification, the following search criteria were selected: (i) trypsin as digestion enzyme; (ii) up to one missed cleavage allowed; (iii) fixed methylthio-cysteine and variable methionine oxidation, deamidation at asparagine and glutamine. The false discovery rate (FDR) for protein identification in PLGS was set to 1% threshold. Only proteins identified with at least two unique peptides were further considered.Dicistroviridae IAPV and CrPV is given in E coverage is denoted by the darkness of the polyprotein sequences’ background. The coverage differed between the (poly)proteins; the 64 detected peptides covered 50% of the AA sequence of the structural polyprotein (48 peptides), whereas only 12% of the non-structural protein was detected (16 peptides). Within the OrfX protein, none of the seven predicted peptides (at least 6 AA long) after trypsin digestion were detected. The presence of 1A would be confirmed by detecting the Stop-Go translational cleavage at the NPG▼P site, but the resulting non-tryptic CGDWDSILLLLSGDIEENPG peptide was not observed.An overview of the genome structure of the rpl23 and ubi, whereas for CrPV it was only 16 times, even with an infection dose of only 500 particles of IAPV and 106 of CrPV. CrPV replication was confirmed by showing a 11,000-fold increase in viral titers 4 days after infection compared to the input viral titer minutes after injection .Infections with IAPV and CrPV showed a similar relative distribution over the tissues, with higher viral titers in the fat body and for IAPV also in the ovaria, and lower viral titers in brain and midgut . Howeverppia-targeting assays, the RNAi efficiency could be evaluated in both the absence and presence of the viruses. The expression levels of ppia in the RNAi control compared to the baseline control measure RNAi efficiency in a virus-free condition, whereas the levels in the virus treatment give an indication of how the virus influences the RNAi efficiency , fat body  and midgut , but not in the ovaries . In the following sections, the ovaries will not be discussed further. The other tissues will collectively be referred to as ‘responsive tissues’. For the ‘CrPV’ experiment, the ppia levels in the fat body were also statistically different .In the ficiency . In the ppia dropped significantly in the fat body (IAPV: p = 0.002 (Tukey’s HSD); CrPV: p = 0.011 (Tukey’s HSD)). However, the ppia levels dropped by 55% in the ‘IAPV’ experiment, whereas they only declined by 25% in the CrPV experiment (note that there can be no effect of virus presence in this comparison). In the brain and midgut, the ppia levels were slightly lowered, but there was too much biological variation in the IAPV experiment to confirm an RNAi event  and F2,22 = 11.218, p > 0.05 (Tukey’s HSD) respectively).Different silencing efficiencies were observed between the selected bumblebee tissues after 48 h in the absence of viruses ), fat body (p < 0.001 (Tukey’s HSD)) and midgut (p < 0.001 (Tukey’s HSD)) compared to the baseline control, and in the case of the fat body and the midgut also a significant silencing compared to the RNAi control (p = 0.001 (Tukey’s HSD) and p = 0.013 (Tukey’s HSD), respectively) The ppia levels in brain, fat body and midgut were lowered to 51%, 19% and 53% of their original levels, respectively. These results indicate an enhancement of the RNAi effect after IAPV infection in all tissues except for the unresponsive ovaries.When IAPV was administered 24 h before the dsRNA treatment (virus treatment in ppia levels compared to the RNAi control (p = 0.036 (Tukey’s HSD)). Moreover, no significant silencing could be observed compared to the baseline control (p > 0.05 (Tukey’s HSD)), indicating that the RNAi system might have become impaired after CrPV infection.In the case of CrPV, only the fat body was analyzed as it was the only tissue showing a significant silencing effect without IAPV presence, which is necessary for confirming RNAi inhibition. The virus was administered to the bumblebees 48 h before dsRNA treatment (instead of 24 h for IAPV). This later time-point was chosen because injection of CrPV resulted in slower replication and later onset of mortality of the bees. We have determined the onset of death to occur at 4 days post infection (dpi) and 5 dpi for IAPV and CrPV, respectively (data not shown), so this alteration promotes similar virus–host interactions at the moment of dsRNA treatment and RT-qPCR evaluation. Forty-eight hours after dsRNA treatment this resulted in a significant increase in dcr-2, ago-2, ninaC, egghead and sid-1 were evaluated 24 h after IAPV infection . A general linear model (GLM) analysis over the responsive tissues showed a significant effect on dcr-2 over the different tissues . The expression of ago-2 was increased in midgut  and in the GLM . At the endpoint of the assay  and a similar, but more variable and not significant upregulation in the brain and midgut . The GLM over the responsive tissues showed a significant effect of dcr-2 . The expression levels of ago-2 remained unaltered in all tissues.First, the RNAi core genes, nfection A. A signhe assay B, a signninaC, egghead and sid-1 in the fat body, only the former was upregulated . Therefore, the expression levels of ninaC were also determined in the other tissues. However, ninaC was significantly downregulated in the brain  and no alteration was observed in the midgut  nor in the GLM over all responsive tissues .When looking at the systemic RNAi genes dcr-2 and ago-2 in the fat body after 96 h , as well as the systemic RNAi genes ninaC and sid-1 . The dcr-2 upregulation after CrPV infection is over nine-fold, whereas for IAPV over all responsive tissues not even two-fold. The expression levels of egghead remained unaltered   B.p < 0.0001, p = 0.0005, In both cases, there is a clear decrease in viral titers in the fat body compared to the control after pre-infection with the other virus. Pre-infection with CrPV for 48 h reduced the IAPV levels 90%  the viral titers in the ovaries were considerably high; (2) protein extraction from the fat body is challenging; and (3) the ovaries do not contain complex microbiota like the midgut does, which might confound the analysis. The HDMSE on IAPV-infected ovaries resulted in a coverage that is comparable to a similar setup for IAPV in Apis mellifera [A first confirmation of the presence of a VSR in IAPV could come on a proteomic level, from the detection of VSR-specific peptides. The ovaries were selected for HDMSellifera  with a hellifera  needed tE result, by itself, it is not possible to confirm with any certainty the absence of these putative VSR proteins in IAPV-infected cells. However, when coupled with the RNAi efficiency data, where the presence of VSR in IAPV should suppress RNAi efficiency, it can be concluded that, even if the 1A or OrfX proteins are produced in the cell during IAPV infection, and even if they are functional VSRs, their functionality is being outweighed by the RNAi system or that the RNAi system of bumblebees is insensitive towards the VSRs of IAPV.From the HDMSB. terrestris, but also an enhancement of the RNAi activity was observed. The fact that a diminished RNAi effect is seen using the same setup for CrPV ensures the efficacy of the ppia assay. An argument could be made that the difference between the experiments could be explained by the longer incubation of CrPV. We believe it was appropriate to extend the viral pre-infection duration because of the lower virulence of CrPV while a 24-h age difference between the bumblebees will probably not affect their capacity for RNAi. The more important question is determining how this IAPV-induced enhancement of RNAi activity can be explained.Not only could the predicted VSR of IAPV not suppress the RNAi efficiency in ppia levels to drop because of a disturbed cellular machinery. However, we collected ample evidence that this is not the case  was seen. It seems that this upregulation of dcr-2 does not determine the outcome of the RNAi pathway, possibly because the VSR 1A is acting on Ago-2, a component downstream of Dcr-2 [dcr-2 silencing does not diminish IAPV replication in bumblebees, possibly because of the too robust replication of IAPV [ninaC expression but they are variable over the different tissues). Again, for CrPV, more significant positive fold changes are noticed . The fact that an upregulation of ninaC was also seen in the fat body after IAPV treatment might indicate a role of the fat body in triggering a systemic RNAi response, but additional experiments are needed before any conclusions can be drawn on this. The exact mechanism behind the IAPV-induced RNAi efficiency remains inconclusive; possibly one or more genes that drive RNAi efficiency fell out of our selection or a yet unknown RNAi signal-spreading mechanism is activated by the presence of IAPV.A first explanation could be that virus infection causes the the case . Anotherof Dcr-2 . Interes of IAPV . Second,West Nile virus [Dengue virus [The viral influence on the RNAi machinery, a key immune response against viral infections, has great implications for host–virus dynamics. It is important to repeat that IAPV is a known pathogen of various pollinating hymenopterans ,25, wherle virus  and Dengue virus , where iue virus  and we hAnopheles gambiae with salivary glands that are refractory to conventional RNAi [Schistocerca gregaria [Apis mellifera after siRNA injection [Locusta migratoria [Our findings are also interesting in light of RNAi-therapeutics development and the use of RNAi as a research tool in functional genomics. The RNAi efficiency in the absence of a virus varies between the different tissues examined, with a significant silencing effect only occurring in the fat body and ovaries which are unresponsive. It is conceivable that the virus could evade the therapeutic in tissues which are insensitive to RNAi, such as the ovaries which showed relatively high viral titers in this study. Variations in RNAi efficiency over insect tissues have also been found in nal RNAi  and in mnal RNAi ). An insgregaria  in the hnjection , and wasgratoria . It needDrosophila [An interesting question emerging from these results is the functionality of the CrPV 1A in bumblebees, a hymenopteran species, which was previously shown to be active in the dipteran osophila . Althougosophila . Our res


Bemisia tabaci. The insect ingests viral particles with its stylets. Virions pass along the food canal and reach the esophagus and the midgut. They cross the filter chamber and the midgut into the haemolymph, translocate into the primary salivary glands and are egested with the saliva into the plant phloem. Begomoviruses have to cross several barriers and checkpoints successfully, while interacting with would-be receptors and other whitefly proteins. The bulk of the virus remains associated with the midgut and the filter chamber. In these tissues, viral genomes, mainly from the tomato yellow leaf curl virus (TYLCV) family, may be transcribed and may replicate. However, at the same time, virus amounts peak, and the insect autophagic response is activated, which in turn inhibits replication and induces the destruction of the virus. Some begomoviruses invade tissues outside the circulative pathway, such as ovaries and fat cells. Autophagy limits the amounts of virus associated with these organs. In this review, we discuss the different sites begomoviruses need to cross to complete a successful circular infection, the role of the coat protein in this process and the sites that balance between virus accumulation and virus destruction.Begomoviruses are vectored in a circulative persistent manner by the whitefly  Begomovirus, family Geminiviridae) are small ssDNA viruses vectored in a circulative persistent manner by the whitefly Bemisia tabaci . B. tabaci disseminates the viruses very efficiently, causing important damage to vegetables, staple food crops and ornamentals, worldwide . . 44]. ThB. tabaci. Indeed, midgut (within and outside the filter chamber) and internal ileum epithelia have a brush border at the apical membrane ideal for viral endocytosis , thorax , head  and haemolymph. The abdomen contained 200–800 million TYLCV genomes, the thorax and head about one-tenth this amount and the haemolymph about one hundredth the amount in the abdomen. The amount of TYLCV-Mld was about one-tenth the amounts of TYLCV in all four compartments studied. In a parallel experiment, the whiteflies were reared for 3 days on infected tomato, followed by 20 days on virus non-host cabbage plants. At this time, the amounts of TYLCV and TYLCV-Mld in the head, thorax and abdomen were similar to those present after 20 days of continuous virus ingestion. These results suggest long-term storage of TYLCV in the guts, as well as a stable distribution of the virus in the insect organs.In another set of experiments , female B. tabaci B, 4–5 days after emergence, were first caged with TYLCV-infected tomato seedlings for 8 h AAPs, then transferred to cotton seedlings. The amounts of viral RNA and DNA in the insects were measured thereafter for up to 28 days. The amounts of V1, V2 and C3 transcripts increased 2–5-fold up to 1–3 days and decreased to basal levels thereafter. The amount of viral DNA in whole insects increased several fold during the first days on cotton and then decreased until they were barely detectable after 2 weeks. The amounts of TYLCV DNA in the head , the thorax (with the esophagus) and the abdomen (with the guts) increased up to three times, peaking after 2 days and decreasing thereafter, reaching initial amounts after 7 days. In dissected midguts, viral DNA amounts increased about seven-fold, peaking at 3–4 days, remaining stable for another 1–2 days and decreasing to initial levels after 6 days. FISH confirmed the accumulation of viral DNA and viral transcripts in the whitefly midguts. The complementary viral genome strand was detected in the nuclei of midgut epithelial cells, supporting the formation of a viral dsDNA replicative form and suggesting that midgut cell nuclei are the sites of virus replication  and on the complementary strand (C3) also increased several fold and then decreased. Transcript accumulation and decay were correlated with the TYLCV-induced upregulation of the autophagy pathway (see below). Similar results were obtained with TYLCCNV, a monopartite begomovirus with a satellite DNAβ [Papaya leaf curl China virus  was unable to replicate and activate the whitefly autophagy pathway. The genomic DNA of PalCuCNV rapidly decreased and remained at a very low level thereafter, suggesting that this virus cannot replicate in the whitefly.In the second case , whiteflite DNAβ . HoweverB. tabaci B and the non-vector T. vaporariorum was measured during 14 days of AAP of on infected tomato plants [T. vaporariorum remained constant for the 14 days of the experiment, while in B. tabaci, it increased about five times during the next 6 days, then decreased to levels present at the beginning of feeding on cucumber. Hence, TYLCV-Mld may replicate in B. tabaci, but not in T. vaporariorum.In the third experiment, the accumulation of TYLCV-Mld from Japan in the vector o plants . The kinB. tabaci B constantly feeding on infected tomato plants were almost identical. A rapid increase was observed during a 24-h AAP, and each insect contained about 100 million viral genomes. These amounts increased slightly, if at all, with time thereafter. In another experiment, the insects were transferred to a non-host plant after AAPs of 6 and 48 h. No significant changes in the virus amount per insect were detected. The complementary DNA strand of TYLCV and TYLCV-Mld was present in insects from the first day of AAP, accumulated progressively over the 20 days of AAP, then decreased. It was hypothesized that this complementary strand was ingested from the infected plant and allowed a minor and transient replication in insects, before being degraded. The authors suggested that the stability of the insect viral content could be the result of preservation mechanisms without replication, rather than an equilibrium between virus replication and degradation.On the other hand, two studies concluded that there was no evidence for TYLCV and TYLCV-Mild (TYLCV-Mld) replication in the whitefly vector. In the first experiment , the kinB. tabaci B and Q were membrane-fed with DNAase I-treated purified TYLCV and TYLCV-Mld virions. Despite these precautions, both ssDNA and dsDNA were found associated with the particles. Moreover, after virion ingestion, whiteflies were maintained on an artificial diet to eliminate possible plant replication factors. Despite these precautions, viral ssDNA and dsDNA were detected in whiteflies. The ratio of virion/complementary strand remained approximately constant for 4 days of feeding on the artificial diet, indicating that replication was not taking place.In the second experiment , to prevB. tabaci B on TYLCV-infected tomato plants activated the insect autophagy pathway [Atg3, Atg9 and Atg12 also increased after 24–48 h on cotton, then decreased to the initial levels (at 0 h). The number of autophagosomes in the midgut of the viruliferous whiteflies increased and decreased with the same time-course. Inhibiting autophagy with 3-methyladenine induced an increase in TYLCV gene transcription and in virus transmission. Similar results were obtained with TYLCCNV. Immunofluorescence staining of autophagic signals and electron microscopy of autophagosome formation suggested that autophagy may play critical roles in the whitefly midgut, a site where TYLCVs are most abundant  and Cotton leaf curl Rajasthan virus (CLCuV) as bait in a yeast two hybrid screen [A midgut protein (coined MGP) was identified using the CP of the begomoviruses d screen . MGP wasd screen .B. tabaci peptidyl-prolyl isomerases protein genes (PPIases or cyclophilins Cyps) CypB, CypD and CypG, only the expression of CypB was upregulated by two-fold in viruliferous B. tabaci B. CypB and TYLCV CP formed complexes that could be resolved in vitro [From the three in vitro . In addiin vitro . Altogetin vitro ,33,34.B. tabaci B (coined BtPRPG) was upregulated upon ingestion and accumulation of TYLCV and TYLCCNV, but not of the monopartite Tobacco curly shoot virus TbCSV [BtPGRP was expressed in several tissues at all life stages from egg to adult. PGPR and TYLCV CP interactions were detected in vitro by immunocapture PCR and in situ by fluorescent immuno-detection in the midguts of viruliferous whiteflies. Taken together, the BtPGRP gene is involved in whitefly immunity, as well as in begomovirus transmission [A peptidoglycan recognition protein gene (PGRP) from us TbCSV . BtPGRP smission .B. tabaci to degrade the six TYLCV proteins is very high. Ultracentrifuge fractionation of whitefly proteins indicated that the highest proteolytic activities (26S proteasome and autophagy) were associated with fractions containing soluble proteins and much lesser in fractions containing midsize and large protein aggregates [Cauliflower mosaic virus (CaMV)-infected Arabidopsis plants [The vast majority of the virus ingested during feeding remains in the digestive tract, especially in the filter chamber and midgut ,6,7,V. Ogregates . It was gregates . A simils plants . CaMV foB. tabaci B populations [T. vaporariorum.To be transmitted, begomoviruses need to cross the gut haemolymph barrier. This checkpoint was demonstrated to be genetically determined by selecting good and poorly TYLCV transmitting lines from ulations . CompareT. vaporariorum to enter the haemolymph and from there the salivary glands [B. tabaci B and the non-vector T. vaporariorum showed that the virus was present at the brush borders and in the surrounding luminal surface of the midgut epithelial cells of both whitefly species. However, it was exclusively found in the cytoplasm of the midgut cells of B. tabaci, not in that of T. vaporariorum [TYLCV is unable to cross the midgut barrier of y glands . Immuno-rariorum .B. tabaci midgut barrier  harbor the obligatory primary whitefly endosymbiont Portiera aleyrodidarum and a variety of facultative secondary symbionts such as Arsenophonus, Cardinium, Fritschea, Hamiltonella, Rickettsia and Wolbachia. Different biotypes in different geographical regions harbor different sets of secondary symbionts [Rickettsia localizes outside the bacteriocytes and appears in most of the body cavity and infects the digestive, reproductive and salivary systems, where it can reach high concentrations [Rickettsia was shown to be directly involved in begomovirus transmission. Comparing two B. tabaci B populations, one infected with Rickettsia, the other Rickettsia-free, showed that high levels of the bacterium in the midgut pushed the virus out of the gut into the haemolymph. The presence of Rickettsia was correlated with increased TYLCV transmission efficiency suggesting that the invasion of the midgut with Rickettsia accelerated virus translocation out of the midgut [B. tabaci endosymbionts produce GroEL homologue proteins, which are secreted into the haemolymph and ensure virus transmission (see below).Whiteflies and endosymbiotic bacteria have interacted for millions of years . Endosymymbionts . Only Ritrations ,76. Ricke midgut . B. tabaThe haemolymph constitutes an open blood system, bathing the whitefly internal organs ,11. OnceB. tabaci B, is produced by Hamiltonella [Hamiltonella. GroELs produced by Wolbachia and Arsenophonus in both B and Q biotypes poorly interact with the TYLCV CP, indicating that they play a minor role, if at all, in TYLCV transmission [B. tabaci Asia II isolates from Rajasthan, India, contain the primary endosymbiont Portiera, but only the secondary endosymbiont, Arsenophonus. Hence, GroEL from Arsenophonus ensures the transmission of cotton leaf curl virus (CLCuV). The potential interaction between GroEL and CLCuV CP was demonstrated by pull-down, immunoprecipitation and yeast two hybrid assays [The GroEL, which protects TYLCV from Israel from degradation in the haemolymph of ltonella . The poosmission . It is id assays .B. tabaci females [The haemolymph and the viruses it contains can be shuffled by the movement of the guts. It was shown that the midgut loop is able to move through the abdomen-thorax constriction, especially in gravid  females ,53. As aThe primary salivary glands (PSGs), not the accessory salivary glands, are involved in begomovirus circulation ,53. The B. tabaci species-specific receptor-like elements that are able to discriminate between begomoviruses, even as similar as TYLCV and TYLCCNV [B. tabaci B efficiently transmits TYLCV and TYLCCNV, B. tabaci Q ingests and retains both viruses, but is unable to transmit TYLCCNV. The selective transmission of TYLCV/TYLCCNV by Q whiteflies was investigated by following the time course of virus penetration in the PSGs, using CP immunofluorescence of dissected glands. The viruses followed a specific path in the glands. Following a 4–6-h AAP, TYLCV CP spread to the entire PSG; at 12–24 h, virions gradually accumulated to the central region of PSGs; at 36–72 h, the viral CP was observed only in the few cells along the salivary duct , but also in other tissues such as the female reproductive system ,16 and fAutophagy was shown to restrain TYLCCNV invasion of fat cells and ovaries  and were transferred to virus non-host plants, the amount of virus they carried decreases by 1–2% every day ,23,24,25B. tabaci tissues. At every stage along the circulative pathway, begomoviruses have to pass checkpoints located in the cibarium, the lumen/gut interface, the gut/haemolymph interface and the PSGs. At each location, the virus CP plays a major role in recognizing would-be receptors [Translocation of begomoviruses depends on their interaction with many eceptors ,30,31,34B. tabaci has remained elusive, though the technical panoply of researchers has greatly extended in the recent years. Comparative transcriptome analyses and the availability of sequence of the genomes of B. tabaci B [B. tabaci organs such as guts [B. tabaci is not well known and has to be worked out.The identification of begomovirus receptors in tabaci B  and Q [8tabaci B  biotypestabaci B ,87,88. T as guts  and sali as guts  may help as guts  may be mB. tabaci has witnessed new developments. Researchers have been able to select for poor begomovirus transmitters within whitefly populations [The search for putative begomovirus receptors and for proteins regulating the translocation of begomoviruses in ulations . In thesknot-1) was identified as a possible regulator of virus amounts in B. tabaci [knot-1 increased by several orders of magnitude the quantity of virus ingested by the insects and transmitted to tomato plants. Hence, it seems that knottin-1 is involved in restraining the amount of TYLCV particles associated with B. tabaci during the circulation of the virus in the insect body.Recently, several whitefly proteins have been identified, which either increase or decrease begomovirus transmission. Most of these proteins have been immuno-located in the midgut, and some of them bind the CP. The events begomoviruses are subjected to, once ingested, emphasize the tissues and processes that are mobilized to control the virus amounts to avoid a virus overflow that might be lethal to the insect. Even, the invasion of organs other than those in the circulative pathway such as fat cells and ovaries are controlled and restrained. Recently, a gene encoding a knottin protein (. tabaci . SilenciB. tabaci B [B. tabaci vector and the begomovirus intruder. The sequence of the genome of the begomovirus non-vector T. vaporariorum, when available, will help focus our quest for genes related to vectoriality.The recently published sequence of the genomes of the tabaci B  and Q [8tabaci B  biotypes


We studied a recent epizootic of Getah virus infection among pigs in the southern part of Ibaraki Prefecture and the northern part of Chiba Prefecture, Japan, focusing on its possible association with outbreaks in racehorses in 2014 and 2015. The genomic sequence of a Getah virus strain from an infected pig was analyzed to evaluate the degree of identity with the strains from horses.P < 0.01); 4/5 batches had positive sera in 2014 and 7/7 in 2015. In north Chiba, seropositivity rates in 2013 (14.1%), 2014 (17.8%), and 2015 (48.0%) were significantly higher than those in the previous years (P < 0.01); 6/27 batches had positive sera in 2013, 3/9 in 2014, and 5/5 in 2015. Complete genome analysis revealed that the virus isolated from an infected pig had 99.89% to 99.94% nucleotide identity to the strains isolated from horses during the outbreaks in 2014 and 2015.Sera were collected from pigs from September to December 2012 to 2015 in south Ibaraki (380 pigs in 29 batches), and from September to December 2010 to 2015 in north Chiba (538 pigs in 104 batches). They were examined by using a virus-neutralizing test for Getah virus. Seropositivity rates in 2012–2013 in south Ibaraki and 2010–2012 in north Chiba ranged from 0% to 1.6%. In south Ibaraki, seropositivity rates in 2014 (28.8%) and 2015 (65.0%) were significantly higher than those in the previous years (Serological surveillance of Getah virus in pigs revealed that the virus was circulating in south Ibaraki and north Chiba in 2014 and 2015; this was concomitant with the outbreaks in racehorses. The Getah virus strain isolated from a pig was closely related to the ones from horses during the 2014 and 2015 outbreaks. To our knowledge, this is the first convincing case of simultaneous circulation of Getah virus both among pigs and horses in specific areas. Alphavirus in the family Togaviridae . However, detailed information on sample collection, such as numbers of farms, numbers of pigs, and time periods, were not indicated in these studies, and it is not clear whether the magnitude of the epizootic outbreaks was larger in 2014 and 2015 than in the previous years. Hence, the association of pigs with the Getah virus outbreaks in horses was only speculative.Outbreaks of Getah virus infection in horses occurred in autumn 2014 and 2015 at the Miho training center of the Japan Racing Association after 31 years of no reported outbreaks in Japan , 9. The Here, we investigated Getah virus seropositivity among pigs in the southern part of Ibaraki Prefecture and the northern part of Chiba Prefecture, around the Miho training center, in the period 2010–2015, focusing on a possible association with the outbreaks in racehorses in 2014 and 2015. To further clarify the interactive circulation of the virus between the species, we isolated a Getah virus strain from a pig and compared its genomic sequence with those of previous equine isolates.2; there are 27 pig farms rearing about 32,000 pigs  and from September to December 2010 to 2015 in north Chiba (538 pigs in 104 batches). They were kindly provided by South Ibaraki Livestock Hygiene Service Center and Chiba Central Livestock Hygiene Service Center. Each batch represented 2 to 52 serum samples  collected at one farm on one day. Some batches were collected at multiple time points on the same farms, but not in the same years. In this case, the samples collected at different time points were regarded as different batches, because they should reflect different virus-exposure status. The numbers of batches and serum samples collected in each month are shown in Tables The study area in the southern part of Ibaraki Prefecture (south Ibaraki) included the village of Miho and the cities of Inashiki, Ushiku, and Tsuchiura, covering an area of 454 kmigs Fig. . The areFor virus-neutralization (VN) testing and virus isolation we used Vero cells . Cells were cultured in minimum essential medium  containing 10% fetal calf serum , 100 units/ml penicillin, and 100 μg/ml streptomycin (Sigma Aldrich Inc.). MEM containing 2% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin was used as a maintenance medium for VN testing and virus isolation.2, Vero cells were added at a concentration of 2.5 × 104 cells/50 μl/well. After incubation for 3 days at 37 °C with 5% CO2, the cells were stained with crystal violet – formalin solution. The test was performed with single dilution of 1:4, and the sera were defined as positive for VN antibodies when the cytopathic effect was completely inhibited. Statistical significance for seropositive rates between the years was analyzed by z-test.For VN testing, we used Getah virus strain 14-I-605 isolated from a racehorse during the 2014 Getah virus outbreak . Sera wenon-structural protein 1 (nsP1) gene using primer sets M2W-S and M3W-S [To detect Getah virus RNA efficiently, we used seronegative samples collected from farms suspected to be contaminated with Getah virus. Serum batches that were collected in September and October of each year and that included seropositive samples were tested for Getah virus RNA by using RT-PCR. Five sera from each batch were pooled (20 μl for each sample), and each pool included as many seronegative samples as possible. For some batches that included more than five seronegative samples, multiple pools were tested. If the number of seronegative samples was not enough to make a pool, seropositive samples were selected randomly from the batch. In total, 24 pools were tested. RNA was extracted from pooled sera by using a nucleic acid isolation kit , and viral gene detection was performed by using an RT-PCR for the Getah virus nd M3W-S .5 cells/2 ml/well) on each well of a six-well plate (Asahi Glass Co. Ltd.). The next day, the cells were washed three times with phosphate-buffered saline and cultured in maintenance medium. To identify Getah virus–specific nucleotide sequences, the supernatants of inoculated cells that showed cytopathic effects were tested by using RT-PCR as described above and sequenced by Filgen Inc. .In the case of those pooled sera that were positive for Getah virus RNA, 25 μl of original sera that had been used to make the corresponding pool was inoculated simultaneously with Vero cells  and 65.0% in 2015 (P < 0.01). The proportions of batches that had positive sera were also high , 17.8% in 2014 (P < 0.01), and 48.0% in 2015 (P < 0.01). In 2013, 6/27 batches had positive sera; this value was 3/9 in 2014 and 5/5 in 2015 (Table The rates of seropositivity to Getah virus in autumn 2012–2015 in south Ibaraki and 2010–2015 in north Chiba are summarized in Tables n = 24), and each pool included as many seronegative samples as possible. This was because virus recovery might not have been achieved from antibody-positive sera: the presence of antibodies should indicate that the viremic phase has finished. Getah virus RNA was detected in four out of 24 pools: two pools collected on 8 September 2014 in Inashiki city (Ibaraki Pref.), one pool collected on 28 September 2015 in Tsuchiura city (Ibaraki Pref.), and one pool collected on 23 October 2015 in Tomisato town (Chiba Pref.)  showed cytopathic effects; the strain isolated was confirmed as Getah virus by RT-PCR for the To clarify the interactions between the Getah virus strains isolated from the pig and the horses, we compared the complete genome sequence of strain 15-I-1105 with those of other strains, including the ones isolated during the outbreaks in horses. Phylogenetic analysis showed that strain 15-I-1105 was located in the same cluster as the strains isolated from horses during the 2014 outbreak , one strain isolated from a horse during the 2015 outbreak (15-I-752), and one strain isolated from mosquitoes in Nagasaki Prefecture in 2012  was closely related to the strains isolated from horses in 2014 (14-I-605-C1 and 14-I-605-C2) and 2015 (15-I-752), with extremely high nucleotide sequence identities. This finding, together with the serological data, indicates this was the first convincing case that Getah virus circulation among horses and pigs occurred interactively in a specific area. On the basis of the theory that pigs are natural hosts of Getah virus, the outbreak among racehorses in 2014 and 2015 was likely preceded by viral amplification in pigs. However, the virus might not be transferred in a one-way manner from pigs to horses; instead, it might circulate interactively between the two species, because the high-level viremia required for vector transmission has also been observed in infected horses [E2, capsid, and nsP1 genes suggested that this cluster containing 12IH26, 14-I-605-C1, and 14-I-605-C2 was closely related to the recent Chinese and South Korean strains but only distantly related to Japanese strains from the 1970s and 1980s [Our phylogenetic analysis of various Getah virus strains revealed that the strain from a pig (15-I-1105) and strains from recent outbreaks in horses  formed a cluster with a strain isolated from mosquitoes in Nagasaki Prefecture in 2012 (12IH26, [Serological investigation of Getah virus in pigs revealed that the virus was circulating in south Ibaraki and north Chiba in 2014 and 2015, during the same period as outbreaks in racehorses. The Getah virus strain isolated from an infected pig was closely related to those from horses during that period. This was the first convincing case of interactive circulation of Getah virus among both pigs and horses in a specific area. Consistent with the findings of previous studies, our current data suggest that pigs are important natural hosts and amplifiers. Periodic surveillance of pigs over a larger area would be useful for estimating the risk of epizootics in horses.


In December 2014, during the height of the Ebola virus disease (EVD) epidemic in West Africa, we wrote, “EVD is only the beginning and only one disease; even if we are to control the current epidemic, the future introduction of this and other highly contagious and virulent microbes to and from global slums is inevitable” . –12. A. alication . While tIn the developed world, despite the low predicted and observed risk of importation and subsequent ongoing transmission of Zika virus due to the 2016 Olympic and Paralympic Games , we contThe energy, media attention, and research resources revolving around the Zika virus epidemic must be harnessed and used to bring the largely ignored urban slum populations of megacities around the world into the global spotlight. Otherwise, we will continue to have the same inconsequential discussions until the next deadly epidemic is sparked in slums.


The rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signals an urgency to identify therapeutics. Recent efforts to rescreen dengue virus human antibodies for ZIKV cross-neutralization activity showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extracellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments. The 4.0 Å resolution pH8.0 complex structure shows that the antibody binds to E proteins residues at the intra-dimer interface, and the virus quaternary structure-dependent inter-dimer and inter-raft interfaces. At pH6.5, antibody C10 locks all virus surface E proteins, and at pH5.0, it locks the E protein raft structure, suggesting that it prevents the structural rearrangement of the E proteins during the fusion event—a vital step for infection. This suggests antibody C10 could be a good therapeutic candidate. There is a pressing need for therapeutic agents against Zika virus (ZIKV). Here the authors present cryoEM structures of a neutralizing antibody (C10) complexed with ZIKV that show C10 preventing structural changes required for virus entry into the cell, suggesting it might be effective in treating Zika infections. Zika virus2The virus surface consists of 180 copies of E proteinAn E protein contains three domains—DI, DII and DIII6650=0.024 μg ml−1), as demonstrated recently in ZIKV infected cell culture11The recent explosion of the number of ZIKV cases, together with the association of ZIKV with the development of microcephaly in fetuses101110We solved the cryoEM structures of Fab C10 complexed with ZIKV at pH8.0, pH6.5 and pH5.0 mimicking the extracellular, early and late endosomal conditions, respectively, and compared them to the cryoEM maps of the uncomplexed ZIKV controls at pH8.0 ref. , pH6.5  and the Micrographs of the uncomplexed ZIKV control at pH8.0 sample show mostly smooth surfaced spherical particles . In the Micrographs of the ZIKV-C10 complexes at all pH conditions show spiky looking particles, due to the Fab molecules bound to virus surface . The 2D The cryoEM structures of ZIKV-C10 complex at pH8.0, pH6.5 and pH5.0 are determined to 4.0, 4.4 and 12 Å resolution, respectively . In eachIn the 4.0 Å resolution pH8.0 complex cryoEM map , the likThe ZIKV-C10 complex intra-dimer epitope is located on the DII (at and around the fusion loop) on one E protein and on DIII and DI of the other E protein in the dimer . A plot A comparison of the cryoEM maps of pH5.0 complex to the pH8.0 complex shows that the E protein layer has moved to a larger radius , whereasComparing the pH8.0 and the pH5.0 complex structures  shows thAlthough the E protein raft structure stays mostly intact in the ZIKV-C10 pH5.0 complex structure, the inter-raft interactions are disrupted , even thAll antibodies can cause ADE at some concentrations. HMAb C10, similar to other potent antibodies, causes ADE at a much narrower range of concentrations compared to the other weakly neutralizing antibodies20−1 were incubated with equal volumes of virus at 37 °C for 0.5 h. One hundred microlitres of each mixtures were then layered on BHK-21 cells in a 24-well plate and incubated at 37 °C for 1.5 h. The infected cells were washed with phosphate-buffered saline, overlaid with carboxyl-methyl cellulose and incubated at 37 °C for 5 days. Cells were fixed and stained, and the plaques were counted. Percentage neutralization was determined from the comparison of the number of plaques in specific antibody dilutions to the control (without antibody). PRNT50 is the concentration of the antibody that causes 50% reduction in plaque numbers.The neutralization activity of the HMAb C10 on Zika virus strain H/PF/2013 was determined by PRNT. Two-fold serially diluted HMAb C10 starting at 0.5 μg mlAedes albopictus C6/36 cells (ATCC) were grown in RPMI 1640 media supplemented with 2% fetal bovine serum at 29 °C. At about 80% confluency, the cells were inoculated with ZIKV strain H/PF/2013 at a multiplicity of infection of 0.5 and incubated at 29 °C for 4 days. The virus-containing media was clarified by centrifugation at 12,000g for 1 h. Virus was precipitated overnight from the supernatant using 8% (w/v) polyethylene glycol 8000 in NTE buffer  and the suspension was centrifuged at 14,334g for 1 h. The resulting pellet containing the virus was resuspended in NTE buffer and then purified through a 24% (w/v) sucrose cushion followed by a linear 10-30% w/v potassium tartrate gradient. The virus band, visualized by its light scattering ability, was extracted, buffer exchanged into NTE buffer and concentrated using a concentrator with 100-kDa molecular weight cut-off filter. All steps of the purification procedure were done at 4 °C. The concentrations and purity of the E protein were estimated with Coomassie blue-stained SDS-PAGE using different known concentrations of bovine serum albumin solution as standards.Monoclonal antibody (mAb) C10 was synthesized in transfected human cells from cloned plasmids . Briefly, previously published heavy and light chain variable region sequences−1) was incubated overnight with immobilized papain (Thermo scientific) at 37 °C. After digestion, the Fab fragment was purified with anion exchange chromatography  and gel filtration  on an AKTA purifier system.The Fab regions of C10 IgG were produced by papain digestion. Briefly, the whole IgG  for 10 s prior to adjusting the pH. The final pH of the virus was reached by addition of a volume ratio of 1.5 μl of 50 mM MES buffer at respective pH (pH5.0 or pH6.5) to every 1 μl of the virus-Fab mixture. The pH-adjusted samples were left on the grid for another 15 s. The grid was then blotted with filter paper and flash frozen in liquid ethane by using the Vitrobot Mark IV plunger . The corresponding controls (ZIKV without Fab) for each pH were prepared similarly.The images of the frozen ZIKV complexes were taken with the FEI Titan Krios electron microscope, equipped with 300 kV field emission gun, at nominal magnification of 47,000 for pH5.0 Fab C10 ZIKV complex, and 59,000 for pH6.5 and pH8.0 complex samples. A 4096 × 4096 FEI Falcon II direct electron detector was used to record the images.− Å−2 for pH8.0, total exposure of 1.05 s and total dose of 43 e− Å−2 for pH6.5 complex. The pH5.0 complex was collected at single image mode, with the dose of 20 e− Å−2. The frames from each ‘movie' were aligned using MotionCorr− Å−2 to use in 3D reconstruction. The images were taken at underfocus in 0.5∼2.5 μm range. The astigmatic defocus parameters were estimated with GctfLeginonThe pH8.0 and pH6.5 ZIKV-C10 structures were interpreted by fitting in the uncomplexed ZIKV (PDB ID 5IZ7) and Fab C10 (PDB ID 4UT9) first as rigid bodies in ChimeraElectrostatic potentials of protein surfaces were calculated using Adaptive Poisson-Boltzmann Solver(APBS)The cryoEM maps and the atomic models of the ZIKV-C10 complex at pH8.0, 6.5 and 5.0 have been deposited in the Electron Microscopy Data Bank (EMD) and the Protein Data Bank (PDB) under the accession codes EMD-9575, EMD-9573, EMD-9574 and 5H37, 5H30, 5H32, respectively. The data that support the findings of this study are available from the corresponding authors on request.How to cite this article: Zhang S, et al. Neutralization mechanism of a highly potent antibody against Zika virus. Nat. Commun.7, 13679 doi: 10.1038/ncomms13679 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Tables


Dicistroviridae is a family of small non-enveloped viruses with monopartite, linear, positive-sense RNA genomes of approximately 8–10 kb. Viruses of all classified species infect arthropod hosts, with some having devastating economic consequences, such as acute bee paralysis virus in domesticated honeybees and taura syndrome virus in shrimp farming. Conversely, the host specificity and other desirable traits exhibited by several members of this group make them potential natural enemies for intentional use against arthropod pests, such as triatoma virus against triatomine bugs that vector Chagas disease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Dicistroviridae which is available at www.ictv.global/report/dicistroviridae. Virions are roughly spherical, with a particle diameter of approximately 30 nm and no envelope , Fig. 1.The RNA genome is monopartite and contains two main non-overlapping ORFs that are flanked by UTRs and separated by an intergenic region (IGR) . The 5′-Replication occurs exclusively in the cytoplasm of infected cells. Cap-independent translation proceeds directly from two distinct internal ribosomal entry site (IRES) elements located within the 5′-UTR and the IGR. These IRES elements permit production of non-structural proteins early in the infection process before host translation mechanisms are inhibited, and excess molar quantities of structural proteins when capsid proteins are required later. Unusually, the IGR IRES directs translation initiation at a 3′-adjacent non-AUG codon and in the absence of all canonical initiation factors. The conserved three-dimensional structure is crucial to the IGR IRES function . Pseudo-Dicistroviridae is comprised of three genera: Aparavirus, Cripavirus and Triatovirus. Demarcation of the genera is based on phylogenetic divergence and unique characteristics exhibited by the internal ribosomal entry site [The family try site . Dicistrtry site . Nervouswww.ictv.global/report/dicistroviridae.Full ICTV Online (10th) Report:


Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus–insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors. The majority of plant viruses that cause disease in agricultural crops rely on biotic vectors for transmission and survival . The larMyzus persicae .,28.2,28]oviridae . After ioviridae ,31,32,33oviridae ,34,35,36oviridae . Criticaoviridae . Interesoviridae . PinheirGeminiviridae) circulative transmission in their whitefly vector were recently reviewed . ,4. 2,4].ncticeps  involvesncticeps ,47. Thisncticeps ,49. RDV ncticeps . RDV Pnscificity . The reourcifera ,53. Thisurcifera ,55. SRBSFrankliniella occidentalis, even though in insect cell lines tubules can be generated from the non-structural movement protein NSm [Tomato spotted wilt virus (TSWV) and other tospoviruses also generate tubules for viral movement in plants . Howevertein NSm . The routein NSm . Infectitein NSm ,59. Numetein NSm ,61,62. Atein NSm . Gc prottein NSm ,65. Desptein NSm ,67.Nilaparvata lugens involves invasion of the principal and accessory salivary glands but does not involve entry into neural tissues or ovarioles [Laodelphax striatellus [L. striatellus and involves Vg interaction with RSV major nucleocapsid protein pc3 [Tenuiviruses are transmitted by planthoppers and leafhoppers and their vector dissemination route includes diverse organs ,69. Diffiatellus ,71. Inteiatellus  and theyiatellus . Tenuivitein pc3 . Rhabdoviridae, monopartite cyto- and nucleorhabdoviruses with known vectors are transmitted in a persistent, circulative-propagative manner by aphids, planthoppers or leafhoppers [Peregrinus maidis appears to breach the transmission barriers in P. maidis by spreading from the midgut to the anterior diverticulum and esophagus, a region that spans the compound ganglia and salivary glands, and then invades neuronal tissues including brain, compound ganglion and compound eye cells to gain access to the salivary glands [In the fhoppers ,75,76. Mfhoppers . Plant ry glands . Plant ry glands ,75, but Insect vectors play a crucial role in determining the plant host range of a virus. Competency for virus transmission is determined by genetic components and specific molecular interactions of virus and vector, and potentially host plant components taken up during feeding. Better knowledge of these interactions and mechanisms of transmission will be essential for developing more effective control measures in future. Currently available strategies to interfere with virus transmission by vectors include host genetic resistance to virus and/or insect, insecticides and integrated pest management . CriticaAn excellent recent review discusses potential avenues to disrupt or interfere with insect transmission of plant viruses . The maiEmerging evidence suggests that non-circulative, “stylet-borne” virus transmission involves more than passive adhesion to the sucking-feeding mouthparts, and instead results from specific molecular interactions between virus, plant and vector. The concept of “transmission activation” has been proposed, based on the immediate intracellular response of CaMV to the presence of the aphid vector on the plant . Aphid pRhabdoviridae), tospoviruses (family Bunyaviridae), tenuiviruses and reoviruses (family Reoviridae), viruses encounter multiple barriers to acquisition, replication, intercellular movement, cell escape and host plant inoculation [During the infection process of persistent, propagative viruses such as rhabdoviruses  leading to the inhibition of internal virus spread . Anti-viGraminella nigrifrons, the leafhopper vector of the nucleorhabdovirus MFSV, were shown to be differentially expressed when insects fed on virus-infected leaves [P. maidis that transmits MMV shows innate immunity and RNAi pathway genes upregulated [Replicating plant viruses have been shown to activate the immune response of their insect vectors. This was first demonstrated for TSWV through up-regulation of thrips transcripts for antimicrobial peptides, pathogen recognition and innate immune system activation . Immune d leaves ,88. The egulated . Persistegulated . A betteThe salivary gland of transmission-competent sucking vector insects deserves special attention. Saliva plays a crucial role in insect feeding behavior and virus transmission . In thriFrankliniella occidentalis thrips [Plant viruses can modify insect vector behavior directly, and indirectly by manipulating plant hosts, leading to enhanced transmission efficiency and spread . Directls thrips . The nons thrips ,95. The s thrips . FurtherVirus-induced biochemical and physiological changes in the host-plant have been shown to influence vector insect host preference . Virus-iWhile the processes involved in transmission of viruses directly from plant to plant or animal to animal vary substantially, viruses of both plants and vertebrates that are vectored by insects have many parallels in their interactions with their insect host. Insects that vector viruses are mainly hemipteran sap-feeders for plants and mainly dipteran blood-feeders (primarily mosquitoes) for vertebrates. In both cases the virus is acquired during feeding on the non-insect host and then is transferred to a naïve non-insect host again via feeding. The interaction with the insect host for vertebrate viruses is considered either biological (the virus replicates in the vector) or mechanical (not involving replication) . As discFlaviviridae, Togaviridae and Bunyaviridae) with Aedes and Culex species mosquitoes transmitting the majority of these [A major focus of insect vector transmitted vertebrate viruses is the mosquito-borne viruses that cause significant human disease. These include viruses from three RNA virus families  . UnderstMosquito-borne viruses generally cycle between infection of the vertebrate and insect hosts. Many mosquito vectors require a vertebrate blood meal to reproduce. Following mosquito acquisition of virus via feeding on an infected vertebrate host, the virus enters and replicates in the cells of the gut epithelium, spreads via the hemocoel to other tissues and organs, culminating in transport to and infection of the salivary glands. The virus then escapes the salivary glands and is transmitted in the saliva when the infected insect feeds on a new vertebrate host (reviewed in ). FollowDrosophila model system has been used to identify many of these antiviral immune pathways [Drosophila viruses can be highly pathogenic to their host. Persistent infection of Drosophila can be established following integration of DNA copies of fragments of an RNA virus genome, created by reverse transcription by a retrotransposon reverse transcriptase. These integrated sequences then act as the templates for initiation of the RNAi response against the virus [Efficient virus transmission is a balance between virus infection and insect host health. While mosquito-borne viruses are commonly pathogenic to their vertebrate host, biological infection of the insect host has few major pathogenic impacts on the insect host, allowing efficient transmission of the virus by the insect vector . The virpathways ,121, buthe virus . Thus unThe mosquito microbiome also plays a role in the outcome of virus infection. The mosquito gut microbiome is diverse and can influence mosquito vector competence Wolbachia and Spiroplasma, are commonly identified in the mosquito microbiota. While Spiroplasma can be pathogenic in mosquitoes, their role in mosquito competence to vector viruses remains to be determined [Wolbachia pipientis has antiviral effects in both naturally infected and experimentally infected insects. Wolbachia is an endosymbiotic bacteria commonly found in cells of the reproductive tissues, salivary glands, Malpighian tubules, head and muscle [Wolbachia is maternally inherited and is well known for its ability to invade invertebrate populations via modification of the reproductive system of the host insect [Drosophila, flies naturally infected with Wolbachia can be protected from RNA virus infection , artifimission  [Culex mosquitoes with Culex flavivirus reduced susceptibility to West Nile virus (WNV) infection [The advent of the ‘omics’ age has identified a plethora of sequences of important arboviruses within vector mosquitoes. In a number of cases these virus sequences have been matched to infectious viruses, which have been shown to have a host range restricted to insects, and are termed insect specific viruses (ISVs) . ISVs arviruses) . ISVs haviruses) ). There nfection  and in vnfection ,151. TheIn addition to the bacterial and viral microbiota, mosquitoes are commonly associated with yeast and fungi (reviewed in ). ComparWolbachia . Seiewed in ,144) andiewed in ). This fustralia ,155. Howustralia . Other microbes of mosquitoes have the potential as platforms for control of arboviruses. Recently, bacteria have been utilized as a vehicle to generate and deliver double-stranded RNA (dsRNA) to mosquitoes to manipulate gene expression, and adaptation of this approach to manipulate arbovirus infection is an interesting  para-transgenic approach ,158. OthAnopheles stephensi [Recently the exciting CRISPR/Cas9 genome editing system has been adapted to introduce refractory genes into the Asian malaria vector tephensi . An impotephensi , which wtephensi . In theotephensi . Vector tephensi .Wolbachia has antiviral effects on virus transmission in mosquitoes [Wolbachia is a common endosymbiont of insects and is predicted to be present in over 40% of arthropod species [Wolbachia has not been identified in the vast majority of aphid species, Wolbachia is known to be present in at least some plant feeding insect vectors including the whitefly [Wolbachia was recently shown to play a role in the supply of essential nutrients to the banana aphid [Wolbachia in the mosquito are strongest following experimental transfer of the bacteria into a new host [Wolbachia into plant virus vectors may interfere with virus replication or transmission is yet to be examined. Other parts of the vector insect microbiome may also have an effect and one might imagine use of inhibitory microbes to block plant virus transmission. Insect-associated bacteria could also potentially be used to deliver dsRNA to knockdown vector genes that are essential for insect survival or that code for virus receptors. It may also be possible to use engineered insect or plant viruses to deliver insect genome editing tools based on programmable RNA-guided nucleases such as zinc-finger nucleases or CRISPR/Cas9. Interestingly, although potential virus receptors for bunyavirus early interactions with host cells were demonstrated in vertebrates, none appear to have been identified in their arthropod vectors [Application of molecular technologies to the investigation of virus–vector interactions can be expected to provide deeper insights in the future. Some of these may be transposed from advances in the understanding of virus-mosquito interactions, although there are both parallels and differences between plant and animal/human systems. The bacterial endosymbiont squitoes ,144 but  species . Althougwhitefly ,164,165.na aphid . Antibionew host , the pos vectors . On the  vectors ,168 and  vectors ; clearlyThe use of green fluorescent protein tagged viruses will aid visualization of virus uptake, tissue tropism, intercellular movement in the vector, and transmission. This will require the use of reverse genetics systems to generate infectious virus clones that are available for many DNA and positive-sense RNA viruses, but are lacking for negative-sense RNA viruses such as TSWV. Only recently has the first reverse genetics system been developed for a plant rhabdovirus, which may serve as a model to develop such tools for other negative-sense RNA viruses . MutatioImmunofluorescence confocal laser scanning microscopy has been used to identify a neurotropic route for MMV in its planthopper vector  and coulhttp://www.aphidbase.com), including the genomes of the pea aphid (Acyrthosiphon pisum) and green peach aphid (M. persicae). Draft genomes for the western flower thrips  and brown planthopper (N. lugens) are also now available and others are in progress through the i5K project (http://arthropodgenomes.org/wiki/i5K). Transcriptome analysis of transmission-competent vectors using RNA sequencing and quantitative RT-PCR has been used to identify insect defense response genes and pinpoint expression in relevant tissues like insect gut, salivary gland and nervous cell tissues [RNAi is increasingly used for functional genomic studies of vector insects including leafhoppers, aphids and thrips, and to knock down insect proteins that interact with virus proteins . These a tissues ,87,89.F. occidentalis are available, and differentially expressed (DE) genes in response to TSWV infection including antiviral defense are being annotated [A foundational toolbox of sequence-based resources has been developed to elucidate thrips gene function and understand thrips-plant interactions and tospovirus transmission  that maynnotated . Global nnotated . Primarynnotated . The binding and functional properties of virus acquisition determinants could be exploited in translational research approaches for improved disease control . This coProtein–protein interactions between plant viruses and their insect vectors are an essential molecular interface that determines acquisition from infected host plants and transmission to new hosts. The apparent specificity of these interactions in both non-persistent and persistent plant virus transmission opens avenues for interference and control of the vector populations as well as virus transmission. While several viral interacting proteins are known, insect vector receptor-like molecules are only beginning to be identified. Increasing application of ‘omics’ technologies has the potential to contribute significantly to the identification and validation of virus-interacting proteins in the vector, as well as, a better understanding of innate immune responses to viral infection. Much knowledge can be gained from the application of emerging technologies including reverse genetics systems applied in insects and insect cells, high resolution imaging techniques, functional genomics and gene editing. Direct and indirect effects on vector behavior in relation to the infected plant host is another important research area impacting on virus acquisition and dissemination that once better understood, may lend itself to adoption of novel control strategies.