Here, we present a new tool, VirusFinder, for the identification of viruses and their integration sites in host genomes using NGS data, including whole transcriptome sequencing (RNA-Seq), whole genome sequencing (WGS), and targeted sequencing data. VirusFinder’s unique features include the characterization of insertion loci of virus of arbitrary type in the host genome and high accuracy and computational efficiency as a result of its well-designed pipeline. The source code as well as additional data of VirusFinder is publicly available at  Viral infection, especially from tumorigenic viruses, is one of the leading causes of deaths worldwide. Some viruses, e.g. the hepatitis B virus (HBV), can fuse into a host genome ("integrated") to interrupt gene functions or induce chromosomal instability With the rapid advances in next generation sequencing (NGS) technologies over the past several years and their increasingly widespread applications in clinical settings, recent large-scale investigations of virus-host interactions were carried out to shed light on virus-related cancers To address the above limitations, here, we introduce VirusFinder for efficient and accurate identification of viruses and their integration sites (if present) from NGS data. Specifically, VirusFinder aims to: (i) detect the presence of viruses of arbitrary types in a host sample, whether they are integrated or unintegrated; (ii) detect virus insertion sites as long as the virus fuses into the host genome and its integration sites are captured by sequencing technologies; and (iii) work on WGS, RNA-Seq or targeted sequencing data. VirusFinder does not require virus sequences as a prerequisite input. Hence, it can not only work with NGS data with a specified virus type, like VirusSeq and ViralFusionSeq, but also be applied readily to samples infected with undiagnosed viruses, to which neither VirusSeq nor ViralFusionSeq is directly applicable.http://www.ncbi.nlm.nih.gov/projects/mapview/) or UCSC hg19/hg18 (http://hgdownload.cse.ucsc.edu/downloads.html#human). VirusFinder runs Bowtie 2 in its sensitive end-to-end mode, in which Bowtie 2 does not trim (or "soft clip") characters from short reads in order to achieve high alignment speed. With the alignment file generated by Bowtie 2 or provided by the user, VirusFinder then garners all reads unmapped to the human reference genome for downstream analysis.VirusFinder’s input can either be raw sequencing reads (in Fastq format) or an alignment file (in BAM format). If user provides only raw sequencing reads, VirusFinder will first use the alignment tool Bowtie 2 Here in this step, user is allowed to provide the sequence of the virus being examined as an input parameter of VirusFinder. VirusFinder will skip step (2) of the pipeline if user provides the virus sequence.http://khavarilab.stanford.edu/resources.html) http://gib-v.genes.nig.ac.jp/), which collects 25,525 virus reference sequences, or a smaller set of viruses of user interest.This step is used to detect the specific type(s) of virus(es) present in the sample. This step will be skipped if user supplies the virus sequence to VirusFinder. If the virus type is unknown, however, VirusFinder first aligns the unmapped reads collected in step (1) to a virus database (virus DB). The current version of VirusFinder (release 1.0) uses the same virus DB, virus.fa, as the one included with the RINS package  identified in previous step (2) or provided by the user. It then uses the mapping tool BWA From the resultant alignment file, VirusFinder calls inter-chromosomal structural variants (SVs) using CREST When the pipeline terminates, three files, ‘virus.txt’, ‘contig.txt’ and ‘integration-sites.txt’, are created in the working directory of VirusFinder. These files contain candidate viruses identified by VirusFinder, contigs mapped to these virus sequences, and detected virus insertion sites, respectively. For each virus insertion event, VirusFinder provides its breakpoints in both the virus sequence and the human genome. For detailed explanations of these files, please read our user manual in the The entire pipeline of VirusFinder, from the initial preprocessing step to the final virus integration site detection, is fully automated. As far as we know, this is the first fully automatic pipeline combining virus detection (step 2) seamlessly with virus integration site identification (step 3) and, thus, the first NGS software enabling the automatic detection of virus integration sites in samples for which viruses may not necessarily be determined beforehand. The aforementioned tool VirusSeq provides both virus detection script and virus integration site identification script too. Unfortunately, in VirusSeq, they are separate programs and cannot work directly together. Another advantage of VirusFinder is that it is capable of analyzing large-scale NGS data efficiently by improving significantly its computational pipelines for viruses and their integration sites detection. VirusFinder further improves its analysis speed by blending a fast aligner Bowtie 2 in the time-consuming step (1) with a slower yet more sensitive aligner BWA on a smaller subset of reads in step (3).VirusFinder was implemented in Perl programming language and has been tested on various Linux platforms. It depends on several third-party tools, including BLAST+ (or BLAST) To evaluate VirusFinder, we compared VirusFinder with the aforementioned VirusSeq We firstly tested the ability of VirusFinder to detect the presence of viruses in human samples. As indicated in We utilized the samples in et al.et al.et al. validated 22 out of 176 WGS samples. Limited by computational resources, we picked 10 out of the 22 validated samples so that: (i) the hotspots of the integration breakpoints in both the human genome, e.g. genes TERT, MLL4 and CCNE1, and the HBV virus genome, e.g. locus 1800, are covered; (ii) our test data includes not only tumor samples but also normal tissues – 200N, 268N, and 180N are three normals; (iii) different sequencing depths, which range from 31.7× to 121.2×, are represented; (iv) our test data includes the aforementioned two samples, 198T and 268T, so as to use them to compare VirusFinder with ViralFusionSeq .Finally, we compared the computation time of the 3 tools on 10 WGS samples infected with HBV virus . All thehttp://www.accre.vanderbilt.edu/), with the same configuration of CPUs in each node. The memory we requested for each job of VirusFinder, ViralFusionSeq, and VirusSeq was 20 GB, 40 GB and 35 GB, respectively.The computation time of the three tools on these samples were analyzed on Vanderbilt Advanced Computing Center for Research & Education Click here for additional data file.


Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes.Soybean cyst nematode , into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+ locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development.We report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.The nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the  Heterodera glycines) is an obligate, sedentary endoparasite that is consistently the most damaging pest of soybean in the U.S. , the results for rhg1E98076/rhg1E98076, Rhg4Ina/Rhg4Ina genotypes transformed with empty vector or vector + Rhg1 locus LRR-K gene were even more similar .The henotype . SCN-susRhg1 locus LRR-kinase gene toward SCN resistance. To facilitate this work, we constructed a new vector for gene silencing in transgenic soybean roots. The binary vector pSM103  was designed to meet the following requirements: 1) efficient cloning of artificial microRNAs, 2) constitutive strong amiRNA expression in legume roots as well as other tissues [Agrobacterium, and 5) high transformation efficiency. The steps taken to develop the vector are shown in Figure in planta, pSM101 and pSM103 were infiltrated into N. bentamiana leaves using A. tumefaciens GV3101. Positive and negative controls  did or did not produce green fluorescence respectively, as expected  relid Figure  and 3B. d Figure  and 3D.Rhg1 locus from the more widely used PI 88788 source of Rhg1 was tested using the soybean cultivar Fayette. Cotyledons of Fayette were transformed with A. rhizogenes containing pSM103 derivatives carrying one of two different amiRNAs that each were designed to specifically target the Gm18 Rhg1 locus LRR-K gene. To determine the effectiveness of the two amiRNAs, RNA was extracted from three independent transgenic hairy roots for each construct. Semi-quantitative RT-PCR was performed in each of two independent biological replicate experiments as shown in Figure Rhg1 locus LRR-kinase gene, and using Fayette GFP-positive roots to verify that unmodified pSM103 does not interfere with expression of the Rhg1 locus LRR-kinase gene. No significant differences were found between the two Fayette controls . No statistical difference was found for the amiRNA LRR-K II, but a trend of partial down regulation was observed , while a significant difference was observed for amiRNAGmLRR-K I (χ2 P = 3.56e-10) and amiRNA GmLRR-K II (χ2 P = 1.156e-4) relative to the expected rate established from Fayette controls.Transformation efficiency was evaluated 3 weeks after s Figure . Howevers Figure  and 5B. Medicago truncatula. The Fayette TC204550 sequence has only 81% identity with M. truncatula Chromosome 5 clone mth2-155g22 (CU302335). M. truncatula seeds were transformed with A. rhizogenes to deliver the same silencing constructs tested in soybean. Three plates, each with 12 germinated plants, were used for each construct. Three weeks after the treatment, no difference in root development was observed between M. trucatula seedlings transformed with the two amiRNA GmLRR-K constructs in comparison to the unmodified pSM103 vector. The transformation efficiencies were not substantially different, ranging from 10% to 30% amongst the plates (data not shown). These results indicate the amiRNAs designed to silence the soybean Rhg1 locus LRR-kinase gene have a root development impact specifically in soybeans.To evaluate potential artifactual impacts of the amiRNA vector in plants lacking the target gene, a transformation experiment was performed using Rhg1 locus LRR-K gene (or empty vector controls) were sub-cultured by propagating the young growing tips (2-3 cm), and were infected with ~250 sterile J2 SCN for each root. The nematode demographics assay was then used to detect relative SCN resistance. The experiment was repeated with three independent biological replicates each using 4-10 separate transgenic hairy roots of each treatment-construct. Two weeks after nematode inoculation, all nematode stages (except eggs) were observed in the transgenic roots tested, and the number of nematodes at each stage was recorded. Roots carrying five or fewer nematodes were excluded from further analysis. As expected, Fayette control roots allowed significantly fewer nematodes to molt and progress to an apparent J3 or J4/adult female stage within 14 days relative to Williams 82 control roots , miRNAs and amiRNAs in plants reduce gene expression primarily by eliciting cleavage of homologous RNA transcripts. The primary benefit of adopting amiRNA technology is specificity -52. Longorg/ and , the amiAgrobacterium plant transformation vector, strong expression of the amiRNA in multiple tissues in soybean, and presence of an intron-disrupted GFP to allow identification of transgenic tissues. Each of these ideas has ample precedent but they had not been brought together for soybean. Gmubi is a strongly expressed promoter - offering potential improvements over CaMV 35S and other promoters for use in soybean and other legumes [Physcomitrella patens and other species [We constructed the vector pSM103 to address three goals: ready acceptance of newly generated amiRNA constructs into an  legumes ,55. Alth species ,57, and  species ,59. AnotA. rhizogenes [The nematode demographics assay was developed as a modification of existing protocols that monitor SCN development on transgenic soybean roots generated using izogenes ,38. Therizogenes ,60,61. Gizogenes . A seconRhg1-locus LRR-K gene on SCN resistance. Using complementation with a full-length gene under control of its native promoter, and gene knockdown using amiRNA technology, we were unable to detect a contribution to SCN resistance by the Rhg1-locus LRR-K gene from either Peking/PI 437654 or PI 88788, the most commonly used sources of SCN resistance in U.S. soybean cultivars. An impact on root development was observed when amiRNA for the LRR-K gene were expressed in transgenic roots, and effects on root development have also been preliminarily reported by the Lightfoot research group http://hdl.handle.net/10101/npre.2008.2726.1. It remains possible that this Rhg1-locus LRR-K gene may in some way impact SCN infection of or development on soybean roots, but no such effect was detected in our studies.In addition to reporting amiRNA use for gene silencing in soybean, a vector to facilitate this approach, and the nematode demographics assay for tests of genes that may contribute to soybean SCN resistance, we also report negative data regarding impacts of the Rhg1 locus is of extremely high economic significance to soybean cultivation in the United States, and has received attention from multiple research groups. It is notable that more than ten years after the filing of patent applications claiming SCN resistance function for the Rhg1-locus LRR-K gene, no groups have published evidence (beyond its presence as one of many candidate genes at the Rhg1 locus) for a functional contribution to SCN resistance by the Rhg1-locus LRR-K gene. We note that, simultaneous with the present study, a group led by Drs. Khalid Meksem and Melissa Mitchum has completed tests for functional contributions of the Rhg4-locus LRR-K gene to SCN resistance and, similar to our work, has not detected a contribution for this Rhg4 candidate gene . We are also aware of work led by Dr. Brian Diers [Rhg1 locus has revealed chromosomal recombination events that genetically separate rhg1-b (PI 88788-source) SCN resistance function from the Rhg1-locus LRR-K gene. Hence evidence is accumulating that the Rhg1 locus LRR-K genes seem unlikely to be the primary source of SCN resistance polymorphism. A tone of caution is appropriate, however, as our present study only provides negative data on this subject. For example, gene silencing is often partial , and the expression knockdown we achieved with amiRNA may have been insufficient to detectably block hypothesized SCN resistance contributions . As another example, the quantitative nature of the functional contribution of any given gene to SCN resistance may be difficult to detect in A. rhizogenes-generated soybean roots. Hence we are cautious with our conclusion, but we can state that when SCN-susceptible sources were complemented with an Rhg1-locus LRR-K allele from an SCN-resistant source, or when expression of the gene in resistant plants was reduced by amiRNA-mediated silencing, no significant impacts on SCN resistance were detected.The ubmitted), in whicRhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance, suggesting that one or more other genes at the Rhg1 locus may be the primary determinant of differences in SCN resistance among different soybean types.Our findings suggest that the nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs may have widespread use in legume biology, and we have developed the vector pSM103 that drives interchangeable amiRNA constructs through a Gmubi promoter, with an intron-GFP marker for detection of transgenic roots. Studies in which expression of the A. tumefaciens GV3101 and A. rhizogenes strain Arqua1 were transformed by freeze-thaw [A. rhizogenes strain Arqua1 was received from Dr. Jean-Michel Ane, University of Wisconsin Madison.eze-thaw  or by eleze-thaw . The celRhg1Ina or rhg1E98076, and for Rhg4Ina, or rhg4E98076, using micro satellite markers Satt309 and Satt424 [http://soybeanbreederstoolbox.org/. DNA sequencing was used to confirm nucleotide identity between the Rhg1-locus LRR-kinase gene from cultivars Ina and Peking.Soybean cultivars Fayette and Ina (SCN-resistant lines), and Williams 82 and E98076 (susceptible lines) were used; E98076 is an SCN-susceptible experimental line from Dechun Wang, Michigan State University. Genotyped Ina × E98076 progeny lines were kindly provided by Dr. Brian Diers, University of Illinois at Urbana-Champaign. Those lines were inferred to be homozygous for  Satt424 ; http://Medicago truncatula seeds (approximately 150 per batch) were scarified in concentrated sulfuric acid for 8 min, washed 3 times in sterile water, plated on 1% deionized water agar plates, and then stratified for 24 h or 48 h at 4°C and germinated by incubating at room temperature overnight [After visual screening for obvious signs of fungal or viral contamination, soybean seeds were surface-sterilized for 16-20 h in a desiccator jar with chlorine gas by adding, to a beaker within the jar, 3.5 ml 12 N HCl into 100 ml household bleach (6% sodium hypochlorite) immediately before closing the jar. At least 20 seeds per experiment were plated onto germination media , 2% sucrose, 1 × Gamborg's B5 vitamins, 7% Noble agar, pH 5.8) in 100 × 25 mm Petri plates. Plates were wrapped with Micropore tape  and incubated at 26°C in a growth chamber (18/6 light/dark hours) for about a week. vernight .A. rhizogenes suspension (OD600 0.7 in Luria broth). The cotyledons were then placed abaxial-side down on a co-culture medium  in 100 × 15 mm Petri plates with a piece of 70 mm filter paper  on the surface of the agar to prevent A. rhizogenes from overgrowing. Plates were wrapped with parafilm and incubated in the dark at room temperature for three days. The explants were then transferred to a hairy root medium (HRM) of 4.3 g/L MS salts , 3% sucrose, 1 × Gamborg's B5 vitamins , 7% Noble agar, 0.15 g/L cefotaxime, 0.15 g/L carbenicillin, pH 5.6 in 100 × 15 mm Petri plates, wounded side up. Plates were wrapped with Micropore tape and incubated in the dark at room temperature until roots emerged, usually in around 3 weeks.Soybean cotyledons were harvested about 7 days after germination by gently twisting them off the hypocotyls. With a sterile forceps and scalpel, several shallow slices were made across the abaxial surface of the cotyledons after dipping the scalpel in M. truncatula with transgenic roots, A. rhizogenes Arqua1 carrying pSM103 with one of the two different amiRNA for the Rhg1 GmLRR-Kinase (amiRNA GmLRR-KI and GmLRR-KII), or the empty vector, were used for M. truncatula transformation [M. trucatula composite plants were watered with sterile water every other day.To generate ormation . FollowiM. truncatula transgenic roots were detected based on GFP expression, using a fluorescence stereomicroscope (LEICA MZ FL III with GFP2 filter). Transgenic soybean root tips were cut into 2-3 cm segments and transferred to HRM. Roots that were expressing incomplete strips of fluorescence (chimeras) or exhibiting overall low levels of GFP fluorescence were avoided. GFP-positive as well as negative control roots were generally used within two months of the initial transformation event, after maintenance by transferring 2-3 cm root tips every two weeks. Independent transgenic events, generated from different inoculation sites or different cotyledons, were maintained separately for use in RT-PCR and nematode demographic assays.Soybean or Rhg1 locus was cloned from Ina genomic DNA. Primers were designed based on BAC sequences from soybean cultivar A3244 [The LRR-kinase gene Glyma18g02680.1 from the ar A3244 , using tRhg1LRR-KF: TTCACCCGTGATACATGTTAATTC;Rhg1LRR-KR: GGAACTTGGAAGTCATTATCTTGGRhg1 locus that includes >2 kb of upstream sequence, the coding region, and 0.8 kb of downstream sequence, which was then cloned into the Invitrogen plasmid pCR8/GW/TOPO according to the manufacturer's instructions. Multiple independent clones were isolated and sequenced in order to avoid clones with PCR-induced mutations. The binary vector pGWB1::erGFP7INT for full-length gene complementation experiments was constructed from pGWB1 [Rhg1 locus were moved in to pGWB1::erGFP7INT using the Invitrogen Gateway™ system  after linearizing the pCR8/GW/TOPO-derived plasmids with XbaI. To obtain a negative control transformation vector lacking the Gateway ccdB negative selectable marker, a 167 bp segment of nonsense DNA was cloned and transferred into pGWB1::erGFP7INT.to amplify a 6.9 kb PCR product from the om pGWB1  by additom pGWB1 , taken fH. glycines per container, and grown in a water bath at 28°C in a 28°C greenhouse. Cysts were collected 6 weeks after infection, and were extracted from infested pots using sieves and centrifugation. Briefly, soil from infected pots was placed in a pitcher of water and the roots were massaged to dislodge attached cyst. The soil-cyst-water slurry was passed over a 595 μm - 250 μm sieve tower, and the mixture from the 250 μm sieve was backwashed into a 50 mL plastic conical tube. The tubes were centrifuged at 2000 RPM for 4 minutes then the supernatant was poured off. A 60% sucrose solution was added to the tubes, stirred, and centrifuged at 2000 rpm for 2 min. Cysts in the supernatant were then collected over a 250 μm sieve. Collected cysts were stored at 4°C in plastic bags in damp sand.A Wisconsin SCN population (Hg type 7), collected by A.E.M., was maintained on the susceptible soybean cultivar Williams 82. Seeds were germinated in a wet 70 mm filter paper (Whatman) in a Petri plates for 4-5 days, planted in autoclaved 2:1 sand:soil mixture, inoculated with 2000-3000 eggs of H. glycines eggs were collected by breaking open cysts and collecting the eggs on a sieve stack consisting of 250 μm - 74 μm - 25 μm sieves (USA Standard Testing Sieve). Eggs were collected from the 25 μm sieve and rinsed. Eggs were placed in a hatch chamber [2 for hatching at room temperature in the dark for 5-6 days. Hatched J2 nematodes were surface-sterilized for 3 min in 0.001% mercuric chloride and washed three times with sterile distilled water [For nematode demographics assays,  chamber  with 3 med water , then sued water , typicalTo construct the pSM103 vector for amiRNA-mediated gene silencing in legume roots Figure , the 35SPlmnc95_Kpn1F: CCAGGTACCCAGGTCCCCAGATTAGCC;Plmnc95EcoR1_R: GCCAGTGAATTCCCGATCTA.The amiRNA constructs from pRS300 derivatives (see below) were added to pCAMBIA 1300::35SP-erGFP7INT-NOS using BamHI/PstI sites. The soybean polyubiquitin (gb|EU310508.1|) promoter (GmUbi) ,55 was aUbi1_HindIII_Fa: CCAAAGCTTGGGCCCAATATAACAACGAC;Ubi_PstI_Ra: CCACTGCAGCTGTCGAGTCAACAATCA.The Gmubi product was cloned into pCR2.1 TOPO and then in pCAMBIA 1300::35SP-erGFP7INT-NOS::miR319a using HindIII/PstI sites.A. tumefaciens GV3101 into Nicotiana benthamiana leaves, along with GV3101 without binary plasmid as a negative control. A. tumefaciens GV3101 strains carrying pGWB1::erGFP7INT, pSM101 and pSM103 were grown overnight at 28°C in 5 ml LB medium with 2.5 μg/ml of rifampicin and 50 μg/ml of kanamycin (the negative control A. tumefaciens GV3101 without binary vector was grown only with rifampicin). A. tumefaciens cells were harvested by centrifugation and resuspended to OD600 0.4 in MMA induction buffer  corresponding to the Glyma18g02680.1 putative LRR-kinase gene from the Rhg1 locus was used to design PCR primers for isolation of the Rhg1 locus receptor-like kinase-encoding allele from the resistant source Fayette, which carries the Rhg1 resistance allele rhg1-b from PI 88788. The primers RTPCR_TC204550F CAA CTT CAA GGC CTC AGG AA; RTPCR_TC204550R GCT ACC CAA AGA AGC AGG AA, amplified a PCR product of 450 bp. The DNA sequences of PCR products were determined by the dideoxy chain-termination method using ABI big dye cycle sequencing kit (3.1) and the ABI 377 automated sequencing service at the University of Wisconsin - Madison Biotechnology Center. Blast analysis of the Fayette version of TC204550 (the EST for this gene in earlier WMD versions) was conducted in March 2008 using the Phytozome database http://www.phytozome.net/soybean. For amiRNA design, the sequence of this Fayette LRR-kinase gene was submitted to http://wmd2.weigelworld.org. The wmd2 tool identified a total of 85 candidate amiRNA sequences to potentially silence the Rhg1 locus LRR-kinase gene, with the first 31 candidates classified by wmd2 as having excellent (most promising) properties (data not shown). The two amiRNA target sequences selected for primer design to modify pRS300 for silencing of the Rhg1 locus LRR-kinase gene were identical to Fayette chromosome 18 sequence as well as the paralogous chromosome 11 sequence from the Williams 82 genome: amiRNA GmLRR-KI: TAAGACTATAAGGGATTGCTG; and amiRNA LRR-KII: TAAGACTATAAGGGATTGCTC. The Arabidopsis miR319a sequence from pRS300 was used as a template to create three PCR products  with soybean target transcript specificity. These products were used as template for a fourth PCR using overlapping primers to create gene specific amiRNA [EST TC204550 UP|Q8L3Y5 (Q8L3Y5)  and RTPCR_TC204550R GCTACCCAAAGAAGCAGGAA (reverse), were used to amplify a 450 bp product. As a control for equivalent starting pools of cDNA, the primers Actin forward GTTCTCTCCTTGTATGCAAGTG and Actin reverse CCAGACTCATCATATTCACCTTTAG were used to amplify a 700 bp segment of soybean actin gene cDNA (emb|V00450.1|) The RT-PCR was performed in triplicate at 55°C, testing three different numbers of cycles (30-35-40) to confirm non-saturation of amplification. PCR products were visualized by ethidium bromide staining after separation on 1.5% agarose gels and band intensity was quantified using the Kodak 1D Image Analysis Software v3.6.Soybean genomic DNA was extracted from young leaves using a CTAB method previously described . Total RData were analyzed by ANOVA using Minitab (v.14) with the General Linear Model and Tukey Simultaneous Test; nematode demographics data for the amiRNA experiments were analyzed using a mixed model .SM, ALH and DC planned, conducted and analyzed most of the experiments and were centrally involved in writing the manuscript. BWD developed soybean germplasm and provided significant ideas and critical review of the manuscript. AEM contributed nematode populations, methodological suggestions and biological insights. AFB conceived the overall project, analyzed results and planned experiments, and was a primary author of the manuscript. All authors read and approved the final manuscript.


Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection.Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species  Hirudo medicinalis is a leech used medically to extract blood, and it is also valued for its capacity to avoid coagulation by secreting hirudin, an anticoagulant substance discovered in leech saliva by Haycraft in 1884. It took another century for the substance to be successfully isolated and characterized by Markwardt, in 1986 [Hirudo with Aeromonas sp. has been well known for years [A. hydrophila have been reported when used in microsurgery. In the natural environment, the interactions between leeches and micro-organisms have not been well characterized, even when other associated bacteria have been identified in the animal. Indeed, some Ochrobactrum sp., Bdellovibrio sp. and Sphingobacterium sp. were detected in the bladders of medical leeches [Rikenella-like bacterium was isolated from the gut of a H. verbena species [The natural habitat of leeches is mostly fresh water from puddles, and the progressive disappearance of this habitat, as well as the intensive collection of wild leeches for medical purpose in the eighteenth century in Central Europe, led to a dramatic decrease in the population and the concomitant classification of the species on the International Union for Conservation of Nature (IUCN) Red List of endangered species . Collector years , and sev leeches , and a R species . Mimiviridae from a wild specimen of a medical leech, Hirudo medicinalis.The role of leeches as a potential reservoir for viruses has also been suspected. Even if they are not known to be natural carriers of viruses, the persistence of the classical swine fever virus and the Myxoma virus in their digestive tract was demonstrated after leeches were fed with swine and rabbit blood contaminated with those viruses ,10,11. IHirudo medicinalis  and Mamavirus (805), whereas 435, 402, 361 and 338 bona fide orthologs were found from other mimiviruses of amoeba including Megavirus chiliensis [Mimiviridae. Of the Hirudovirus predicted proteins, 774 (78%) and 190 (19%) found a protein from Mimivirus or Mamavirus, respectively, as top hit in the GenBank non-redundant database. Proteins from other mimiviruses were identified as top hits for other Hirudovirus proteins, and among them, 18 were detected in the genome of Lentillevirus, which is classified within lineage C of amoebal mimiviruses [Bona fide orthologs from these viruses showed a mean amino acid identity of 99.1±2.6% , respectively. In addition, mean nucleotide identities for these bona fide orthologs was 99.3±1.8% , respectively. These values are in the same order of magnitude than those described for bona fide orthologs of Mimivirus and Mamavirus that is another strain of Mimivirus [The sequenced genome assembly resulted in two scaffolds of 1,155,382 and 25,660 base pairs, with a low (28%) mean GC content, which is similar to those of other mimiviruses of amoebae . A totaliliensis , LBA111 iliensis , Moumouviliensis  and Monviliensis , respectiviruses . FinallyPhylogenic reconstructions based on a concatenated alignment of three conserved genes shows that Hirudovirus strain clusters with Mimivirus and Mamavirus in the lineage A, previously defined for mimiviruses of amoebae , which aHirudo medicinalis analyzed, led to the isolation of a giant virus from the internal part of the animal. The virus was absent from the flesh, and the presence of the virus in the internal organs and the gastrointestinal tract suggests that the giant virus may have been ingested with food. Indeed, giant viruses can naturally be found in several ecological niches, such as fresh water, seawater , and soil [Hirudo species can explain the presence of a mimivirus in the digestive tract of the leech. Several studies have shown that viruses persist in the digestive tract of leeches fed with contaminated blood [Aeromonas, and with several proteobacterial genera [Hirudinae. However, other mimiviruses have been isolated from humans, whereas marseilleviruses, other giant viruses that infect amoebae, have been isolated from humans and Eristalis tenax, an insect member of the order Diptera [One single specimen, among the 5 and soil ,20,21. Hed blood ,5,6,9. Ml genera ,9,20. Thl genera . Alternal genera . Notablyl genera . Our worl genera . Notably Diptera . These dSamples and inoculation of amoeba. Animals tested for the presence of giant viruses came from two small streams at two locations in Tunisia and France. A total of five leeches of the species Hirudo medicinalis were isolated, three in Tunisia and two in France. Wet bottles were used for transporting live samples to the laboratory. The method used to prepare the animals and the procedure for isolating the virus have been recently detailed elsewhere [Hirudo medicinalis were rinsed with 96% ethanol for 20 min and then washed with sterile PAS . The washing solution was saved. The animals were dissected to obtain samples of flesh (F) and internal organs in digestive tract (IO+DT). These samples were crushed mechanically before inoculation using the high throughput isolation system previously described [lsewhere . Brieflyescribed .Preliminary characterization of viruses. After the appearance of an amoebal lysis plaque, indicates the presence of a giant virus, the agar plate was cut into small pieces around the plaque, resuspended in 500 µl sterile PAS, centrifuged at 15000 rpm for 30 minutes, and re-inoculated onto a fresh amoebal monolayer. The presence of the giant virus was then assessed by Hemacolor staining after cytocentrifugation of a 100 µl sample of culture supernatant. The preliminary classification of the virus was performed by observation of the culture supernatant, which was negative stained with a 3% ammonium molybdate solution and evaluated with electron microscopy. Whole genome sequencing. For whole genome sequencing, 25 culture flasks (150 cm2) were prepared with 5 ml of virus, 10 ml of amoebae and 30 ml of nutritive medium PYG . After amoebal lysis, viruses were purified and concentrated; genomic DNA extraction was performed with phenol-chloroform, and the whole genome was pyrosequenced on the 454-Roche GSFLX as previously described [escribed .Sequence assembly, gene prediction and annotation. The genome assembly was performed with the Newbler software [Acanthamoeba polyphaga Mimivirus [Acanthamoeba castellanii mamavirus [Acanthamoeba polyphaga moumouvirus [Megavirus chiliensis [Pandoravirus dulcis and Pandoravirus salinus [P. dulcis and P. salinus. Bona fide orthologs, which are orthologs identified through the widely used approach consisting in the identification of reciprocal best hits between two gene repertoires, were determined here by the Proteinortho program with an e-value cut-off of 1e-3 and minimum amino acid identity and coverage of 30% and 70%, respectively [software . Genes wsoftware . All ORFimivirus , Acanthaamavirus , Acanthamouvirus , LBA111 mouvirus , Megaviriliensis , Moumouv salinus . The Hirectively . In addiectively . Phylogenetic analysis. The phylogenetic analysis was based on three concatenated core genes  of the nucleocytoplasmic large DNA viruses [ viruses . Multipl viruses , informa viruses  and a ph


Polyomaviridae recently detected in respiratory tract specimens by shotgun sequencing. Intriguingly, viral genome has been detected in 0.4% to 11.5% of respiratory tract specimens from children with respiratory disease. The levels of co-infection with established respiratory viruses were in the range between 30.8% and 91.7%. Moreover, some studies report detection of WUPyV in stool or serum. So far, WUPyV infections can not be distinguished from other viral infections by means of clinical symptoms. Respiratory tract disease like pneumonia or bronchitis is frequently observed in patients harbouring WUPyV. Detection of viremia suggests systemic infections. However, the available data do not prove WUPyV to be a human pathogen. Further investigations are necessary.The WU polyomavirus (WUPyV) is a novel member of the family  They are caused by a broad spectrum of established respiratory tract pathogens, particularly by viruses including the respiratory syncytial virus (RSV), the most important cause of severe respiratory tract infections in young children, influenza viruses, parainfluenza viruses, rhinoviruses, coronaviruses 229E, OC43), and adenoviruses. Studies investigating the entire spectrum of established respiratory tract pathogens are rare and mostly based on the detection of pathogen specific antibodies. Only in recent years, a few studies became available presenting data based on the direct detection of relevant infectious agents. Creer and co-workers  identifi29E, OC43By now, the pathogenic impact of the human metapneumovirus and its relevance on respiratory tract infections has been well established. It is a common cause of upper (URTI) and lower respiratory tract infections (LRTI) in children, elderly, and immunocompromised persons; asymptomatic infections seem to be uncommon . hMPV haRecently, the WU polyomavirus was detected in respiratory tract specimens .2.The first polyomaviruses were isolated in 1971 and named JC virus and BK virus. In 2007 and 2008 3 further viruses of this particular family were described for the first time: WU polyomavirus, KI polyomavirus, and Merkel cell polyomavirus . WUPyV wPolyomaviridae. Thus, the identification of a novel virus belonging to this viral family has been proposed, exclusively based on molecular data. So far, no system for in vitro or in vivo propagation has been discovered, neither a cell line nor an animal model. Thus, until now Koch’s postulates could not be fulfilled. However, the molecular data form reasonable evidence to propose the existence of the novel virus WUPyV.The combination of these technologies led to the identification of a DNA-sequence distant to all sequences included in the sequence database, but related to viruses of the family Large-scale screening based technologies as the one used for the discovery of WUPyV may be a powerful tool in discovering new infectious agents causing respiratory tract diseases particularly with regard to the etiological gap of up to 40%.3.The novel virus has a 5229 bp dsDNA genome with a GC-content of 39%. The genome contains an early coding region encoding the T-antigens, a late coding region encoding structural proteins, and a non-coding control region . All fea4.Polyomaviridae form small, non-enveloped, icosahedral particles with 40–45 nm in diameter containing a supercoiled dsDNA genome [Typical members of the family A genome . The virBased on the genome organisation, two subclasses of the polyomavirus family have been postulated: the primate-like group and the mouse polyoma-like group . The gen5.Polyomaviruses have a restricted host range. The BK virus and the JC virus, the closest relatives of WUPyV within the well established members of the virus family, are human-specific. This suggests a host range restricted to humans for WUPyV, too. Infection with BKV takes place in early childhood; 90% of 5–9 years old children are seropositive. WUPyV infections have been reported predominantly in children <3 years of age  suggestiJC virus and BK virus primary infections are typically subclinical or linked to mild respiratory illness ,15. ViraWUPyV is made responsible for upper and lower respiratory tract diseases, e.g. bronchiolitis, croup, and pneumonia . Besides6.For detection of WUPyV-infections several conventional PCR formats as well as real-time PCR protocols were developed reporting sensitivities up to 100% and specificities up to 97.7% –25. AntiThe diagnostics of established respiratory viruses is based on a broad spectrum of available assays, aiming at the detection of virus-specific antibodies, viral protein, or viral nucleic acids. Acute respiratory tract infections commonly have a short incubation period. Thus, antibodies are usually not present at the beginning of the acute phase of the disease. For this reason, detection of virus-specific antibodies by complement fixation assays, enzyme linked immunosorbent assays (ELISA), or immunofluorescence assays (IFA), all of which are available for established respiratory viruses, is inappropriate for the identification of the etiologic agent causing an acute respiratory tract infection, but useful for retrospective or epidemiological studies. Direct detection of viral protein or viral nucleic acids is the mode of choice for respiratory virus diagnostics. Viral cell culture represents the gold standard for the well established respiratory viruses, but is too slow for in time diagnostics. Less time consuming methods have been developed. These comprise polymerase chain reaction (PCR), antigen-specific ELISA, antigen-specific IFA, and, available only for a few respiratory viruses, very fast immunochromatographic assays. Sensitivity and specificity of these assays vary considerably. The PCR displays the highest sensitivity. Thus, being restricted to PCR formats for detection of WUPyV is no bias to sensitivity. Particularly real time PCR formats have an established track record in both, very sensitive detection and differentiation between colonisation and acute infection based on the quantitative data ,27. Avai7.Following the discovery of WUPyV in Australia, the virus was detected in specimens from patients with respiratory tract disease on all continents suggesting a worldwide distribution ,29–31. SThe data available are mainly based on retrospective studies exclusively including symptomatic patients. The detection rate in respiratory samples from children with respiratory disease varies from 0.4% to 11.5% ,35. The et al. [Le and co-workers  presenteet al.  for noso10 copies/ml, but low and medium viral loads were dominating [The real time protocols available allow the quantification of WUPyV. Quantification of viral loads in respiratory tract specimens revealed viral titers up to 10minating ,24. No cFew studies included asymptomatic control groups ,36,42–44Prospective studies have been published only recently. Van der Zalm and co-workers  reported8.To this time there are no studies available concerning therapeutic interventions. This is mainly due to the retrospective nature of most investigations. Further, no causal association between WUPyV and respiratory disease has been shown yet . As the 9.The discovery of novel respiratory viruses has the potential to diminish the diagnostic gap for respiratory tract infections. Creer and co-workers , who omiSo far it has not been finally proven, if WUPyV is a real causative agent for respiratory diseases. Association of virus detection with previously unexplained respiratory disease led to the tempting idea of WUPyV representing a new etiologic agent, particularly in cases where no other respiratory tract pathogen could be identified. Furthermore, the association of mouse pneumotropic polyomavirus with intestinal pneumonia and significant mortality  indicateMany studies correlating the detection of WUPyV with the incidence of respiratory symptoms argue for this hypothesis, but it remains difficult to prove as asymptomatic control groups have not been investigated sufficiently. Additionally, the studies including control groups report dissenting findings. However, in two studies asymptomatic individuals display a lower frequency of virus detection compared to symptomatic patients. One of these is the only prospective study available.The difficulty to prove WUPyV to be a respiratory pathogen may be due to the fact that most studies available so far try to correlate the virus with respiratory tract disease in general. In case of WUPyV being the causative agent of a particular entity of respiratory tract disease, inclusion of patients displaying any kind of respiratory disease may bias the investigation. Focussing on a single entity of respiratory disease proved successful for HCoV-NL63, which was shown to cause croup.No association of WUPyV viral loads and clinical symptoms could be observed so far, and co-infections with other viruses were described frequently. This weakens the hypothesis of WUPyV being a respiratory tract pathogen. However, collection of samples was not performed by means of a standardized protocol among the various groups or at a defined time point, after onset of symptoms. As viral loads decrease during the acute phase of infection, the combination of longitudinal data from different time points in one analysis may bias the correlation of viral loads and symptoms. Furthermore, detection of co-infection does not exclude pathogenic potential for WUPyV, as early childhood is characterized by subsequent episodes of respiratory infections. Co-detection of the declining pathogen responsible for the last episode of respiratory disease and the pathogen responsible for the present, acute disease has to be expected. Only determination of the viral load kinetics would allow defining the clinical impact of a detectable microorganism.The large T-antigen of WUPyV contains binding sites for the RB protein and p53. Thus, tumourigenic potential cannot be excluded. However, the association of WUPyV with other diseases, particularly tumour diseases has not been sufficiently investigated yet.part of the endogenous viral flora without pathogenic potential;an opportunistic pathogen with pathogenic potential in the respiratory tract under conditions still to be defined;a pacemaker for secondary infections;a viral pathogen using the respiratory tract only as an entry route to reach the final target cell; infection of this cell type is the basis of a disease not related to the respiratory tract.Taken together, the currently available data neither prove nor deny WUPyV to be a respiratory tract pathogen. Several scenarios may describe the role of the virus best. WUPyV may beTo reach a final conclusion more powerful, controlled studies need to be performed prospectively. However, retrospective analysis of age- and time-matched control populations also permits conclusions if the data collected at the time are related to respiratory illness. Ideally, prospective studies would be carried out in a multicenter-design including control groups concentrating on the correlation of WUPyV with single entities of respiratory tract diseases. Criteria for inclusion of specimens have to be comparable, as well as the time point and the protocol for extraction of specimens. All known or suspected respiratory tract pathogens should be included and follow-up investigations should be performed. The detection method of choice would be quantitative PCR to allow discrimination between colonisation and productive infection. Additionally, serological investigations should be included to show that the patients seroconvert in response to an infection. The study participants should be evaluated before enrolment. Beside investigation of respiratory tract disease, the cell type hosting the persisting virus has to be identified and a possible association with tumour diseases has to be investigated.


Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot, and mouth diseases (HFMDs), and EV71 is now recognized as an emerging neurotropic virus in Asia. Effective medications and/or prophylactic vaccines against HFMD are not available. The current results from mouse immunogenicity studies using in-house standardized RD cell virus neutralization assays indicate that (1) VP1 peptide (residues 211–225) formulated with Freund's adjuvant (CFA/IFA) elicited low virus neutralizing antibody response (1/32 titer); (2) recombinant virus-like particles produced from baculovirus formulated with CFA/IFA could elicit good virus neutralization titer (1/160); (3) individual recombinant EV71 antigens  formulated with CFA/IFA, only VP1 elicited antibody response with 1/128 virus neutralization titer; and (4) the formalin-inactivated EV71 formulated in alum elicited antibodies that cross-neutralized different EV71 genotypes (1/640), but failed to neutralize CVA16. In contrast, rabbits antisera could cross-neutralize strongly against different genotypes of EV71 but weakly against CVA16, with average titers 1/6400 and 1/32, respectively. The VP1 amino acid sequence dissimilarity between CVA16 and EV71 could partially explain why mouse antibodies failed to cross-neutralize CVA16. Therefore, the best formulation for producing cost-effective HFMD vaccine is a combination of formalin-inactivated EV71 and CAV16 virions. Hand, foot and mouth diseases (HFMDs) caused by enteroviruses like Coxsackievirus A16 (CVA16) and Enterovirus 71 (EV71) infections have become serious public health problems in Southeast Asia . Recent First isolated in 1969, EV71 is a nonenveloped RNA virus of the family Picornaviridae, 25–30 nm in diameter, that contains a single molecule of plus sense ssRNA (7.5–8.5 kb) and four structural proteins VP1, VP2, VP3, and VP4 . Two strE. coli, and formalin-inactivated EV71 virus grown in Vero cell culture with and without animal serum containing media. Therefore, the results obtained from the current studies provide valuable information for future HFMD vaccine development.The concern for potential virulent viruses reversed from attenuated vaccines , 14 has All experiments were conducted in accordance with the guidelines of the Laboratory Animal Center of NHRI. The animal use protocols have been reviewed and approved by the NHRI Institutional Animal Care and Use Committee: NHRI-IACUC-098033-A.African green monkey kidney (Vero) and rhabdomyosarcoma (RD) cell lines were purchased from the American Type Culture Collection (ATCC) . Fetal bovine serum (FBS) was purchased from Moregate Biotech  and was certified by the manufacturer to be free of bovine spongiform encephalopathy (BSE). The E59 strain (genotype B4), the clinical isolate of the EV71 virus, was obtained from the Taiwan CDC. EV71 E59 virus stocks were collected from the supernatants of infected Vero cells at three days postinfection (DPI). Nucleotide sequences of VP1 reported in this study have been submitted to public domain (accession no. GQ150746.1), and the amino acid sequences of all four structural viral proteins reported in this study are available upon request. The EV71 subgenotype isolates used in this study are B4 (N0781-TW-01); B5 (N2838-TW-03); C4 (N3340-TW-02); CVA16 (N5079). These clinical virus isolates further underwent at least one round of plaque purification before growth in Vero and/or RD cells as virus seed stocks for virus neutralization assays.6 cell per mL) grown in each roller bottle containing 200 mL of culture medium were infected with EV71/E59 at a multiplicity of infection (MOI) of 10−5. EV71 was collected from the culture supernatant of each bottle at five DPI. Cell debris were removed by filtration through a 0.65-μm membrane , and the crude virus bulk was concentrated 20- to 40-fold using a 100-kDa cut-off diafiltration membrane in a tangential flow filter (TFF) cassette (Sartorius Stedim Biotech). One liter of EV71 virus concentrate was purified using an AKTA Pilot liquid chromatography system  equipped with Sepharose Fast Flow 6 gel. Fractions were collected and analyzed by immunoblotting, and virus infectivity was measured using the issue culture's infectious dose (TCID50) assay. Fractions containing the virus were pooled, further concentrated, and then inactivated with 0.02% formalin (v/v) at 37°C for 5 days. The vaccine bulk was obtained after sterile filtration using a 0.22 μm filter, subjected to SDS-PAGE and western blot analyses, and stored at 4°C. The VP2 content was analyzed by Q-ELISA, and the total protein concentration of the vaccine bulk was also determined by the BCA protein assay. The production of EV71 virus was done using E59 vaccine strain in the presence of serum-containing medium  or VP-SFM medium  according to the procedures described in , 19. Bri50 as described previously by Liu et al. [50 values were calculated using the Reed-Muench method [Virus titers were determined using the median endpoint of the TCIDu et al. . The TCIh method .E. coli BL21-DE3 and purified using Ni-NTA resin affinity chromatography  as previously described by Liu et al. [μg of either individual recombinant antigen , or formulated with alum, or CFA/IFA adjuvant.The recombinant EV71 antigens  were expressed in u et al. . The purμg of EV71 VLPs in the presence either of alum or CFA.EV71-VLPs were produced using a recombinant baculovirus expression system using the P1 gene derived from the EV71 E59 virus isolate . The EV7SDS-PAGE and Western blot analyses of the purified viral antigens were performed according to the protocols reported previously by Liu et al. .μg of either individual or mixed synthetic peptides formulated in either PBS, or alum, or complete Freund's adjuvant (CFA)/incomplete Freund's adjuvant (IFA). The reactivity of the antibody  to synthetic peptide was analyzed by ELISA according to the protocol previously reported by Panezutti et al. [Peptide synthesis was performed by Kelowna International Scientific Inc.  as previously described by Liu et al. . To testi et al. .μg of EV71 protein formulated with 1.5 mg of alum per dose. Sera were collected two weeks after each immunization and used for immunological analysis. The specificity and anti-EV71 titer of the antisera were tested by western blotting and TCID50-based virus neutralization assay, respectively.Immunogenicity studies were conducted according to the protocols reported previously by Liu et al. . Briefly50 assay according to the protocols reported previously by Liu et al. [Virus neutralization titer of each serum sample was determined using RD cell and in-house standardized TCIDu et al. . The virThe rVP-ELISA was performed according to the protocol reported previously by Liu et al. . Our recent studies have identified two linear immunodominant neutralization epitopes VP1-43 and VP2-28 that, respectively, correspond to residues 211–225 of VP1 and residues 136–150 of VP2 . Althougin vitro  in the presence of CFA/IFA adjuvant also produced weak virus-neutralizing antibody responses against EV71. The titers were found to range from 1/32 to 1/64. Again, these EV71 viral antigen-specific antisera failed to neutralize CVA16 at 1/8 serum dilution. These results suggest that there were no CVA16 cross-neutralizing antibodies elicited from recombinant antigens.A recent report by Liu et al.  indicateμg of EV71 VLPs in the presence either of alum or CFA produced antibodies with virus neutralization titers of 1/128 and 1/160, respectively. Interestingly, EV71 VLP alone also elicited a virus-neutralizing antibody response with a 1/64 titer. The current results are consistent with previous reports by Chung et al. [μg of EV71 VLPs formulated with CFA could elicit virus neutralizing antibody responses with a titer of 1/512 in adult mice and protect newborn mice against lethal EV71 challenges by passive immunization. Again, these EV71 VLP-specific antisera were found to fail to neutralize CVA16 at 1/8 serum dilution. C56BL/6 mouse immunogenicity studies were also performed and the results were very similar to those obtained from BALB/c mice. These results suggest that while EV71 VLPs may fully mimic the structural organization of EV71, the structural similarity does not elicit mouse antibodies that are capable of cross-neutralizing CVA16.Since a handful of prophylactic VLP-based vaccines against hepatitis B virus and human papillomavirus are currently commercially available, many VLP-based vaccine candidates against different diseases are in clinical trials or in preclinical evaluations . To thisμg of formalin-inactivated EV71 virion alone or formulated in alum elicited antibody responses that (1) recognized viral structural proteins in western blot analysis, (2) reacted specifically with VP1-43 neutralization epitope, and (3) neutralized EV71 B4 genotype with average titers ranging from 1/64  to 1/640 , but (4) failed to neutralize CVA16  against different EV71 genotypes  antigen produced and purified from different expression systems could elicit strong virus neutralizing antibody responses and protect newborn mice against lethal EV71 challenge –5. Howevμg of EV71 VLPs could elicit virus-neutralizing antibody responses with a titer of 1/512 in adult mice and protect newborn mice against lethal EV71 challenges by passive immunization. Although these EV71 VLP-specific antisera were capable of cross-neutralizing other EV71 genotypes as determined by peptide-ELISA and Western blot analysis and have higher EV71 neutralization titers than those obtained from synthetic peptide or recombinant VP1, it still failed to neutralize CVA16 at 1/8 serum dilution. These results suggest that EV71 VLP may have structural similarity and fully mimic the EV71 structural organization, but could not elicit mouse antibodies that can cross-neutralize CVA16. Again, the amino acid sequence dissimilarity in CVA16 could explain this observation since mouse antibodies recognize the single immunodominant epitope VP1-43. In fact CVA16 has several substitutions in this location, in particular those lysine residues are found to be critical for neutralizing antibody binding [Recombinant virus-like particles produced from baculovirus or yeast expression systems have structural organization mimicking the conformation of authentic native viruses, but lack the viral genome to potentially provide safer vaccines. In addition, prophylactic VLP-based vaccines against hepatitis B virus and human papillomavirus are currently commercially available. Our results are consistent with previous reports by Chung et al.  that 10  binding .μg) of inactivated EV71 virion strongly cross-neutralized different EV71 genotypes and weakly cross-neutralized against CVA16, with average titers of 1/6400 and 1/32, respectively. Our current results are consistent with results reported by both Bek et al. and Dong et al. [The concern over potential virulent viruses reversed from attenuated vaccines , 14 has g et al. , 31 whic


Hylomyscus simus) captured in Sangassou, Guinea. Sequence and phylogenetic analyses of the genetic material demonstrate a novel hantavirus species, which we propose to name "Sangassou virus."Hantaviruses are rodentborne, emerging viruses that cause life-threatening human diseases in Eurasia and the Americas. We detected hantavirus genome sequences in an African wood mouse ( Bunyaviridae, are emerging viruses that cause 2 life-threatening human zoonoses: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome . The virus genome consists of 3 segments of negative-stranded RNA; the large (L) segment encodes viral RNA-dependent RNA polymerase, the medium (M) segment encodes glycoprotein precursor, and the small (S) segment encodes nucleocapsid protein. In contrast to other members of the Bunyaviridae, hantaviruses are not transmitted by arthropods but are spread by aerosolized excreta of rodents of the family Muridae, their natural hosts , which causes a mild form of HFRS in Europe, and the less pathogenic Tula virus (TULV) are Arvicolinae–associated hantaviruses. In 1993, Sin Nombre virus (SNV) was discovered in the United States as the first member of the third group, Sigmodontinae–associated hantaviruses. SNV from North America and Andes virus (ANDV) from South America are the most prominent examples of viruses causing HPS , Seoul virus (SEOV), and Dobrava virus (DOBV), which cause HFRS in Asia and Europe, are examples of Hylomyscus simus) in Sangassou, Guinea.Hantaviruses cause human diseases predominantly in Asia, Europe, and the Americas. Few studies have considered hantaviruses in Africa; such reports originated from serologic surveys of human populations. In this study, we report detection and initial genetic characterization of the first indigenous African hantavirus detected in an African wood mouse  were trapped in Guinea from 2002 to 2004 and screened for hantavirus RNA by reverse transcription–polymerase chain reaction (RT-PCR). We used a molecular genetic approach to screen the rodent population because hantavirus RNA (as shown for SNV) can be amplified from the blood of persistently infected mice by RT-PCR over a long period  designed from an alignment of all available nucleotide sequences of the highly conserved L segment. For the RT-PCR, total RNA was extracted from wild-trapped rodent blood (preserved in liquid nitrogen) with the Blood RNA kit  and reverse transcribed with random hexamers as primers.In a survey for rodentborne hemorrhagic fever viruses, 612 small rodents representing 17 different genera (most abundant were H. simus) generated an L segment–derived PCR product of expected size. This rodent was trapped in January 2004 in a forest habitat near the village of Sangassou, near Macenta, Guinea . Its karyotype was determined  and the complete cytochrome b gene was sequenced and compared with the genes of other Hylomyscus species recognized in the most recent revision of the genus (A sample (designated SA14) obtained from 1 of 4 investigated African wood mice (The 412-nucleotide (nt) sequence of the first PCR product was determined by amplification, cloning, and sequencing of overlapping fragments generated by 2 seminested PCRs (GenBank accession no. DQ268652). Additional S and M segment–specific nested PCR assays were developed to further characterize the novel virus. PCR fragments of 837 nt and 694 nt could be analyzed .Hantavirus. Murinae-associated hantaviruses  showed the highest similarity to the SA14 sequence in all 3 genomic segments . This similarity is consistent with the evolutionary relationship of their putative hosts. On the amino acid level, corresponding sequences of deduced viral proteins showed highest similarity with those of other Murinae-associated hantaviruses . The amino acid sequence divergence between SA14 and most related hantaviruses corresponds to that typically found between different virus species, e.g., SNV and ANDV.The Murinae-associated hantaviruses. PUUV, TULV, SNV, and ANDV sequences were used as outgroups. In the S segment ML phylogenetic tree . Within this well-supported cluster, the SA14 sequence is most closely related to the DOBV clade. The M segment analysis showed an identical placement of SA14 with strong statistical support .The S-, M-, and L-segment–derived nucleotide sequences of SA14 were subjected to maximum likelihood (ML) and neighbor-joining (NJ) phylogenetic analyses with available nucleotide sequences of other tic tree , SA14 cl  divergent from those of other hantaviruses, they form a distinct clade in phylogenetic trees, and they were detected in a rodent species previously not recognized as a natural host of hantaviruses. We propose to name the new species Sangassou virus (SANGV) after the locality where it was detected.–Apodemus agrarius, the natural host of HTNV, is not found in Africa. Based on the putative cross-reactivity of antigens from HTNV, SANGV, and other Murinae-associated viruses, human infections, at least in tropical forest parts of Africa where Hylomyscus species are prevalent, could be caused by SANGV or other Murine-associated hantaviruses.Although hantaviruses are emerging viruses circulating in Asia, Europe, and the Americas, our study represents the first genetic evidence for hantaviruses in Africa. Suspected human hantavirus infections have been reported in various African countries (Sigmodontinae-associated hantaviruses were not recognized until 1993, even in such a highly developed country as the United States.To our knowledge, 1 case of HFRS has been reported in central Africa (Further studies are needed to verify the presence and distribution of hantaviruses in Africa and their potential impact on human health. These studies should focus on areas with forest activities, such as logging, which may bring humans into contact with viral reservoirs (


Latimeria chalumnae). Phylogenetic analyses place CoeEFV basal to all known foamy viruses, strongly suggesting an ancient ocean origin of this major retroviral lineage, which had previously been known to infect only land mammals. The discovery of CoeEFV reveals the presence of foamy-like viruses in species outside the Mammalia. We show that foamy-like viruses have likely codiverged with their vertebrate hosts for more than 407 million years and underwent an evolutionary transition from water to land with their vertebrate hosts. These findings suggest an ancient marine origin of retroviruses and have important implications in understanding foamy virus biology.Little is known about the origin and long-term evolutionary mode of retroviruses. Retroviruses can integrate into their hosts' genomes, providing a molecular fossil record for studying their deep history. Here we report the discovery of an endogenous foamy virus-like element, which we designate ‘coelacanth endogenous foamy-like virus’ (CoeEFV), within the genome of the coelacanth . We provide evidence suggesting that foamy viruses and their hosts share a coevolutionary history of more than 407 million years, and that foamy viruses accompanied their vertebrate hosts on the evolutionary transition from water to land. These findings indicate that the retroviruses originated in the primeval ocean millions of years ago.The deep history of retroviruses is still obscure. Retroviruses can leave integrated copies within their hosts' genomes, providing a fossil record for studying their long-term evolution. Endogenous forms of foamy viruses, complex retroviruses known to infect only mammalian species, appear to be extremely rare, so far found only in sloths and the aye-aye. Here, we report the discovery of endogenous foamy virus-like insertions within the genome of a so-called ‘living fossil’, the coelacanth ( Foamy viruses are complex retroviruses thought exclusively to infect mammalian species, including cats, cows, horses, and non-human primates Latimeria chalumnae), which we designate ‘coelacanth endogenous foamy-like virus’ (CoeEFV). The discovery CoeEFV offers unique insights into the origin and evolution of foamy viruses and the retroviruses as a whole.The continual increase in eukaryotic genome-scale sequence data is facilitating the discovery of additional ERVs, providing important insights into the origin and long-term evolution of this important lineage of viruses. In this study, we report the discovery and analysis of an endogenous foamy virus-like element in the genome of the coelacanth . Hence, it provides decisive evidence that CoeEFV originated from a foamy-like virus.We screened all available animal whole genome shotgun (WGS) sequences using the tBLASTn algorithm using the protein sequences of representative foamy viruses  and iden viruses . Two add viruses . Notably viruses . A Conseses  is uncertain Endogenous retroviruses are likely to undergo a gradual accumulation of neutral mutations with host genome replication after endogenization 2 = 0.7115, p = 1.10×10−5, 2 = 0.7024, p = 1.41×10−5, 2 = 0.7429, p = 4.26×10−6, To further evaluate the relationship of foamy viruses, we reconstructed phylogenetic trees based on the conserved region of Pol proteins of foamy viruses and Class III retroviruses, the conserved region of foamy virus Pol and Env protein concatenated alignment, and the conserved region of foamy virus Env protein alignment, respectively. The three phylogenies have the same topology in terms of foamy viruses , and S6.There are two alternative explanations for these phylogenetic patterns. One is that the exogenous progenitor of CoeEFV is not truly the sister taxon to the mammalian foamy viruses, but a more distant relative. The robust posterior probability (1.00) placing them in the same clade and the absence of evidence for viruses or virus-like elements from other species disrupting this clade argue against this view, as does the significant similarity between the Env proteins of CoeEFV and the foamy viruses . MoreoveThe other alternative to the hypothesis that these viruses have co-diverged over more than 407 million years is that they somehow moved, in more recent times, from terrestrial hosts to sarcopterygian hosts that inhabited the deep sea, and that the similarity of the coelacanth virus to the mammalian viruses is due to cross-species (in fact cross-class) transmission, rather than shared history. However, as illustrated by the significant correlation between host divergence times and viral distances , and S6,Moreover, the habitat isolation of the coelacanth and terrestrial vertebrates would have provided limited opportunities for direct transfer of foamy viruses to coelacanths. Taken together, these lines of evidence strongly suggest that foamy viruses and their vertebrate hosts have codiverged for more than 407 million years, and that foamy viruses underwent a remarkable evolutionary transition from water to land simultaneously with the conquest of land by their vertebrate hosts.Our analyses provide compelling evidence for the existence of retroviruses going back at least to the Early Devonian. This is the oldest estimate, to our knowledge, for any group of viruses, significantly older than the previous estimates for hepadnaviruses (19 million years) Dating analyses provide the clearest evidence for when and where retroviruses originated. There is strong evidence that foamy viruses shared a common, exogenous retroviral ancestor more than 400 million years ago . The discovery of endogenous lentiviruses demonstrates that lentiviruses, a distinct retroviral lineage that includes HIV, are also millions of years old All available animal whole genome shotgun (WGS) sequences from GenBank were screened for endogenous foamy viruses using the tBLASTn algorithm and the protein sequences of representative exogenous and endogenous foamy viruses . SequencLatimeria chalumnae tissue sample was obtained from Ambrose Monell Cryo Collection (AMCC) at the American Museum of Natural History, New York. Genomic DNA was extracted using the DNeasy tissue kit  following the manufacturer's instructions. Amplification of ∼680 bp gag gene and ∼650 bp env gene fragments was performed with the degenerate primer pairs, FVpol-F (5′-AACAGTGYCTYGACCMAACC-3′) and FVpol-R (5′-TAGTGAGCGCTGCTTTGAGA-3′), FVenv-F (5′-CTGGGGATGACAAYCAGAGT-3′) and FVenv-R (5′-CCACTCRGGAGAGAGGCAAC-3′). PCR was performed in 25 µl of final volume reactions with 0.1 µl Platinum Taq HiFi enzyme , 1 µl primer mix (10 µM each), 0.5 µl of 10 mM dNTP mixture, 1 µl of 50 mM MgSO4, 2.5 µl of 10× PCR buffer, and 1 µl of template DNA. The PCR reactions were cycled under the following conditions: initial denaturation at 94°C for 2 minutes, 45 cycles of , and final elongation at 72°C for 5 minutes. The PCR products were purified using QIAquick spin columns . Purified PCR products were cloned into the pGEM-T Easy vector . Cloned products were sequenced by the University of Arizona Genetics Core with an Applied Biosystems 3730XL DNA Analyzer. The sequences have been deposited in GenBank (Accession Nos. JX006240-JX006251).Ethanol preserved All protein sequences were aligned using Clustal Omega For the phylogenetic tree based on the foamy viruses and Class III endogenous retroviruses Pol protein, Class III endogenous retroviruses were used to root the foamy viral phylogeny . Because−9 substitutions per site per year, standard deviation = 0.1×10−9) was used as the rate prior http://beast.bio.ed.ac.uk) was employed for Bayesian MCMC analysis with a strict clock model The nucleotide sequences were aligned using MUSCLE Dataset S1The conserved region of Pol proteins of CoeEFV and various representative exogenous and endogenous retroviruses.(TXT)Click here for additional data file.Dataset S2The conserved region of the foamy viruses and Class III endogenous retroviruses Pol protein.(TXT)Click here for additional data file.Dataset S3The conserved region of foamy virus Pol and Env protein concatenated alignment.(TXT)Click here for additional data file.Dataset S4The conserved region of foamy virus Env protein alignment.(TXT)Click here for additional data file.Dataset S5The alignment of five sequences  used to estimate the age of the CoeEFV invasion.(TXT)Click here for additional data file.Dataset S6The alignment of two sequences (contig243355 and contig219087) used to estimate the age of the CoeEFV invasion.(TXT)Click here for additional data file.Figure S1Schematic mapping of CoEFV fragments identified in this study onto the CoeEFV consensus genome.(PDF)Click here for additional data file.Figure S25′-TGGCACCCAACGTGGGG-3′. The PBS sequence of human foamy virus is 5′-TGGCGCCCAACGTGGGG-3′ . There is only one substitution.CoeEFV consensus genomic sequence. The ambiguous nucleotides were filled according to contig187425, contig187426, and contig178313 of coelacanth genome. The CoeEFV PBS is nearly identical to the PBS of extant foamy viruses. The CoeEFV PBS sequence is (PDF)Click here for additional data file.Figure S3Phylogenetic relationships among retroviruses. The phylogeny was reconstructed with the Bayesian method via MrBayes 3.1.2. The posterior probabilities are shown on the nodes. The foamy virus and lentivirus clades were highlighted in red and blue, respectively. Branch lengths are in expected amino acid changes per site.(PDF)Click here for additional data file.Figure S4Alignment of the two sets of sequences used for dating CoEFV invasion. Flanking sequences are shown for each sequence set with consensus genomic sequence of CoeEFV.(PDF)Click here for additional data file.Figure S5A) Midpoint rooted phylogenetic tree of foamy viruses. The phylogeny is the 50% majority-rule consensus tree inferred from conserved region of foamy virus Pol and Env protein concatenated alignment with MrBayes 3.1.2. Posterior probabilities are shown at the nodes. Branch lengths are in expected amino acid changes per site. B) A plot of the correlation between foamy virus divergence and their vertebrate hosts' divergence times. The virus branch lengths are derived from the virus tree in A.(PDF)Click here for additional data file.Figure S6A) Midpoint rooted phylogenetic tree of foamy viruses. The phylogeny is the 50% majority-rule consensus tree inferred from conserved region of foamy virus Env protein alignment with MrBayes 3.1.2. Posterior probabilities are shown at the nodes. Branch lengths are in expected amino acid changes per site. B) A plot of the correlation between foamy virus divergence and their vertebrate hosts' divergence times. The virus branch lengths are derived from the virus tree in A.(PDF)Click here for additional data file.Table S1The representative retrovirus sequences used for genome screening and phylogenetic reconstruction.(PDF)Click here for additional data file.Table S2The matching contigs identified in coelacanth genome.(PDF)Click here for additional data file.


Climate change can cause arthropodborne diseases to emerge. Arboviruses have evolved a number of strategies to survive environmental challenges. This review examines the factors that may determine arbovirus emergence, provides examples of arboviruses that have emerged into new habitats, reviews the arbovirus situation in western Europe in detail, discusses potential arthropod vectors, and attempts to predict the risk for arbovirus emergence in the United Kingdom. We conclude that climate change is probably the most important requirement for the emergence of arthropodborne diseases such as dengue fever, yellow fever, Rift Valley fever, Japanese encephalitis, Crimean-Congo hemorrhagic fever, bluetongue, and African horse sickness in the United Kingdom. While other arboviruses, such as West Nile virus, Sindbis virus, Tahyna virus, and Louping ill virus, apparently circulate in the United Kingdom, they do not appear to present an imminent threat to humans or animals. This page was updated on July 19, 2006 to incorporate the corrections in Vol. 12, No. 8Flavivirus that will be discussed in this review include yellow fever virus (YFV), dengue virus (DENV), Israel turkey meningoencephalomyelitis virus, and Bagaza virus. European human and animal pathogens in the genus Alphavirus include SINV, Ockelbo virus, and Chikungunya virus and in the family Bunyaviridae, sandfly fever Naples virus (often referred to as Toscana virus), sandfly fever Sicilian virus, Crimean-Congo hemorrhagic fever virus (CCHFV), Inkoo virus, and Tahyna virus, which is widespread throughout Europe. Rift Valley fever virus (RVFV) and Nairobi sheep disease virus (NSDV) could be introduced to Europe from Africa through animal transportation. Finally, the family Reoviridae contains a variety of animal arbovirus pathogens, including bluetongue virus, which is currently circulating in Europe, and African horse sickness virus, which has been found in Europe but is not currently circulating. This review considers whether any of these pathogenic arboviruses are likely to emerge and cause disease in the United Kingdom in the foreseeable future.More than 550 arboviruses have been identified . Many arTransmission of arboviruses between invertebrates and vertebrates imposes constraints on evolution and dispersal, which are reflected in their phylogenetic relationships . HoweverIxodes spp. vectors that inhabit the moist forest undergrowth. Nymphal ticks may infect newly hatched larvae when they co-feed on forest animals. Since these vertebrates do not become sick or have detectable viremia, this direct method of vector infection is known as nonviremic transmission. In contrast, LIV and related viruses in Spain, Turkey, Greece, and nearby regions produce viremia and fatal encephalomyelitis in domesticated animals when infectious ticks feed on them  is transmitted to sheep by Rhipicephalus appendiculatus and causes fever and gastroenteritis; the infection is often fatal. NSDV is distributed widely throughout Africa . The WNV that was introduced into New York was related to an Egypt/Israel strain that is also present in the Volga River Delta. Despite its high virulence in birds (particularly corvids), horses, and alligators in North America, WNV circulates relatively harmlessly in the Old World, presumably reflecting co-evolution of virus and host over a long period.Since birds are highly mobile and can travel great distances in a relatively short time, they play a role in arbovirus dispersal. SINV, for example, is carried annually between South Africa and Scandinavia by migrating birds . SINV isOther emerging flaviviruses include Usutu virus, which appeared unexpectedly in Austria in 2000 , killingBunyaviridae, genus Phlebovirus) is usually transmitted by Aedes spp. mosquitoes and occasionally infects livestock, producing epizootics with high death rates in sheep and other ruminants. Humans in areas where RVFV is epizootic may also become infected. Until 2000, RVFV was most commonly found only in eastern, western, and southern Africa. However, in September 2000, an outbreak was reported in Saudi Arabia and subsequently in Yemen, representing the first cases identified outside Africa and showing the potential for global dispersal.RVFV  is transmitted to domestic and wild ruminants by culicoide midges. Bluetongue virus epidemics can threaten entire livestock populations and were first described in South Africa after Merino sheep were introduced from Europe in the late 18th century. The virus subsequently dispersed globally, recently reemerging in Italy, Spain, and Portugal  are vectors of animal diseases, such as bluetongue virus  and African horse sickness. According to Campbell and Pelham-Clinton .Some species of -Clinton , 41 CuliSandflies are not known to be endemic in the United Kingdom, but they are present in all Mediterranean countries. As climate change takes effect, sandflies are expected to become established in the United Kingdom. Thus, sandfly fever/Toscana virus might emerge later in this century.Finally, direct transmission of arboviruses between vertebrates  has been recognized for many years. For example, LIV and TBEV may be transmitted through the milk of sheep and goats . MoreoveArboviruses rarely cause disease in their maintenance hosts or vectors; consequently, viremic blood is not available for vectorborne transmission. Arboviruses have therefore developed other transmission strategies to ensure their survival. For years, the favorite mechanisms to explain virus survival in the absence of disease were vertical transmission, overwintering, or persistent infection in the arthropod or vertebrate host. We now recognize that the process of nonviremic transmission between co-feeding ticks or co-feeding mosquitoes, and perhaps ticks co-feeding with mosquitoes, is a powerful force for virus transmission even in the presence of immunity. Therefore, this repertoire of transmission strategies can ensure arbovirus perpetuation in the absence of disease.Armed with this versatility, arboviruses can adapt to virtually any situation. Most arboviruses have evolved a sylvatic existence that is infrequently associated with disease in vertebrate hosts. However, in the presence of susceptible hosts, an environment that favors rapid arthropod amplification, an efficient mechanism for dispersal, and a suitable climate for transmission, DENV, RVFV, Japanese encephalitis virus, and YFV may cause epidemic outbreaks of immense proportions.This variety of critical factors must come together at an appropriate time for an arbovirus to emerge as an epidemic pathogen, a situation that has rarely happened in the United Kingdom. Of course, if climate change has the effects that experts predict it will, the situation could change during the 21st century. In conclusion, despite compelling evidence that potentially highly pathogenic arboviruses are circulating or, alternatively, are being introduced annually into the United Kingdom, no imminent threat exists that they will cause epidemic outbreaks of serious concern to public or environmental health.