Arboviruses are (re-) emerging viruses that cause significant morbidity globally. Clinical manifestations usually consist of a non-specific febrile illness that may be accompanied by rash, arthralgia and arthritis and/or with neurological or hemorrhagic syndromes. The broad range of differential diagnoses of other infectious and non-infectious etiologies presents a challenge for clinicians. While knowledge of the geographic distribution of pathogens and the current epidemiological situation, incubation periods, exposure risk factors and vaccination history can help guide the diagnostic approach, the non-specific and variable clinical presentation can delay final diagnosis. This case report summarizes the laboratory-based findings of three travel-related cases of arbovirus infections in Uganda. These include a patient from Bangladesh with chikungunya virus infection and two cases of dengue fever from Ethiopia. Early detection of travel-imported cases by public health laboratories is important to reduce the risk of localized outbreaks of arboviruses such as dengue virus and chikungunya virus. Because of the global public health importance and the continued risk of (re-) emerging arbovirus infections, specific recommendations following diagnosis by clinicians should include obtaining travel histories from persons with arbovirus-compatible illness and include differential diagnoses when appropriate. Febrile illnesses in travelers have been reported in 20 to 70% of cases returning from tropical and sub-tropical regions, and although associated with low mortality rates, significant morbidity has been observed . Fever iflavivirus, family Flaviviridae, and chikungunya virus (CHIKV), genus alphavirus, family Togaviridae, are transmitted to humans via bite from the Aedes (Ae.) aegypti and Ae. albopictus mosquito. Dengue fever (DF) and chikungunya are typically characterized by fever, myalgia, arthralgia, rash, with dengue being more likely to cause severe disease including hemorrhagic complications. Globally, the incidence of DF has increased 30-fold in the last 50 years [Dengue virus (DENV), genus 50 years , with fo50 years . Because50 years , symptomAe. aegypti was the principal vector; however, in recent epidemics in Réunion Island and southern India, Ae. albopictus has been co-implicated [Chikungunya is normally associated with acute diffuse polyarthralgia with recovery usually within weeks. The virus is antigenically and genetically most closely related to onyong-nyong virus (ONNV) and to a lesser extent, Mayaro and Ross River viruses . A numbeplicated . In Afriplicated . Severalplicated –14. Dengplicated . With inLaboratory diagnosis of arboviral infections at Uganda Virus Research Institute (UVRI) is accomplished by serologic methods, virus isolation, and group- and virus-specific reverse transcription (RT)– polymerase chain reaction (PCR). A typical serologic algorithm involves testing acute-, and whenever possible, convalescent-phase serum specimens for anti-viral immunoglobulin M (IgM), followed by a plaque reduction neutralization test (PRNT) to confirm a presumptive IgM-positive sero-status. Virus isolation and RT-PCR are used with acute-phase specimens (before day 5 post-onset) because duration of viremia is typically 2–4 days. Herein, we report two serologically confirmed infections of DENV in patients traveling from Ethiopia and, one CHIKV positive infection confirmed by virus culture and RT-PCR in a traveler from Bangladesh, over a one-month period.The Department of Arbovirology, Emerging and Re-Emerging Infectious Diseases at UVRI, established arbovirus surveillance in 2013 to obtain clinical public health data about the causes of acute febrile illness (AFI). In the period between September and October 2017, three cases of suspect arbovirus infection in international travelers coming from endemic areas were registered. Serology, RT-PCR, and virus isolation were performed by the arbovirus laboratory at UVRI, Entebbe. Based on the patients’ journeys from endemic areas for arbovirus infections and on the clinical manifestations, serum samples were collected and divided into three aliquots: 1 for IgM ELISA, 1 for virus isolation and 1 for storage and future testing. Heat inactivated sera were tested for IgM antibodies as described . Virus iTotal nucleic acid from the CHIKV CPE positive cell culture sample was sequenced using the SeqPlex WTA Kit , the Apollo 324 System , and the MiSeq platform . Quality trimmed reads were filtered against coliphage phi-X174 (NC_001422.1) and the human H19 reference genome (NC_000001 - NC_000024), and the remaining reads were de novo assembled and BLAST analyzed for identification. The trimmed and filtered reads were mapped against the top BLAST-identified sequence (GenBank MF773566) and the initial draft genome to generate a final genome sequence. All sequence analysis used CLC Genomics Workbench (Qiagen). Sequence alignments and phylogenetic trees (Neighbor joining with Jukes-Cantor and 1000 bootstrap replicates) were generated using the final draft sequences generated and all near full length sequences for each organism available in GenBank.A 40-year-old male traveling from Bangladesh was admitted with fever and joint pain at Nakasero Hospital on 09 Sep 2017. Date of initial symptom onset of illness was 08 Sep 2017. The patient had no significant past medical history, was on no regular medication, and had received yellow fever specific travel-related vaccination. The patient, whose final destination was the Democratic Republic of Congo (DRC) where he worked, became ill while in transit in Dubai before traveling to Uganda. Once in Uganda and at Nakasero Hospital, a blood sample was collected on 09 Sep 2017 and transported to the arbovirus laboratory at UVRI.Less detailed information is available for case report 2 involving two sisters who traveled to Ethiopia to visit their father for about 6 weeks. They became ill but got better after a week with treatment in Ethiopia. Their father had been diagnosed with a case of DF 2 months prior to them falling sick. They traveled back to Uganda on 27 Aug 2017. They arrived at the UVRI clinic on 03 Oct 2017, and a blood sample was collected from each person, approximately 5 weeks post-travel. They looked well but requested a DF test to confirm test results performed in Ethiopia.A total of 284 serum samples collected in 2017 were tested for yellow virus (YFV), West Nile virus (WNV), DENV1–4, CHIKV and Zika virus (ZIKV) antibodies by IgM capture ELISA . Only thVirus culture on Vero cell monolayers was performed in T25 flasks. The serum sample from the Bangladeshi traveler exhibited prominent and characteristic 100% CPE on day 2 post-infection. RNA extracted from harvested virus supernatant and tested by group-specific RT-PCR was positive for alphavirus and negative for flavivirus and bunyavirus. Further testing by real-time RT-PCR using CHIKV, ONNV and Semliki Forest virus (SFV) specific primers, yielded positive results for CHIKV. Next generation sequencing of the RNA isolated from cell culture supernatant of the CPE-positive culture sample identified a CHIKV genome that genetically clustered within a CHIKV clade containing predominantly Asian CHIKV strains (Fig. In many malaria-endemic countries, the clinical practice has always been to presumptively treat febrile patients for malaria. However, with improved and easily accessed malaria diagnostics in recent years, a substantial proportion of acutely ill febrile patients in sub-Saharan Africa have been shown not to have malaria , 19. CliCase report 2, involving two sisters who traveled to Ethiopia, tested IgM positive for more than one virus Table . SubjectUganda is a country with environmental conditions conducive to the spread of arboviruses and entomology studies have reported the presence of transmission-competent mosquito vectors –14. The The arbovirus surveillance program at UVRI has the capability to detect, identify and report both endemic and travel-associated arbovirus infections. The rise in incidence of mosquito-borne infections in international travelers highlights the portability of these diseases and the ease with which they can rapidly spread around the world. Therefore, specific recommendations following diagnosis of imported arbovirus infections by clinicians, should include obtaining travel histories from persons with arbovirus-compatible illness and include differential diagnoses when appropriate. Data collected during routine surveillance of febrile illness and associated trends, improve health outcomes and the ability to detect and respond to unusual events of potential public health consequences .Comprehensive and integrated disease surveillance and laboratory systems can generate information used to minimize inefficiencies of disease-specific silos and assure that limited public health resources are leveraged appropriately to improve health security. Also, such systems can identify and detect epidemic-prone illnesses whether existing or novel.The 2030 Agenda for Sustainable Development [The criteria for arbovirus surveillance and diagnosis must therefore be based on extensive clinical, epidemiological and laboratory findings. Laboratory confirmation is as important in order to distinguish infections with similar clinical manifestations such as dengue fever and other arboviruses. Most low- and middle-income countries do not have systematic processes for collecting, analyzing and putting these data to use, and as such reliable routine data has been identified as one of the main challenges for effective public health action at country level . To bridelopment . Impleme


Rhipicephalus microplus parasitizing cattle in the southern region of Brazil using metagenomics. Our study revealed the presence of viruses that had not been previously described in the region, including lihan tick virus (Phenuiviridae family) and wuhan tick virus 2 (Chuviridae family), as well as expands the biogeography of jingmen tick virus (Flaviviridae family) in Brazil. Also, we described three novel tymoviruses , named guarapuava tymovirus-like 1 to 3. We described the genomic and phylogenetic characterization of these viruses. Our study sheds light on the viral diversity of Rhipicephalus microplus in South America, and also expands the biogeography of tick viruses that were previously described only in Asia.Ticks are ectoparasites spread worldwide and are well known as vectors of many viruses of great importance to human and animal health. However, the viral diversity in ticks is still poorly understood, particularly in South America. Here we characterized the viral diversity present in  Currently, approximately 900 species of ticks have been identified and taxonomically classified into three families: Argasidae, Ixodidae, and Nuttalliellidae3. Viruses transmitted to humans by bites of infected ticks are found mostly in the family Ixodidae, especially in the genera Ixodes, Haemaphysalis, Hyalomma, Amblyomma, Dermacentor and Rhipicephalus1.Ticks are haematophagous ectoparasites that live by feeding on vertebrates, and they are known to be vectors of important human and animal pathogensAsfarviridae, Arenaviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae, Flaviviridae, Nairoviridae, and Phenuiviridae5. Many tick-borne viruses are emerging and re-emerging pathogens, which have caused outbreaks of devastating and often fatal diseases, representing a serious public and animal health problem in many regions of the world. Thrombocytopenia syndrome virus, Heartland and Louping ill7 are examples of emerging viruses, while Crimean-Congo hemorrhagic fever, African swine fever virus and Nairobi sheep disease virus are re-emerging viruses4.To date, at least 38 viral species are known to be transmitted by ticks. They are distributed in eight viral families: Rhabdoviridae, Reoviridae, Flaviviridae, and orders Bunyavirales and Mononegavirales10. Also, novel viruses that have not been classified can represent important pathogens in human or veterinary health, as well as viruses that may be regarded as commensals14.Recently, studies based on high-throughput sequencing (HTS) have revealed an unprecedented diversity of viruses in ticks, including new viral species classified into families Ixodidae ticks have been shown to be distributed in South America, including 21 Amblyomma, two Dermacentor, two Haemaphysalis, one Boophilus, one Ixodes and one Rhipicephalus species3. Despite the widespread of ticks in this region and the high importance of tick-borne viruses, there are only two viruses associated with ticks described in South America: Cacipacoré virus and Mogiana tick virus (MGTV)17.Currently, twenty-eight species of Rhipicephalus microplus parasitizing cattle in the southern region of Brazil. In addition to the virome analysis, we performed experimental infections using different host cell lines to assess the in vitro host range of the viruses that were identified.Here, we applied a metagenomics approach to determine the diversity of viruses present in de novo assembly, the viral contigs represented 0.30 to 24% of all contigs in each library  and five pools of bovine blood (4 to 15 per pool) generated a total of 7,634,294 to 34,753,840 paired-end reads with 68.56% to 95.47% of bases ≥ Q30 with a base call accuracy of 99.90% , 6 to 16 × (segment 2), 5 to 31 × (segment 3) and 10 to 71 × (segment 4). To determine the frequency and geographic distribution of JMTV in cattle, the individual cattle samples were screened by RT-PCR as previously described9. JMTV was detected in 67% (4/6) of tick pools and 14% (5/36) of bovine sera. Interestingly, our phylogenetic analysis revealed that JMTV strains identified in cattle were closely related to strains from ticks sampled from the same geographical regions  of JMTV was detected by RT-PCR at day six post-infection in the first and second passage on C6/36 cells, and in the first passage on BME26 cells. A gradual detachment of the cell layer was observed in C6/36 and BME26 cells during the first passage, although a typical cytopathic effect (CPE) was not observed. Collectively, these results showed that JMTV is present in Phenuiviridae family). Based on BLAST analysis, we identified that this virus shared 97 to 98% amino acid identities with a LITV described in Rhipicephalus microplus collected in China8. Using RT-PCR, we confirmed the HTS results, which LITV was present in 5 out of 6 of tick pools from all states sampled in the southern region of Brazil. However, not a single sample derived from cattle was positive for this virus. Phylogenetic analysis of both RdRP and N proteins reveals that LITV strains form a monophyletic group with other phlebovirus-like viruses with uncertain M segment, showing a clade that is basal to the Phlebovirus genus in the Phenuiviridae family  and large (L) segments of two strains and partial genomes of four strains of a unique species of a phlebovirus-like virus in the tick pools. The nearly full L and S segments presented 6,495 and 1,535 nucleotides in length, respectively , guarapuava tymovirus-like 2 (GTV-like 2) and guarapuava tymovirus-like 1 (GTV-like 3). The nearly complete genome of GTV-like 1 was 6,625 nt long, and the partial genome of GTV-like 2 and GTV-like 3 were 5,256 nt and 4,433 nt long, respectively , and we did not find any positive in cattle sera. Collectively, these results showed that novel lineage of tymoviruses, comprised of viruses referred to as GTV-like 1, 2 and 3, is circulating in the central region of Paraná State in Rhipicephalus microplus ticks.We identified two nearly complete sequences and two partial genomes of three novel species of tymoviruses-like in pools of ely Fig. . The measid Fig. . Phylogeily Fig. . FurtherAnimalia kingdom18. Recently, metagenomic studies have shown that invertebrates exhibit a greater viral diversity than vertebrates, and such ‘virosphere’ might in part reflect the enormous species number and diversity of arthropods19. Ticks are globally spread ectoparasites that act as vectors of human and animal pathogens because they feed on a wide range of animal hosts, including humans, in both wild and urban areas4. Here, we explored and described the virome of Rhipicephalus microplus ticks from cattle in Brazil, including the characterization and molecular epidemiology of viruses from Flaviviridae, Phenuiviridae, and Chuviridae as well as the Tymovirales order.Viruses are the most abundant infectious agents present in all domains of life, and invertebrates comprise the vast majority of the Rhipicephalus microplus ticks in China9. This virus possesses a genome organization composed of four segments, two of which are related to the nonstructural genes of the Flavivirus genus, suggesting the first evidence of a segmented flavivirus-like species. Based on recent studies where other viruses associated with JMTV were identified, the genus Jingmenvirus was proposed within the Flaviviridae family21. Our genomic analysis revealed that JMTV and MGTV, another virus previously detected in Rhipicephalus microplus ticks in Central-West and Southeast region of Brazil, shared 97% to 99% nucleotide identity in all segments, suggesting that they might be the same viral species16. The frequency of JTMV in ticks collected in Brazil was similar to that previously described in China, but we observed a higher prevalence of JMTV in bovine samples in our study compared to the previous report in China with ~14% versus 3.44%, respectively9. Therefore, considering MGTV as the same viral species as JMTV, our results expanded the geographic distribution of JMTV. We also describe for the first time the presence of JMTV in cattle in the American continent. This finding highlights the need for further studies to elucidate the host range of JMTV. Interestingly, the phylogenetic trees showed that the JMTV strains described in Brazil clustered within a monophyletic clade that is distinct from the strains identified in China and Africa, suggesting that JTMV strains circulating in Brazil probably belong to a new genotype21.JMTV is a recently discovered virus identified in 15. On the other hand, although we performed experimental infections on several invertebrate and vertebrate cell lines, JMTV could only be detected in the invertebrate cells C6/36 and BME26. However, further studies must be conducted to elucidate the role of JMTV as a potential tick-borne pathogen.We attempted to isolate JMTV, but it was unsuccessful due to the lack of plaque formation. Furthermore, we could not visualize CPE on different cell lines. However, this is not surprising because issues to grow JMTV in cell lines have been previously reportedPhenuiviridae family. The strains of LITV identified in our study shared high levels of identity at the amino acid level with the prototype virus described in Rhipicephalus microplus in China8. Although we recovered a nearly complete genome of both large and small segments of LITV, we were unable to identify any sequence with similarity to segments that encode the GPC encoded in the medium segment. The same observation was previously described for the prototype virus8. Our molecular epidemiology analysis showed that LITV has a high-frequency distribution in ticks in the southern region of Brazil, while it was unable to be detected in cattle. From the viral propagation attempts, we have detected the genome of LITV only in invertebrate cell lines, suggesting that replication of LITV might be restricted only to invertebrate host, but the attempts of viral isolation were unsuccessful in this study. Interestingly, our phylogenetic analysis showed that LITV strains were clustered in a monophyletic group with other phlebovirus-like viruses with an unrecognized medium segment, which can be a potential new genus in Phenuiviridae family. Recent studies have described some new phlebovirus-like viruses that, like LITV, apparently do not harbor a medium segment. These viruses were detected in ticks collected in the United States of America (USA) and in China22. Several explanations have been suggested in order to understand the detection of a phlebovirus-like virus with only the small and large segments, including: (i) these viruses may have an atypical M segment that is undetected by HTS approach due to its great sequence divergence; (ii) they could actually encode only for the large (RdRP) and small (N protein) proteins while coping with other means for cellular entry, since they do not code for an envelope glycoprotein; (iii) small and large segments may exist in an episome-like form in tick cells, thus may not form an infectious virion, but may instead use transovarial transmission or some other means as a vehicle for their dissemination to new hosts11. Also, LITV could be a retrotransposon-like or an endogenous viral element, which is unlikely because we showed that LITV genomes could be detected in invertebrate cell lines after serial passages, suggesting that the virus is replicating in vitro. We also did not detect the LITV viral genome in BM26, a tick lineage. Unfortunately, we were unable to isolate LITV by plaque-forming assay due to the inability of the virus to replicate in the vertebrate cell lines we used. Therefore, further studies should elucidate an atypical M segment or a new alternative mechanism for viruses with different glycoprotein coding strategies in the Bunyavirales order.LITV is a segmented negative-stranded RNA virus with a linear genome classified into the Chuviridae is a putative novel viral family with representatives of both segmented and unsegmented viruses8. Here, we expanded the geographical distribution of this family, describing a wide circulation of WTV-2 in Rhipicephalus microplus ticks in the southern region of Brazil. This virus is very similar at the amino acid level with the prototype species and formed a monophyletic clade with other chuviruses described in ticks from China and USA13. This viral family is constituted only by viruses described in invertebrates, but we detected the genome of WTV-2 in invertebrate and vertebrate cell lines, as observed in our viral propagation attempts, but the attempts of viral isolation were unsuccessful in this study, and the genome detection can be residual viral RNA from inoculum.The Tymoviridae family , a predominantly plant-infecting virus family and transmitted by phloem-sucking insects23. These viruses, tentatively named GTV-like 1, 2 and 3 constitute a divergent monophyletic group with Bee Macula-like virus and Varroa Tymo-like virus, both unassigned tymoviruses24. The Bee Macula-like virus and Varroa Tymo-like have been observed with high prevalence in honeybees (Apis mellifera L.) and varroa mites (Varroa destructor) in Europe and USA, but the probable vector is Varroa destructor24. Although these viruses could be considered as contaminating plant viruses in our tick samples, the Varroa destructor is an obligate parasite that only feeds on bee hemolymph and has no contact with plants, suggesting that GTV-like 1, 2 and 3 could also be invertebrate viruses that share the same common ancestor. However, further studies are needed to reveal the role of these viruses in the pathogenesis of invertebrate hosts.Our virome analysis also identified three novel viruses characterized as having a similar genome organization from viruses of the 18. Here, although the sample size of ticks used in this study was relatively small, we extended the current viral diversity with the notable characterization of viruses from Rhipicephalus microplus and its parasitized cattle in the southern region of Brazil. We supposed that these viruses identified in our study and previously reported in Asia could result from a deep association or co-evolution with Rhipicephalus microplus, and possibly can be found worldwide in subtropical and tropical regions in this tick species26. This region comprises three different states, covers 576,774 km², and is considered a major production hub of bovine meat for export. Finally, our results showed that viruses from ticks could be spread on multiple continents and also highlight the role of ticks as important viral reservoirs in South America.It has already been reported that the known viral diversity is under-sampled and underestimated27. All samples were stored at −80 °C. All procedures, protocols, and methods performed in this study were approved based on guidelines and regulations of animal research by the Ethics Committee for Animal Research of the UNICENTRO (001/2016).Paired samples (engorged ticks and blood) from 36 cattle were collected between October 2015 to June 2016 from four locations in the southern region of Brazil. The sites of collection were cattle farms in the municipalities of Guarapuava and Manoel Ribas in Paraná State, Lages city in Santa Catarina State, and Ronda Alta county in the Rio Grande do Sul State, all from the southern region of Brazil , date and collection site. Tick pools (~50 engorged ticks per pool) were homogenized with a mortar and pestle in 1 ml of Hank’s buffered salt solution (HBSS). Ticks and bovine sera pools were centrifuged at 10,000 g for 10 minutes at 4 °C, and supernatants were filtered using 0.22 μm-pore-size polyvinylidene difluoride filters  to minimize the presence of cells, debris, and bacteria. Subsequently, the pools were ultracentrifuged at 180,000xg for 3 hours at 4 °C in a 30% sucrose solution , and resuspended in 500 μl of HBSS. To remove the naked DNA and RNA, 200 μl of the resuspended pellet from each pooled sample were digested in a cocktail with 20U of Turbo DNase , 25U of benzonase , and 0.1 mg/ml of RNase A  at 37 °C for 2 hours in 20 μl of 10X DNase buffer . Then, the capsid-protected viral genomes were extracted with a QIAamp viral RNA mini kit . Subsequently, RNA was quantified using a Qubit® 2.0 Fluorometer  and the purity and integrity of RNA samples were measured using an Agilent 2100 Bioanalyzer .de novo using the metaViC pipeline (https://github.com/sejmodha/MetaViC)28. This pipeline is divided into two major components. The first step of the cleaning pipeline is to remove the adapters and short reads (<80 nucleotides) using Trim Galore . The ribosomal RNA (rRNA) sequences are removed using RiboPicker29. After this, DIAMOND30 can be run for each read against the refseq protein database for each file. Krona charts31 are generated for each DIAMOND output file that describes the read based classification of the sample. The DIAMOND results are then converted to a BLAST tabular output and the Genbank Identifier (GI) column from the output is extracted. GIs are mapped back to NCBI taxonomy databases to extract the corresponding taxonomy and division ID. In our case, we are only interested in the sequences that match viruses, environmental sequences and the sequences that do not match anything in the database. Therefore, any read that is matching protein sequences originating from bacteria, invertebrates, mammals, rodents, phages, plants, vertebrates, primates, and synthetic constructs are identified based on the division ID and are filtered from the sample files. Finally, the sample files are properly paired using Prinseq-lite.pl32. These reads can then be submitted to the next stage of the pipeline. Sequence reads from the previous step of the pipeline can be submitted to this script that carries out the de novo assembly using SPAdes34 and IDBA-UD35 with the k-mer values 31, 55, 77, and 99 for SPAdes and a range of the k-mer values starting with minimum k of 31 to maximum k of 99 for IDBA-UD.cDNA was generated from total RNA using the Superscript II kit  according to the manufacturer’s instructions. cDNAs were prepared for high-throughput sequencing using the TruSeq RNA Universal  protocols and standard multiplex adaptors. A paired-end, 150-base-read protocol in the RAPID module was used for sequencing on an Illumina HiSeq. 2500 instrument as recommended by the manufacturer. Sequencing was performed at the Life Sciences Core Facility from University of Campinas, Brazil. The resulting sequencing reads were assembled 36, an assembly merging pipeline that uses mummer to find overlaps between two assemblies and joins them. Reads are aligned back to the contigs (>200 nucleotides), and supercontigs using bowtie237 and unmapped reads are extracted using bam2fastq. In order to check the assembly quality, a QUAST38 analysis is performed for each contig assembly and supercontigs. Contigs longer than a length of 200 and supercontigs generated by GARM are merged and classified using DIAMOND against refseq protein database. Krona tools are used to create interactive HTML output to visualize the BLAST tabular output formatted results generated by DIAMOND. After, we also performed the reference genome with viruses found using Bowtie237.The assembled contigs from two assembly tools are then merged using GARM39 for identification of transmembrane regions and signal peptide, and to NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for identification of glycosylation sites. Annotations of protein domains were performed with InterPro 60.040.Viral genomes were evaluated regarding size and ORF prediction with Geneious 9.1.2 . Protein sequences were submitted to the TOPCONS web server41, and manually edited to removing all ambiguously aligned regions. ML trees were inferred using IQ-TREE version 1.4.3 applying 1,000 bootstraps, substitution models were determined by the best-fit model based on Bayesian Information Criterion considered 144 reversible amino acids substitution models and 88 nucleotides substitution models43. Phylogenetic trees were visualized using FigTree v.1.4.2.Maximum likelihood (ML) phylogenetic trees were inferred using amino acids and nucleotides alignments of viral genomes described in this study with representative members of each viral family. Multiple sequence alignments (MSA) were generated using RevTrans 2.09. Amplicons were visualized by gel electrophoresis in 1.5% agarose gels. All PCR products were verified by dideoxy sequencing using ABI 3730 genetic analyzer .To determine the authenticity and frequency of viral sequences in tick pools and individual cattle samples, we designed primer sets to specifically amplify for each virus identified by HTS , BME26 , BHK-21 (baby hamster kidney), LLC-PK1 (pig kidney cell line), MDBK (bovine kidney), UMNSAH/DF (chicken embryo fibroblast), HEK-293 (human embryonic kidney), Vero (African green monkey kidney) and Vero E6 (a Vero cell line deficient for type I interferon). C6/36 and BME26 cells were propagated as previously described using Leibovitz’s L-15 medium supplemented using 10% heat-inactivated fetal bovine serum, 50 mg/ml of gentamicin and 2 mg/ml of amphotericin B, and maintained at 28 °C or 34 °C, respectively45. Vertebrate cell lines  were propagated as previously described using D-MEM containing 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO249.Various vertebrate and arthropod cell lines were used in experimental infections including: C6/36 (Virus samples were filtered through a 0.22-μm filter, and 250 μl was inoculated onto cell monolayers in T25 flasks. Flasks were gently rocked for 1 hour at 37 °C before 7 ml of the respective culture media containing 4% FBS was added. Inoculated cells were incubated for six days. Viruses were passaged three times in each cell line, and for each passage, RNA was extracted from cells and supernatant. Virus infection was assessed by RT-PCR and Sanger sequencing, as described above.Supplementary Information


Thrips palmi is a widely distributed major agricultural pest in the tropics and subtropics, causing significant losses in cucurbit and solanaceous crops through feeding damage and transmission of tospoviruses. Thrips palmi is a vector of capsicum chlorosis virus (CaCV) in Australia. The present understanding of transmission biology and potential effects of CaCV on T. palmi is limited. To gain insights into molecular responses to CaCV infection, we performed RNA-Seq to identify thrips transcripts that are differentially-abundant during virus infection of adults. De-novo assembly of the transcriptome generated from whole bodies of T. palmi adults generated 166,445 contigs, of which ~24% contained a predicted open reading frame. We identified 1,389 differentially-expressed (DE) transcripts, with comparable numbers up- (708) and down-regulated (681) in virus-exposed thrips compared to non-exposed thrips. Approximately 59% of these DE transcripts had significant matches to NCBI non-redundant proteins (Blastx) and Blast2GO identified provisional functional categories among the up-regulated transcripts in virus-exposed thrips including innate immune response-related genes, salivary gland and/or gut-associated genes and vitellogenin genes. The majority of the immune-related proteins are known to serve functions in lysosome activity and melanisation in insects. Most of the up-regulated oral and extra-oral digestion-associated genes appear to be involved in digestion of proteins, lipids and plant cell wall components which may indirectly enhance the likelihood or frequency of virus transmission or may be involved in the regulation of host defence responses. Most of the down-regulated transcripts fell into the gene ontology functional category of ‘structural constituent of cuticle’. Comparison to DE genes responsive to tomato spotted wilt virus in Frankliniella occidentalis indicates conservation of some thrips molecular responses to infection by different tospoviruses. This study assembled the first transcriptome in the genus Thrips and provides important data to broaden our understanding of networks of molecular interactions between thrips and tospoviruses. Frankliniella occidentalis is world-wide the most devastating invasive species, with a broad host range, transmitting multiple tospoviruses  including the economically important tomato spotted wilt virus (TSWV)  are multi-subunit storage proteins with phenol oxidase activity that are involved in insect innate immunity, humoral immune response to pathogens and disease resistance [2 fold change 1.75  vs 13.31 ] was shown to have phenol oxidase activity in melanogenesis [2 fold change 2.08  vs 2.88 ] has a physiological role in pH and ion regulation pathways. Endoglucanase [log2 fold change 1.2–1.3  vs 1.95 ] is a salivary gland-associated cell wall digestive enzyme [T. palmi transcriptome. Deoxyribonuclease I [log2 fold change 1.31  vs 2.62 ] is an endonuclease that cleaves DNA and has been suggested to play a role in apoptosis [2 fold change of 2.82 associated with vector response to TSWV infection in F. fusca adult transcriptome [The up-regulated genes common to both virus-vector interactions are associated with innate immunity and salivary glands. Hexamerins [logsistance . Similarogenesis . Carbonipoptosis . This ge2 fold change 5.0–7.1  vs 4.5 ], which is a structural constituent of cuticle and alpha-tocopherol transfer-like [log2 fold change 7.0  vs 4.9 ] which has been shown to be involved in antiviral immunity in Drosophila melanogaster [F. occidentalis larvae [T. palmi adults.When down-regulated genes in adult transcriptomes were compared, none of the most highly down-regulated TP transcripts were shared between the two systems. However, genes of endocuticle structural glycoprotein [lognogaster  were dows larvae  and T. pThrips. We analysed T. palmi transcriptome in response to CaCV infection to identify DE transcripts and to classify these genes by sequence homologies (ontologies) to known proteins to begin to dissect the global response of this thrips species to tospovirus infection. Here, we present hypotheses about the effect of virus infection on three dynamic processes in vector biology: innate immunity, growth and development, and fitness.Quantifying changes in global gene expression is one means of inferring cellular and physiological responses of insects to infection by insect-pathogenic viruses or insect-vectored plant-viruses . This isT. palmi. Cathepsins are known to be involved in various biological processes including protein degradation, apoptosis and signalling activated in late endosome and lysosome and implicated in virus entry and replication [Based on Blastx descriptions and GO terms for DE transcripts, we identified 86 up-regulated transcripts putatively associated with innate immune response that may have been triggered by CaCV infection . The majlication , 106. Thlication .T. palmi. Besides pigmentation, melanin deposits and encapsulates foreign targets such as nematodes, parasitoids and virus-infected tissues and inhibits progression of infection [T. palmi transcriptome, we identified several PO genes and a CLIP domain-containing SP that were up-regulated in virus-exposed thrips. Previous studies have shown that PO cascade is activated in mosquitoes in response to Semliki Forest virus infection resulting in inhibition of virus spread in cell culture [Genes associated with melanisation seem to play a significant role in virus-exposed nfection , 107. Ennfection , serine nfection  and laccnfection  are the nfection . In T. p culture , 110. CL culture , 60. In  culture –65.Genes involved in other innate immune system pathways such as Toll, antigen processing and presentation, and complement and coagulation cascades  were also found up-regulated. Among the up-regulated transcripts in CaCV-exposed thrips, 63 encoded proteolytic enzymes, including 30 serine protease and trypsin genes. According to GO assignment all serine proteases and trypsins in this thrips transcriptome were predicted to exhibit serine-type endopeptidase activity. The role that these proteases may play in virus-exposed thrips is not known. Previously it was shown that insect serine proteases and trypsin-like serine proteases are generally involved in hemolymph coagulation, activation of antimicrobial peptide synthesis, and melanin synthesis , 112. ThT. palmi identified activation of different immunity-related pathways than the results reported by Zhang and collaborators [F. occidentalis-TSWV, such as Toll, JAK-STAT and RNA interference. Either these pathways have no significant role in adult T. palmi against CaCV or they were active only in larvae at the time of virus acquisition and initial replication and intercellular movement, which was not captured by our adult transcriptome. In another study, Schneweis and colleagues [F. occidentalis exposed to TSWV, 75% of innate immune response related transcripts were annotated as insect storage proteins, hexamerins which are also associated with humoral immunity. We did not identify hexamerins among the DE transcripts of T. palmi suggestive of activation of different set of genes in T. palmi’s innate immunity. When compared with F. fusca-TSWV adult DE genes, we identified up-regulation of several similar immune system-related genes such as cathepsin B, aminopeptidase N, serine proteases and heat shock protein 70. Therefore, it is likely that more generally in adult thrips infected with tospoviruses, immune reactions like apoptosis, phagocytosis and proteolysis are activated.Our study of borators  for F. olleagues  showed tin silico. All lipase 1 sequences lacked a signal peptide, hence were considered unlikely to be components of saliva and more likely involved in innate immunity. Of the 15 lipase 3 genes, nine sequences contained a signal peptide and a predicted cleavage site at their N-terminus, suggestive of secreted proteins. Lipases have previously been identified to be associated with salivary glands of other insects, including the potato leafhopper (Empoasca fabae) [Bemisia tabaci) [F. occidentalis [T. palmi, contained predicted signal peptides and cleavage sites. Similar proteins were identified in salivary gland transcriptomes of E. fabae and F. occidentalis [Salivation is an essential process in insect feeding , 114 anda fabae) , whitefl tabaci)  and F. odentalis . Other ldentalis , 88. Onedentalis  and the dentalis , 119. UpF. occidentalis [E. fabae [Lygus hesperus [T. palmi transcriptome is consistent with findings in other insects. Based on signal peptide predictions, some of these enzymes may be secreted, a requisite for extra-oral digestion.Plant-feeding insects secrete a range of cell wall-degrading enzymes to release cell contents and to facilitate subsequent ingestion . Pectin dentalis , E. fabaE. fabae  and the hesperus . Other chesperus , 123–125Apis melifera) [Ades aegypti) [Bombyx mori) [Tribolium castaneum) [F. occidentalis [E. fabae, [F. occidentalis salivary gland transcriptome [T. palmi transcriptome.A α-amylase that may be involved in sugar metabolism was identified among up-regulated transcripts in virus-exposed thrips. This sequence lacked an obvious signal peptide, but its GO term ‘extracellular exosome’ suggests extracellular localization and a putative digestive role. α-amylases have been identified in the saliva of many insects including honeybee (elifera) , mosquitaegypti) , silkworyx mori)  and red staneum) , and alsdentalis  and E. f. fabae, . Genes ecriptome  but wereT. palmi ACE may have similar functions.Among the other salivary gland-associated genes identified in this study were angiotensin-converting enzyme-like (ACE) genes which may have a role in inhibition of plant defence , 91. Wan2 fold change >9) in adult and pupal transcriptome of F. fusca infected with TSWV [2-fold change of 2.0. Abundance of Vg transcripts and their receptors in virus-exposed T. palmi implies transovarial transport of egg yolk protein components to the growing oocytes had been promoted in the presence of CaCV infection. Enhanced vitellin and vitellogenin levels in response to a plant virus infection was first reported from whitefly, MEAM1 (B biotype) B. tabaci that fed on tomato yellow leaf curl China virus-infected plants [F. occidentalis [Recilia dorsalis that fed on rice gall dwarf virus (RGDV)-infected plants showed significantly reduced longevity and fecundity associated with reduced levels of Vg compared to non-viruliferous individuals [Among the up-regulated genes in CaCV-exposed thrips, 54 transcripts represented vitellogenin (Vg) homologues . Vg contith TSWV . The priith TSWV . In femaith TSWV . Accumulith TSWV . Among td plants . These adentalis . In contividuals .Dialeurodes citri) [A. aegypti [Apis mellifera) [T. palmi may play a role in innate immunity to CaCV infection. In support, Vg was found to be 9-fold up-regulated in pupae of F. fusca exposed to TSWV [Laodelphax striatellus) [T. palmi, Vg was the most abundant transcript in virus-exposed small brown planthopper transcriptome [T. palmi cannot be excluded due to a current lack of knowledge in tissue tropism and virus localization in the body of T. palmi. Furthermore, Vg was not among the most abundant unigenes and DE genes in F. occidentalis transcriptome infected with TSWV [T. palmi interaction, but the exact role that Vg may play needs to be investigated further.There are other reports that have shown involvement of Vg in immune responses of insects including citrus whitefly (s citri) , A. aegy aegypti  and honellifera) . Based o to TSWV . Anotheratellus) . Similarcriptome . Previoucriptome . Furtherith TSWV . TherefoT. palmi and estimated differential abundance of transcripts when exposed to CaCV infection. The majority of DE transcripts did not contain Blastx annotations or conserved functional domains, but their differential abundance reflects unique roles in T. palmi-virus interaction and T. palmi biology in general. Although we cannot directly compare our data with studies of F. occidentalis [F. fusca [T. palmi appeared to be species-specific. Aside from the inherent differences across thrips species and tospoviruses species, differences in the experimental methods and computational protocols and parameters used in data analyses could explain the weak overlap in transcripts responsive to virus across the three studies. For example, DE analyses for F. fusca [T. palmi were performed using de novo transcriptome assemblies, whereas Schneweis and colleagues [F. occidentalis.In summary, we have assembled a whole-body transcriptome of adult dentalis  and F. fF. fusca  exposed F. fusca  and T. plleagues  used a dWe identified virus-responsive up-regulated DE transcripts that have putative roles in thrips innate immunity, feeding and fecundity. Down-regulated transcripts that are likely associated with structural component of cuticle and integral components of membrane may be responsible for delaying thrips development and increased longevity to ensure virus persistence and transmission. However, functions of those genes need to be experimentally validated in future. For example, transcripts associated with thrips feeding and fecundity could be candidates for potential targets in thrips and tospovirus management. In addition, transcriptomic data generated in this study will enrich genomic information of thrips and will allow functional studies on other economically important thrips and insects more broadly.S1 Table(DOCX)Click here for additional data file.


Caliciviridae family comprises four other accepted genera: Sapovirus, Lagovirus, Vesivirus, and Nebovirus. There are six new genera proposed: Recovirus, Valovirus, Bavovirus, Nacovirus, Minovirus, and Salovirus. All Caliciviridae have closely related genome structures, but are genetically and antigenically highly diverse and infect a wide range of mammalian host species including humans. Recombination in nature is not infrequent for most of the Caliciviridae, contributing to their diversity. Sapovirus infections cause diarrhoea in pigs, humans and other mammalian hosts. Lagovirus infections cause systemic haemorrhagic disease in rabbits and hares, and vesivirus infections lead to lung disease in cats, vesicular disease in swine, and exanthema and diseases of the reproductive system in large sea mammals. Neboviruses are an enteric pathogen of cattle, differing from bovine norovirus. At present, only a few selected caliciviruses can be propagated in cell culture (permanent cell lines or enteroids), and for most of the cultivatable caliciviruses helper virus-free, plasmid only-based reverse genetics systems have been established. The replication cycles of the caliciviruses are similar as far as they have been explored: viruses interact with a multitude of cell surface attachment factors (glycans) and co-receptors (proteins) for adsorption and penetration, use cellular membranes for the formation of replication complexes and have developed mechanisms to circumvent innate immune responses. Vaccines have been developed against lagoviruses and vesiviruses, and are under development against human noroviruses.Besides noroviruses, the  Caliciviridae family. Murine norovirus (MuNoV) has received particular attention, since it can be propagated in cell culture [Caliciviridae family other than noroviruses*.Noroviruses (NoVs) are a major cause of epidemic acute gastroenteritis (AGE), and most chapters of this Special Issue are devoted to the structure, replication, pathogenesis, immune responses, epidemiology/evolution, and vaccine development relating to viruses of this genus of the  culture , can be  culture ,3,4,5 an culture ,7,8,9. H culture  or in hu culture . Reverse culture . Vaccine culture . The preCaliciviridae other than noroviruses complements the many contributions to NoV research in this Special Issue of Viruses which relate to:NoV structure ;The molecular biology of NoV replication ;NoV replication in the immunocompromised host ;Factors affecting host susceptibility to NoV infection ;In vitro propagation of NoVs ;Innate and acquired immune responses to NoV infections ;Molecular epidemiology of HuNoVs ;Use of animal models of NoV infection ;Treatment and development of antivirals ;Vaccine development .*The review on Caliciviridae family contains five genera: Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus. Their taxonomy including prominent virus species is presented in Recovirus, Valovirus, Bavovirus, Nacovirus, Minovirus and Salovirus, have been proposed  was found in MuNoV, encoding a virulence factor, VF1. The other genera  possess a large ORF1 and a standard ORF2 ; ORF3  has been proposed for sapovirus.The genome organisation of the different calicivirus genera  are members of the only other and bats . Like Noand bats ,38, fourand bats . Pairwisand bats   can be propagated in LLC-PK cells in the presence of intestinal content or bile acids , attemptSaVs of different genogroups and genotypes co-evolve in humans, preferential infection with particular genotypes, such as with HuNoV GII.4, has not been observed . The oveRecently, a nucleoside acting as a viral polymerase inhibitor has been identified, which blocks the transcription of MuNoV, rotavirus, and SaV genomes in cell culture and—for rotavirus—acts as an effective antiviral in an adult mouse model .Lagovirus contains two main species, Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV). Due to a relatively close genomic relationship of RHDV and EBHSV, it has recently been proposed to subsume them under one species, subdivided into two genogroups  and several genotypes within genogroups [in vitro [Lagovirus has been described [The genus nogroups . Lagovirnogroups ,61,62,63nogroups . After enogroups ; the binnogroups . Followinogroups , and intnogroups . The virnogroups . RHDV cain vitro . A reverescribed .RHDV grows in multiple organs of infected animals, mainly in the liver, but also in the spleen and blood mononuclear cells, and causes disseminated intravascular coagulation, leading to multiple organ haemorrhages and necroses ,72. RHDVFeline calicivirus (FCV) is the major representative of this Calicivirus genus, causing mainly upper and rarely lower respiratory tract diseases. Some virulent strains cause systemic disease, including subcutaneous oedema, necroses in multiple organs  and interstitial pneumonia [Vesicular exanthema of swine virus, which causes vesicular disease and foetal damage in swine [San Miguel sea lion virus, which produces exanthemas and diseases of the reproductive system in large sea mammals [neumonia ,85. Untineumonia , and alsneumonia . Other vin swine ,89, and  mammals ,91.FCV enters cells by first binding to α2,6-linked sialic acid as an attachment receptor and then interacts with the feline junctional adhesion molecular A (fJAM-A), which serves as a functional co-receptor ,92. ReceDue to its longstanding cultivability, there is more detailed information on the FCV replication cycle. Conserved amino acid residues in the capsid protein of FCV were identified which were essential for interaction with fJAM-A . KnockdoVesiviruses have also been recognized as infectious agents for household dogs in Italy  and moreThe Hom-1 calicivirus, isolated from a human laboratory worker in 1998 as an inadvertent transmission of the San Miguel sea lion virus , was fouSome authors have suggested that vesivirus infection in humans may be quite common and possibly be associated with hepatitis of unknown but suspected infectious cause ,114.Nebovirus, of the Caliciviridae [Caliciviridae. Infection of calves with bovine NoV and nebovirus appears to be common in the USA [In 2006 the sequence of a cattle-pathogenic enteric virus, Newbury agent-1, was determined and proposed as a new genus, iviridae . Neboviriviridae . Recombiiviridae ,118, sim the USA , Brazil  the USA , Turkey  the USA , Iran [1 the USA , Korea [ the USA , and Chi the USA ,125.Recovirus [A rhesus macaque was the source of a novel calicivirus, named Tulane Virus (TV), which although most closely related to NoVs, was sufficiently different in nucleotide sequence from other caliciviruses to justify classification in a new genus, ecovirus . Recovirecovirus , and difecovirus . Tulane ecovirus ,129,130.ecovirus . Biologiecovirus . Recovirecovirus . Recovirecovirus , and rececovirus . A recovecovirus .Valovirus [Between 2005 and 2007, caliciviruses isolated from pigs in Canada were proposed as a new genus, alovirus   and Nacovirus (turkey) were proposed [Between 2009 and 2011 caliciviruses were isolated from chicken and turkey from German farms. Phylogenetic analysis of partial deduced amino acid sequences of the non-structural protease and polymerase genes (320–350 aa) and of the VP1 gene (530–700 aa) strongly suggested the presence of new genera, and the names proposed . Of 24 cproposed . A naco-proposed   and Minovirus (from bitterfish) [Novel caliciviruses have been isolated from fish and have been proposed as new genera: terfish) ,31.The ability to propagate viruses in cell/enteroid culture;The availability of helper virus-free (plasmid only-based) reverse genetics systems;The availability of suitable animal models.Major progress in calicivirus research has been made by the availability of a combination of tools:Caliciviridae is summarized in Lagovirus and Vesivirus, with the other genera requiring further investigation. Recombination in nature has been documented for Sapovirus, Lagovirus, and Nebovirus, and thus seems to be a general feature of the caliciviruses. Since many genera of the Caliciviridae contain important human and animal pathogens, future research will be driven by scientific curiosity as well as public health and economic needs.These tools have been achieved for MuNoV and are close to being fulfilled for HuNoVs. The degree to which these pre-conditions have been accomplished for the other genera of the


Gaussia luciferase (Gluc) gene. Using reverse genetics, NY-r19-Gluc, a replication competent reassortant influenza A subtype H3N2 virus expressing reporter Gluc was successfully generated. This reporter virus is stable during replication in Madin-Darby canine kidney (MDCK) cells, and preliminary studies demonstrated it as a useful tool to evaluate antivirals. In addition, NY-r19-Gluc virus will be a powerful tool in other studies including the application of diagnostic and therapeutic antibodies as well as the evaluation of novel vaccines.Reporter influenza A viruses (IAVs) carrying fluorescent or luminescent genes provide a powerful tool for both basic and translational research. Most reporter IAVs are based on the backbone of either subtype H1N1 viruses, A/Puerto Rico/8/1934 (PR8) or A/WSN/1933, but no reporter subtype H3N2 virus is currently available to our knowledge. Since the IAV subtype H3N2 co-circulates with H1N1 among humans causing annual epidemics, a reporter influenza A subtype H3N2 virus would be highly valuable. In this study, the segments of A/Wyoming/3/03  virus encoding hemagglutinin and neuraminidase, respectively, were reassorted with the six internal genes of PR8 where the NS gene was fused with a  Orthomyxoviridae of enveloped viruses, and the genome consists of 8 negative-sense, single-stranded viral RNA (vRNA) segments, coding for at least 11 proteins  here) and parental virus A/PR8 stocks were produced as previously described .The rescue plasmids for the internal segments of the PR8 backbone, including pDZ-PR8/PB2, -PR8/PB1, pDZ-PR8/PA, -PR8/NP, -PR8/M, -PR8/NS, and -PR8/NS-Gluc were used as previously described . The resThe plasmids pDZ-NY/NS-Gluc were constructed as follows:(i) Construction of pDZ-NY/NS-SAnull in which the splicing acceptor site was disruptedSap I linearized pDZ plasmid, and the resulting plasmid carries what is herein referred to as the NS ‘Splice Acceptor null’ (SA-null) gene.The NY/NS-SAnull-left and NY/NS-SAnull-right fragments were amplified from pDZ-NY/NS using primer pairs 5′-INFU-NCR-NYNS1-F/3′-SDM-NYNS-SAnull-R and 5′-SDM-NYNS-SAnull-F/3′-INFU-NYNEP-NCR-R respectively. Using an In-Fusion cloning kit, the NY/NS-SAnull-left and NY/NS-SAnull-right fragments were cloned into (ii) Construction of pDZ-NY/NS-GlucUsing pDZ-NY/NS-SAnull as a template, fragments NY/NS-Gluc-1 and NY/NS-Gluc-3 were amplified by primer pairs 5′-INFU-NCR-NYNS1-F/3′-INFU-NYNS1-nostop-R and 5′-INFU-NYNEP-F/3′-INFU-NYNEP-NCR-R respectively.NY/NS-Gluc-2 was amplified from pDZ-PR8/NS-Gluc using primers 5′-INFU-Gluc-F and 3′-INFU-Gluc-2A-R.Sap I linearized pDZ plasmid.Using an In-Fusion cloning kit, the NY/NS-Gluc-1, NY/NS-Gluc-2 and NY/NS-Gluc-3 fragments were cloned into All primers used in this process are listed in As described previously, wild-type A/NY (H3N2) was generated by using the reverse genetics system with indicated rescue plasmids . BrieflyIn order to generate reporter IAV H3N2, three strategies were employed respectively.Gaussia luciferase, which is the same as the strategy used to construct PR8/NS-Gluc previously described [First, pDZ-NY/NS-Gluc instead of pDZ-NY/NS was used to generate a recombinant virus in which the NS gene of the NY virus was fused with the reporter gene escribed . The recSecond, the NS segment of the NY virus was replaced with PR8/NS-Gluc, and the virus was designated as NY-r18-Gluc.Third, the six segments other than HA and NA of the NY virus were replaced with those of PR8-Gluc, and the virus was designated as NY-r19-Gluc. In parallel, a reassortant NY-r19 virus was generated by replacing the six internal segments of NY with those of wild-type PR8 virus.50 values were determined using MDCK cells and the titer was calculated by the Reed–Muench method [The TCIDh method .50 per cell. After incubation at 37 °C for 1 h, the cells were washed and fresh medium was added. The viruses were harvested respectively when most cells showed the cytopathic effect (CPE) and titrated for TCID50 values.The viruses of NY, NY-Gluc, NY-r18-Gluc, NY-r19, and NY-r19-Gluc were propagated in MDCK cells. MDCK cells of 80% confluence grown in 10 cm dishes were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.01 TCIDIn order to test the genetic stability of reporter IAV NY-r19-Gluc, the virus was serially passaged in MDCK cells eight times, labeled as P1 to P8 accordingly.A multicycle replication assay was performed to compare the replication properties of the indicated viruses in vitro. Briefly, MDCK cells were seeded into 24-well plates 1 day before inoculation with the indicated viruses at a MOI of 0.01. After incubation at 37 °C for 1 h, the cells were washed, followed by addition of fresh medium. At various time points, aliquots were removed for determination of Gluc activity and viral titers.Gaussia luciferase assay was performed as previously described [The escribed . BrieflyMDCK cells grown in 24-well plates were infected with NY-r19-Gluc virus at a MOI of 0.1. After 1 h of incubation at 37 °C, the cells were washed, and fresh Opti-MEM containing 2 µg/mL TPCK-trypsin was added. For antiviral determination, indicated concentrations of TBHQ or arbidol were present all through the process. At around 72 h post-infection (p.i.), aliquots were removed for the luciferase assay.Previously, a reporter influenza A subtype H1N1 virus PR8-Gluc was successfully generated by fusing a Gluc gene to the NS1 open reading frame . In thisTo generate the reporter influenza A subtype H3N2 virus, another strategy was explored based on previous work by Fukuyama et al. . They suAs shown in However, an altered CPE upon NY-r18-Gluc infection into MDCK cells was observed. As shown in X-31, which is a 6:2 genetic reassortant encoding the HA and NA genes of A/Hong Kong/1/1968 (H3N2) in a backbone comprising the six internal genes of PR8, is often used in studies requiring an IAV H3N2 subtype ,41,42,43in vitro. The CPEs in MDCK cells respectively infected with NY-r19-Gluc of early and late passages were also compared, and no difference was detected (The stability of Gluc in the reassortant virus NY-r19-Gluc was determined by serial passages in MDCK cells. As shown in detected cTogether, these results show that reassortant NY-r19-Gluc virus is a good candidate as a reporter influenza A subtype H3N2 virus for further characterization and application.To determine the replication properties of the recombinant virus, the growth kinetics of NY-r19-Gluc virus was evaluated and compared to that of wild-type NY and NY-r19. As shown in Collectively, these data demonstrate that the reporter influenza A subtype H3N2 virus NY-r19-Gluc is replication-competent, and viral proliferation can be monitored by the Gluc assay.Considering that HA is a promising target for antiviral development and given the subtype-specificity of HA inhibitory small molecules, we tested NY-r19-Gluc against some group-2-specific HA inhibitors. To achieve this, MDCK cells were infected with NY-r19-Gluc at a MOI of 0.1, in presence of TBHQ, an inhibitor specifically targeting group-2 HAs, and arbidol, a broad spectrum HA inhibitor, respectively. At 72 h p.i., the expression level of Gluc was determined to monitor virus replication.50 of 3.4 μM and 0.9 μM, respectively. In contrast, PR8-Gluc virus, which possesses a group-1 HA, was inhibited by arbidol but not by TBHQ. These results indicate that the NY-r19-Gluc virus can be used to evaluate IAV antivirals.As shown in Reporter viruses provide a powerful tool for basic virology studies as well as antiviral development. However, the introduction of foreign genes or modifications of existing viral genes can lead to altered the virological properties, such as delayed replication kinetics or reduced virulence; moreover, such alterations can create an evolutionary pressure, leading to loss of the reporter genes . In the A second strategy using the PR8 NS gene instead of that of the NY virus to express NS1-Gluc chimeric protein was also explored in this study, and a reporter influenza A subtype H3N2 virus NY-r18-Gluc was successfully rescued. This reporter virus can express a high level of Gluc protein, and the reporter gene is adequately maintained upon passaging in MDCK cells b. Howevein vitro; furthermore, the expression level of Gluc protein can be used to monitor virus replication accurately (Using a third strategy, we successfully generated a reporter influenza subtype H3N2 virus NY-r19-Gluc, which is a 6:2 genetic reassortant encoding the HA and NA genes of the NY virus in a backbone comprising the six internal genes of PR8, where a Gluc gene is fused to the NS segment as previously described . Althougcurately .50 values comparable to the results using traditional methods [The usefulness of the NY-r19-Gluc virus as a tool to evaluate subtype-specific HA inhibitors was validated with TBHQ, an inhibitor specifically targeting group-2 HAs, and arbidol, a broad-spectrum HA inhibitor ,22,24. T methods ,46,47. SIn summary, a stable replication-competent reassortant influenza A subtype H3N2 virus carrying a Gluc gene was generated here. This reporter virus can be adapted as a powerful tool, providing simple and robust assays especially for IAV studies of subtype-specificity.


Sand fly–borne phleboviruses are associated with febrile diseases and nervous system infections in the Mediterranean basin. Sandfly fever was first reported in the Balkan Peninsula at the end of the 19th century. Since then, accumulating data show that the Balkan Peninsula, as a transboundary region between Asia and Europe, plays a major role in the emergence of vectorborne diseases in Europe. To provide an inclusive approach, we collected published data on phleboviruses in the Balkan countries and used them to evaluate the impact of these pathogens from virologic, epidemiologic, and public health perspectives. Recent findings show a high diversity of phleboviruses belonging to 3 species or serocomplexes circulating heavily in the Balkans. Focusing on undisputable human pathogens, we found direct and indirect laboratory documentation for Toscana virus, Sandfly fever Sicilian virus, and Adria virus. These data demonstrate that the Balkans are a hotspot for phleboviruses transmitted by sand flies. Phlebovirus, family Phenuiviridae, order Bunyavirales) are 80–120 nm in length and display helical symmetry. Their genome consists of 3 segmented negative-sense single-stranded RNA: the large segment encodes the viral RNA polymerase (RdRp), the medium segment encodes envelope glycoproteins (Gn and Gc), and the small segment encodes nucleocapsid protein (N) and nonstructural protein (NS) , which is still a tentative species. SFSV and SFNV are both responsible for sandfly fever, a self-limiting but incapacitating febrile illness. Toscana virus (TOSV), discovered in 1971, was incriminated as causing central and peripheral nervous system infections in 1983  to collect all the sand fly–borne phlebovirus data from the Balkan region. In addition, we investigated libraries and other national resources to identify books and conference reports that are not accessible on web-based resources. We used the keywords “sand fly,” “Phlebovirus,” “Bunyaviridae,” “Phenuiviridae,” “sand fly fever,” “pappataci fever,” “three-day fever,” “sandfly fever,” “Toscana virus,” “Sicilian virus,” “Naples virus,” “SFSV,” and “SFNV” matched with “Balkan,” “Balkan Peninsula,” “Yugoslavia,” “Slovenia,” “Croatia,” “Bosnia-Herzegovina,” “Macedonia,” “Republic of Macedonia,” “Former Yugoslav Republic of Macedonia,” “FYROM,” “RoM,” “Albania,” “Bulgaria,” “Greece,” “Montenegro,” “Romania,” “Moldova,” “Serbia,” and “Kosovo” for the research. After gathering all the data, we discarded the irrelevant publications. We put the collected data in order based on country, year, and the phlebovirus species complex.We searched global web-based resources (PubMed [https://www.megasoftware.net/). We constructed a phylogenetic tree using the neighbor-joining method , which was clearly distinct from lineages A and B  Table. S Croatia  Table. Salehabad phlebovirus species. Adria virus RNA was detected in the blood of a 2.5-year-old patient with febrile seizures in Greece  was detected from 2 pools of  in 2015  Table. Sd Serbia  Table. P. perfiliewi sand flies from the Republic of Macedonia in 2015. Sequence analysis demonstrated that BREV is most closely related to Adana virus, which was isolated in Turkey from field-collected sand flies in 2012. In Croatia, Zaba virus (ZABAV) was isolated from P. neglectus sand flies. ZABAV is most closely related to Adria virus and Salehabad virus. Both BREV and ZABAV belong to the Salehabad phlebovirus species  was isolated in  species  Table.SFSV and SFNV are both responsible for a febrile illness that is self-limited but incapacitating, with signs that are commonly observed in arboviral diseases, such as fever, headache, malaise, photophobia, myalgia, and retroorbital pain. From a clinical perspective, it is impossible to distinguish SFNV from SFSV infections, and also to discriminate between SFNV/SFSV and other arboviral infections. As mentioned previously, historic records were based on clinical and epidemiologic evidence, but virological documentation was lacking for studies before the 1950s  Table. Although SFSV and SFNV infections are clinically indistinguishable from each other, they are caused by genetically and antigenically different viruses. Infection with SFNV does not induce cross-protection against SFSV and vice versa , future actions should be directed at implementing direct and indirect diagnosis of Salehabad phlebovirus species in clinical microbiology laboratories to better understand their potential public health impact.Adria virus was the first member of the The fall of communism, the breakup of the former Yugoslavia, and the following civil war and other climatic and environmental changes resulted in an increase of zoonotic infections that emerged or reemerged in the Balkans (In summary, when historical and recent data are compiled, it appears that the Balkan region is a hotspot for viruses transmitted by sand flies, including those that cause diseases in humans. The variety of different viruses is higher than in other regions that were investigated, and certain areas display sympatric circulation of several viruses. Circulation of these viruses must be assessed by studies conducted in human populations and vertebrates, and diagnosis of human infections caused by sand fly–borne viruses must now be implemented using molecular and serologic tools in clinical microbiology laboratories.Results of literature search for study of sand fly–borne phleboviruses in the Balkan region.


Citrus tristeza virus (CTV) produces a non-coding subgenomic RNA referred to as low-molecular-weight tristeza 1 (LMT1), which for a long time has been considered as a by-product of the complex CTV replication machinery. In this study, we investigated the role of LMT1 in the virus infection cycle using a CTV variant that does not produce LMT1 (CTV-LMT1d). We showed that lack of LMT1 did not halt virus ability to replicate or form proper virions. However, the mutant virus demonstrated significantly reduced invasiveness and systemic spread in Nicotiana benthamiana as well as an inability to establish infection in citrus. Introduction of CTV-LMT1d into the herbaceous host resulted in elevation of the levels of salicylic acid (SA) and SA-responsive pathogenesis-related genes beyond those upon inoculation with wild-type (WT) virus (CTV-WT). Further analysis showed that the LMT1 RNA produced by CTV-WT or via ectopic expression in the N. benthamiana leaves suppressed SA accumulation and up-regulated an alternative oxidase gene, which appeared to mitigate the accumulation of reactive oxygen species. To the best of our knowledge, this is the first report of a plant viral long non-coding RNA being involved in counter-acting host response by subverting the SA-mediated plant defense.During infection,  Adeno- and Herpesviridae families and non-coding subgenomic RNAs (sgRNAs) that are generated by positive-sense RNA viruses of the family Flaviviridae (reviewed in [Cauliflower mosaic virus (CaMV), a pararetrovirus from the family Caulimoviridae with a nicked, double-stranded circular DNA genome  ,2,3. WhiIn plants, two major mechanisms mediate antiviral defense. The first is RNA silencing, which is triggered by viral double-stranded RNA that are processed into siRNAs of 21–24 nt in size by the host RNA silencing machinery, which in their turn guide degradation of RNA sequences in a homology-dependent manner ,6. To ovThe second mechanism involves phytohormone , 2-hydroxybenzoic acid)-mediated signaling pathways. SA is a multi-faceted hormone, which plays a role in plant development and response to biotic stress , one of the largest plant RNA viruses, which belongs to the family Closteroviridae, produces a plethora of different RNA species in the infected cells. Those include a positive-strand genomic RNA and its complementary negative-strand copy, more than 30 3′- and 5′-terminal sgRNAs of both polarities, a number of defective RNAs, which sequence composition, size, and amount greatly vary depending on the virus strain and virus-propagating host, as well as a large number of viral siRNAs that are generated by the host RNA silencing machinery in response to virus invasion [invasion ,25,26,27invasion . The othinvasion ,24,25,28invasion ,30; p20 invasion ; and p33invasion ,33. EachBeet yellows virus [In addition to the above sgRNA species, two unusual small (~ 750 nt and ~ 650 nt) 5′ co-terminal positive-strand sgRNAs referred to as low-molecular-weight tristeza 1 and 2  are abundantly produced during CTV infection ,34,35,36ws virus . Althougws virus . Importaws virus . On the ws virus .Nicotiana benthamiana or form proper virions. Yet, the level of the CTV-LMT1d accumulation was reduced compared to that of the parental wild-type (WT) CTV. Moreover, the inability to produce LMT1 resulted in a significant decrease in the virus invasiveness and systemic spread in this herbaceous host. The observed phenotypic differences correlated with a notable difference in the SA-mediated host immune responses to the wild type and the LMT1-deficient viruses, implicating the LMT1 RNA in counter-acting host defenses. Furthermore, LMT1 was found indispensable for the CTV infection of the natural citrus host. Taken together, these results suggest that CTV has evolved the lncRNA LMT1 to evade host immune system in order to facilitate virus infection. To the best of our knowledge, this is the first report for a plant viral lncRNA being involved in subverting the SA-mediated plant defense.In this study, we examined a role of LMT1 in CTV infection using a virus mutant that does not produce LMT1 (CTV-LMT1d). Lack of the LMT1 production did not halt virus ability to amplify in the initially inoculated cells of AscI to Bsu36I restriction endonuclease recognition sites was synthesized with the alteration of six nucleotides  using custom gene synthesis service and cloned into the pUC58 vector . The synthesized fragment in the pUC58 vector was digested using AscI and Bsu36I restriction endonucleases and ligated into the corresponding region of pCTV-GFP, which was digested with the same restriction endonucleases. The virus constructs obtained were screened by restriction endonuclease (EcoRV) digestion pattern and confirmed by sequencing. The engineered CTV variants were used for inoculation of N. benthamiana and Citrus macrophylla plants as described below. Conservation of the parental sequence including introduced mutations in the virus progeny was verified by sequence analysis of the reverse transcription polymerase chain reaction (RT-PCR) products obtained using total RNA extracts from virus-inoculated plants and primers corresponding to the modified region. Sequence analysis was performed at Macrogen Inc. .pCTV-LMT1d was generated using an infectious cDNA clone of the T36 isolate of CTV tagged with the green fluorescent protein (GFP) ORF placed under the CaMV 35S promoter in the binary vector pCAMBIA1380  to cytosine (C) at nt 109 was amplified by overlap-extension polymerase chain reaction (OE-PCR) using two pairs of primers 35S-F and OE-R1 (5′-AGTGTAATTTTTGTTGTGGGAATATTA-3′) for fragment 1 or OE-F2 (5′-TATTCCCACAACAAAAATTACACTACGTCGAAACTC-3′) and LMT1-R for fragment 2. Two overlapping fragments were used as templates to amplify the final product using a pair of primers (35S-F and LMT1-R). The substitution fragment of LMT1M2 containing an alteration of Ts to Cs at nt 109 and 265 was synthesized through custom gene synthesis service and cloned into the pUC58 vector . The synthesized fragment in the pUC58 vector was used as a template to amplify a fragment to substitute the corresponding region of pLMT1. The oligonucleotide primers 35S-F and LMT1-R were used to amplify the fragment. Both LMT1M1 and LMT1M2 fragments were subsequently substituted in pCAMBIA to generate the final product as described above for pLMT1.For the construction of pLMT1, a fragment of the LMT1 region placed under the CaMV 35S promoter sequence was amplified using pCTV-GFP as a template with a pair of primers annealing to the 35S promoter and nt position 800 in the CTV genome  and subsequently substituted using an In-Fusion HD Cloning Kit  for the corresponding fragment excised from the linearized pCAMBIA vector by digestion using restriction endonucleases Agrobacterium tumefaciens EHA105 was transformed by heat shock with the cloned plasmids, and the resulting transformants were selected on the Luria–Bertani agar plates containing two antibiotics (50 µg/mL rifampicin and 25 µg/mL kanamycin). Over-night cultures of a selected single colony were gently resuspended in a buffer containing 10 mM 2-(N-morpholino) ethane sulfonic acid , 10 mM MgCl2 and 150 mM acetosyringone at the optical density (OD)600nm = 0.1 or 1.0. Following three-hour incubation at room temperature without shaking the suspension was infiltrated into three-week-old N. benthamiana plants using needless syringe.Agroinfiltration of CTV constructs was conducted as previously described . BrieflyN. benthamiana plants tested positive for CTV infection by RT-PCR and fluorescence microscopy were harvested at seven dpi and used for virion extraction as previously described [C. macrophylla trees. The concentration of the prepared virions was adjusted based on the enzyme-linked immunosorbent assay (ELISA) to the absorbance value (A)405 nm = 2.5 to ensure the introduction of an equal amount of inoculum. The inoculated citrus plants were maintained under the greenhouse conditions until further analysis.The infiltrated leaves of escribed . The extN. benthamiana plants was observed using a hand-held ultraviolet (UV) lamp  set 6 inches above the plants in a dark room. Leaves of N. benthamiana plants and bark tissue from citrus trees were examined for GFP fluorescence using a Leica MZ10F fluorescence dissecting stereomicroscope with an attached Leica DFC310FX camera  and processed using Leica LAS-X (Leica Microsystems).GFP fluorescence in the infiltrated leaves of C. macrophylla or leaf discs (1 cm in diameter) from N. benthamiana was ground in liquid nitrogen, and total RNA was extracted using the Trizol Reagent  according to the procedure of the manufacturer. RNA extracts were resuspended in 100 µL of RNase-free water. RNA concentration was measured using a NanoDrop Ultraviolet–Visible (UV–Vis) Spectrophotometer . The RNA extracts were diluted with RNase-free water to adjust concentration to 100, 10 and 1 ng/µL. Aliquots were stored at −80 °C until use.150 mg of bark tissue collected from young flushes of Total RNA (0.5 µg) was separated by electrophoresis in formaldehyde denaturing 1% agarose gels in MOPS buffer  and transferred to Biodyne™ B nylon membranes  using Trans-blot SD semi-dry transfer system . Hybridization of digoxygenin (DIG)-labeled riboprobes specific to the positive-strand RNA corresponding to the 5′ or 3′ end of the CTV genome generated as described in Satyanarayana et al.  was carrTM Real-time PCR detection system (BIO-RAD) using the SuperScript® III One-Step RT-PCR kit  with 2 µL of total RNA extract (1 ng/µL), 5 µL of 2× reaction mix, 0.4 µM of the forward  and reverse primers  corresponding to the intergenic region between the RNA-dependent RNA polymerase and p33 ORFs of CTV and 0.1 µM of 6-FAM/BHQ-1 labeled Taqman probe (5′-AGCAACCGGCTGATCGATTGATT-3′) in a total reaction volume of 10 µL. Cycling conditions included reverse transcription at 50 °C for 5 min, incubation at 94 °C for 2 min, and 40 cycles of 94 °C for 10 s and 60 °C for 40 s. Control samples in each run included total RNA from healthy citrus, water control, and at least two RNA transcript dilutions generated for the standard curve.Viral load in the inoculated plants was analyzed by RT-quantitative (q)PCR using total RNA as a template and determined using a standard curve as previously described . BrieflyC. macrophylla or leaf tissue from N. benthamiana plants ground in 5 mL of the extraction buffer  per sample was used for double-antibody sandwich ELISA as per manufacturer’s instructions  to confirm presence of viruses.0.25 g of bark tissue from young flushes of http://rsbweb.nih.gov/ij) directly from the images taken. For each mutant virus, over 100 flexuous virions were measured and up to 10 longest virions were selected to calculate the mean value.10 μL-drops of virion extracts were placed on the 400 mesh copper grids  for negative contrast electron microscopy. After 5 min incubation, the virion solution on each grid was removed using filter paper. The grids were stained using 1% uranyl acetate dissolved in distilled water for 30 s followed by drying in the air. Images were taken using Hitachi H-7000 transmission electron microscope  equipped with a Veleta (2k × 2k) CCD side mount camera  at 100 kV. The length of virions was measured using Image J software (2). Upon the addition of 5 units of micrococcal nuclease , digestion was carried out by incubation in 37 °C water bath for an hour. A “stop solution”  was added to halt the reaction.Virions prepared as described above were diluted (1 mg/200 μL) in nuclease digestion buffer  as per the manufacturer’s instructions. Briefly, 2 µL of total RNA (10 ng/µL) extracted as described above with a pair of 0.5 µM oligomers for PR-1a/c, PR-2 and PR-5 [NbAOX1 [TM Real-time PCR detection system (BIO-RAD). A pair of oligomers amplifying ubiquitin (UBQ) gene  was used as an internal control. The cycling conditions included reverse transcription at 55 °C for 10 min, incubation at 95 °C for 2 min, and 40 cycles of 95 °C for 10 s and 60 °C for 60 s. Ct values were averaged for triplicates of each sample from three independent RT-qPCR reactions prior to calculating relative values using the 2−ΔΔCt method [The relative expression levels of SA-responsive genes were evaluated by RT-qPCR usig Lunaand PR-5  and NbAO [NbAOX1  in totalt method .® homogenizer and resuspended in 250 µL of 0.1 M acetate buffer was incubated in the presence  or absence (for free SA) of β-glucosidase at 37 °C for 90 min. The incubated extract was distributed into three wells (20 µL/well) and mixed with 60 µL LB and 50 µL Acinetobacter sp. ADPWH_lux culture (OD600nm = 0.4) followed by incubation at 37 °C for 60 min. The luminescence was read using a Synergy HTX plate reader .Quantification of SA content in the plants was determined as previously described . BrieflyP. syringae (isolated from tobacco plants in Florida) was resuspended in 10 mM MgCl2 and adjusted to 103 colony-forming unit (CFU)/mL. N. benthamiana leaves pre-inoculated with Agrobacterium cultures harboring pCTV-WT, pCTV-LMT1d, pLMT1 or empty vector were sequentially inoculated with the bacterial suspension using a needless syringe at 4 or 7 days after initial infiltration with pLMT1/empty vector or pCTV-WT/pCTV-LMT1, respectively. Leaf discs were collected from infiltrated areas at 3 or 4 days after challenge infiltration, respectively, and placed into 500 µL of 10 mM MgCl2. Tissue samples were homogenized using 1600 MiniG® high-throughput homogenizer  at 1500 strokes per minute for 2 minutes and serially diluted to 101–105 folds. 20 µL of serially diluted samples were dropped on King’s B agar  plate and incubated at 28 °C for 48 h, and colonies were counted. The number of CFU per collected leaf disc was determined through the formula: CFU/leaf disc = (dilution factor × colony count/20) × 500/8. Statistical analysis was done as described above.A pure culture of N. benthamiana leaves were washed with double distilled H2O three times and stained with 1 mg/mL DAB-HCl solution  overnight. The samples were de-stained in 100% ethanol. The DAB stain intensity was quantified by measuring pixel intensity of infiltrated area using Fiji software [3,3′-Diaminobenzidine (DAB) staining was performed as previously described . Brieflysoftware .N. benthamiana plants by agroinfiltration.To investigate the role of LMT1 in the virus infection cycle, we generated a full-length cDNA clone of a mutant CTV variant that does not produce LMT1, CTV-LMT1d, by introducing a six nt-substitution into the CE driving production of the LMT1 sgRNA during virus replication A. As shoN. benthamiana leaves agroinfiltrated with pCTV-WT or pCTV-LMT1d construct , which suggested that mutations within the LMT1 CE did not affect virion assembly. To further confirm proper packaging of the CTV-LMT1d genome at the LMT1 CE region, purified virions were treated with a micrococcal nuclease (MNase) and examined by RT-PCR to verify whether the genome was protected from the MNase activity , and the CP that encapsidates the rest of the genome ,50,51. Acroscopy D. As we activity F. Using activity A, L1Pro N. benthamiana leaves by Agrobacterium-mediated infiltration of the respective GFP-tagged constructs. For both virus variants, examination of the infiltrated leaves using a hand-held UV lamp revealed production of a strong GFP fluorescence indicating their successful multiplication. In either case, the fluorescence was present in numerous cells distributed throughout the leaf lamina areas located between the vascular bundles . At three months post-inoculation, strong GFP fluorescence was detected in the phloem cells of the newly emerged tissue in most citrus trees inoculated with CTV-WT, indicating the establishment of successful infection. In contrast, none of the trees inoculated with CTV-LMT1d showed signs of infection . Examination of leaves at 21 dpi using a fluorescence microscope revealed formation of GFP-expressing foci composed of few mesophyll cells in the leaves inoculated with the wild-type and the mutant viruses . Nevertheless, to confirm that the phenomenon observed above is mediated at the RNA level, we generated two constructs that expressed mutant versions of LMT1 carrying substitutions within the original start codon for ORF 1a  or the original start codon and the next potential start codon   A. To vernstructs B. Next, ve genes , the Agrnfection E and a sacterium F. Taken As shown previously, SA can promote plant resistance to viruses (reviewed in ). SA inhAOX-1a gene in the leaves infiltrated by the two virus variants as well as the constructs expressing the LMT1 RNA and its mutants. Remarkably, the expression level of AOX-1a in the leaves infected by CTV-WT was significantly higher compared to that in the CTV-LMT1d-infected leaves . AOX was reported to function as a suppressor of ROS accumulation upon virus infection ,65. Hered leaves C. Furtheversions D. AltogeCompared to plant and animal genomes, those of viruses have significantly smaller sizes. For this reason, most if not all virus-encoded factors such as proteins or RNA sequences are justified. Viral proteins are often multifunctional and engaged in many different processes at different stages of the virus infection cycle. The same could be true for viral lncRNAs that represent an exciting yet not well understood area. One of the crucial tasks of a virus is to combat a defense response that the host mounts upon pathogen perception. To this end, in addition to a long list of viral proteins shown to counter-act host immune receptor signaling or antiviral RNA silencing, many of the studied viral lncRNAs appear to be active participants of the virus–host arms race as well. Such RNAs, perhaps less immunogenic, could carry or form a number of secondary/tertiary structures that bind cellular proteins, thus, effectively modulating their involvement in the cellular processes, including antiviral defense. For instance, adenovirus lncRNA VAI binds to the host protein kinase R, which is known to block cellular translation in response to virus infection through phosphorylation of eIF2, and inhibits its activity, thereby allowing viral protein synthesis ,67. FurtN. benthamiana, there was an impediment in the establishment of systemic infection, compared to that by the wild-type CTV. This correlated with less efficient cell-to-cell movement and low occurrence of the mutant virus variant in cells forming the vascular bundles, suggesting that hindered ingress of CTV-LMT1d into the vascular system in the initially inoculated leaves affected the development of systemic infection. Importantly, virion formation was not altered in the mutant virus, and, thus, such virus property cannot be attributed for the phenotype observed. On the other hand, apparent deficiency in the accumulation and movement of the LMT1-deficient virus was accompanied by the elevated levels of SA and the SA-responsive genes, which were significantly higher than those upon the wild-type infection, implicating LMT1 in modulation of SA signaling. The results of further experiments using transient expression of the LMT1 RNA proved its ability to suppress the accumulation of this hormone compound and pointed to the role of LMT1 in facilitating virus accumulation and movement by influencing the host-virus interplay.Our study shows that LMT1, a lncRNA of CTV, facilitates virus infection by some means, which include modulation of the host immune response. LMT1 greatly increases invasiveness of CTV in the herbaceous host and is absolutely required for its infection of the natural citrus host. Although the LMT1-deficient virus was able to spread in N. benthamiana and tomato ringspot virus in tobacco and hindered the systemic translocation of TMV in N. benthamiana, CMV in tobacco and squash, and potato virus X (PVX) in tomato [n. tabacum, CMV-, turnip crinkle virus- or turnip mosaic virus-Arabidopsis, PVY-potato, and bamboo mosaic virus- or TMV-N. benthamiana pathosystems [The observations made in this work are in agreement with previous studies, which examined incompatible as well as compatible plant host-virus systems and demonstrated that induction of phytohormonal signaling could inhibit virus accumulation and/or movement and hamper virus host invasion ,22,74,75n tomato ,18,75,78osystems ,79,80.As we demonstrated recently, infection by CTV triggers a signaling cascade resulting in ROS accumulation, and three viral proteins—p33, p23, and p20—contribute to this reaction . Here, wN. benthamiana [AOX-1a levels, which was accompanied by lowering of ROS accumulation. We, therefore, hypothesize that this chain of events promoted virus movement from the initially infected cells to the neighboring cells and further into the vascular system allowing the virus to effectively spread throughout the plant. The proposed model of the effect of LMT1 on the plant innate immunity, which takes advantage of the established innate immunity pathways involving SA, AOX, and ROS , is Our study examined the role of LMT1, a lncRNA of one of the largest RNA viruses in its pathogenicity. Although LMT1 was discovered more than two decades ago, its functional importance was not understood, and for a long time this RNA has been considered as a by-product of a complex transcription strategy in a closterovirus. Here, we show that LMT1 functions in modulation of the host immune response to promote virus infection. Specifically, LMT1 antagonizes the SA-mediated signaling. To the best of our knowledge, CTV LMT1 is the first example of a viral lncRNA implicated in regulation of this branch of the plant innate immunity. We hypothesize that LMT1 has evolved as a consequence of virus adaptation to a perennial woody host with complex innate system barriers.