Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus . After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS.Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus  was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the  Initially in 2007, and again between 2008 and 2010, cases of a life-threatening disease with sudden fever, thrombocytopenia, and leukopenia were reported in Henan Province, China. Patient reports of tick bites suggested that this disease could be infectious or tick-transmitted. Many patients were provisionally diagnosed with human granulocytic anaplasmosis (HGA). However, only 24 of 285 (8%) had objective evidence of HGA, suggesting that other pathogens likely contributed to fever, thrombocytopenia and leukopenia syndrome (FTLS). Illumina sequencing was used for direct detection in clinical samples of pathogens possibly associated with FTLS. A novel bunyavirus was found only in samples from FTLS patients. Further epidemiologic and laboratory investigation confirmed that the novel bunyavirus was associated with FTLS. The results illustrate that metagenomic analysis is a powerful method for the discovery of novel pathogenic agents. Combined with epidemiologic investigation, it could assist in rapid diagnosis of unknown diseases and distinguish them from other diseases with similar symptoms caused by known pathogens. Anaplasma phagocytophilum) in neighboring Anhui province Orientia tsutsugamushi) in this area Ehrlichia chaffeensis), and Tsutsugamushi disease. Specific methods such as polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) for these pathogens were then used to determine if these cases were attributable to HGA or HME A. phagocytophilum based on serology and DNA testing. Thus, the disease was initially considered at least partly caused by A. phagocytophilum, and cases were provisionally diagnosed as suspected HGA based on clinical and epidemiological data A. phagocytophilum confirmation (6 of 206 patients) and no pathogen was isolated. Similar cases were also reported in the mountainous and hilly areas of nearby Shandong, Jiangsu, Hubei and Anhui provinces, indicating that the disease already existed for some time and was widely distributed In May 2007, a county hospital in Xinyang City, Henan Province treated three patients with fever, abdominal pain, bloating, nausea, vomiting, gastrointestinal bleeding, and elevated aminotransferases. The local hospital diagnosed the disease as acute gastroenteritis. A family member of one patient reported the disease to the Henan Center for Disease Control and Prevention (CDC), which sent a team to investigate. The investigation revealed that the disease had the following characteristics: (1) acute onset with fever; (2) low white blood cell and platelet counts; (3) high levels of alanine and aspartate transaminases; (4) positive urine protein. On the basis of these features, the Henan CDC excluded the possibility of gastrointestinal disorders. In order to identify the disease etiology, the Henan CDC team used the above clinical characteristics as the case definition to search for similar cases in local hospitals in this and neighboring counties, while establishing a disease surveillance system that required all medical institutions to report cases that met the above case definition. Altogether, 79 cases were found in 2007 in Henan, with 10 fatalities . All patients were farmers and resided in mountainous or hilly villages, and many had reported tick bites 7–9 days before illness, further suggesting an infectious etiology. In recent years, patients with similar clinical symptoms were reported with human granulocytic anaplasmosis  detection, the research team intensified its virus search to take into account arthropod-borne viruses, including Flaviviridae , Togaviridae , and Bunyaviridae  On the basis of epidemiological and clinical characteristics, we considered two types of diseases to be possible: rickettsial and arthropod-borne viral disease. Because of the low rates of Culture followed by serological and molecular tests is a standard approach for identifying an unknown virus. However, culture of an unknown virus is time-consuming, even taking several years to confirm a novel infection like HIV This research was approved by the Review Board of the Center for Disease Control and Prevention of Henan Province, the Review Board of the Center for Disease Control and Prevention of Xinyang city, the Review Board of Beijing Institute of Genomics, the Review Board of Beijing Genomics Institute in Shenzhen, and the Review Board of the Institute of Microbiology and Epidemiology. All participants gave written informed consent for use of their samples in research.Given the serious nature of FTLS, it was decided to handle all clinical specimens and perform all experiments involving live virus in a biosafety level-3 (BSL-3) facility.We studied 285 Henan province patients with FTLS whose samples were submitted to the Henan Province CDC between May 2007 and July 2010. Acute-phase serum samples from all patients were collected. Paired convalescent sera were available from 95 patients.A. phagocytophilum, Ehrlichia chaffeensis, Dengue fever virus, Japanese encephalitis virus, Chikungunya virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Sandfly fever Naples Sabin virus, and Hantavirus A. phagocytophilum) were purchased from Focus Diagnostics . Antigen slides for diagnosis of other pathogens were prepared by our laboratories. Fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG (Fc) was purchased from Sihuan Sci-Technics Company .Sera were tested by reverse transcription (RT)-PCR, PCR, and/or indirect IFA serological assays for a number of microbial agents, including Equal quantities (100 µL) of acute-phase sera from 10 FTLS patients who had a history of tick bite were pooled and centrifuged at 1000 x g for 10 minutes. The supernatant was collected for DNA and RNA extraction. The same was done for 10 sera from healthy subjects (control).DNA was extracted from 140 µL of each sample using the QIAamp DNA mini Kit  according to the manufacturer's instructions. DNA was eluted from the columns with 50 µL water containing 20 µg/mL RNaseA. After incubation at 37°C for 15 minutes to eliminate RNA, DNA was used immediately or stored at −80°C.Total RNA was extracted from 140 µL of each sample using the QIAamp viral RNA mini Kit  according to the manufacturer's instructions. RNA was eluted from the columns with 50 µL of diethyl pyrocarbonate (DEPC)-treated water containing 1 U DNaseI. Samples were incubated at 37°C for 15 minutes to eliminate human DNA, followed by DNase inactivation at 95°C for 10 minutes. RNA was used immediately or stored at −80°C.2, 10 mM DTT, 100 ng of random hexamer primers, 200 U of Superscript II , 25 U of RNasin , and 0.5 mM dNTPs.cDNA was synthesized from 6 µL of RNA by reverse transcription at 45°C for 50 minutes in a 20-µL solution containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 100 µM dNTPs, 1 U Taq DNA Polymerase  and 100 ng of random hexamer primers containing a linker (5'-GCCGGAGCTCTGCAGAATTCNNNNNN-3'). After denaturing at 95°C for 5 minutes, targets were amplified by 45 cycles of 95°C for 30 seconds, 40°C for 30 seconds, 50°C for 30 seconds, and 72°C for 90 seconds. The amplified products were detected by agarose gel electrophoresis. Pure water was used as a negative control.Random hexamer PCR was carried out in a 25-µL mixture containing 4 µL of cDNA or DNA, 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgClFTLS patient and control genomic DNA/cDNA libraries were constructed according to the manufacturer's instructions (Illumina). In brief, RT-PCR and PCR products were roughly quantified by UV absorption and equal amounts of each sample were mixed. Nucleic acids within the mixtures were sheared by sonication, and fragments in the 150–180-bp range were collected by cutting bands from an agarose gel after electrophoresis. The sheared DNA and cDNA ends were repaired using Klenow DNA polymerase, after which 5' termini were phosphorylated and 3' termini were polyadenylated. The adaptors were added, PCR enrichment was performed, and 150–180-bp fragments were collected for sequencing by the Illumina method.The sequencing procedure was performed according to the manufacturer's instructions (Illumina). In this process, template library DNA was hybridized to the surface of the flow cells and multiple copies of DNA were made to form clusters using the Illumina cluster station. Workflow steps included template hybridization, isothermal amplification, linearization, and final denaturation and hybridization of sequencing primers. Paired-end sequencing (100 cycles) was performed using a four-color DNA Sequencing-By-Synthesis (SBS) technology following the manufacturer's instructions.ftp://ftp.ncbi.nih.gov/genbank/). The resulting alignments were filtered to identify unique sequences by examining alignment (identity ≥80%) and E-value scores (e≤10−2). Filtered unique alignments were examined in the taxonomy database (NCBI) using a custom software application written in Perl (BioPerl version 5.8.5). Unmapped reads were examined in GenBank nucleic acid and protein databases using BLASTn and BLASTx, respectively −5 was applied to both BLASTn and BLASTx analyses. Sequences phylotyped as viral were placed in the “viral” category.Short Oligonucleotide Analysis Package (SOAP) was used to handle the large amounts of short reads generated by parallel sequencing Parvoviridae family. PCR performed to detect human bocavirus tentatively identified in sera from FTLS patient samples amplified a 291-bp fragment of the NS1 gene, as described previously Our mass sequencing data revealed that some sequences showed possible infection with human bocavirus, which belongs to the Tehran virus which belongs to the Phlebovirus genus of the Bunyaviridae family. We developed a PCR strategy based on the 168-bp sequence identified in sera from FTLS patient samples to detect the novel bunyavirus using forward (PF: 5'-GAC ACG CTC CTC AAG GCT CT-3') and reverse (PR: 5'-GCC CAG TAG CCC TGA GTT TC-3') primers designed with Primer3 , 0.25 µM forward primer, and 0.25 µM reverse primer. Thermocycling conditions were as follows: 95°C for 4 minutes (denaturation), followed by 35 cycles of 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds. Amplified products were detected by agarose gel electrophoresis and sequenced using an ABI 3730 DNA Sequencer.Our mass sequencing data revealed a 168-bp sequence  indicating the possible presence of a novel virus with closest identity  to The Vero E6 cell line (African green monkey kidney cell) was selected for isolation of the novel bunyavirus associated with FTLS because it supports the growth of many bunyaviruses Cells showing CPE and containing novel bunyavirus RNA were collected for thin-section electron microscopy. After discarding the culture supernatant, virus-infected cells (50 mL) were mixed 1∶1 with 4% glutaraldehyde , placed onto Formvar-carbon-coated grids, and stained with 1% methylamine tungstate. Specimens for thin-section electron microscopy were prepared by dehydrating washed cell pellets with serial dilutions of acetone and embedding in epoxy resin. Ultrathin sections were cut on an Ultracut LKBV ultramicrotome, stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (JEM-1400).Escherichia coli JM109 were transformed with the ligation mixture and cultured on LB agar containing X-gal. White clones were sequenced using an ABI 3730 DNA Sequencer. Contaminating human and extraneous sequences were eliminated using CrossMatch, and the complete sequence was assembled using Phred-Phrap-Consed The medium from 20 mL of novel bunyavirus-infected Vero E6 cells was centrifuged at 1,000 x g for 10 minutes and then at 4,000 x g for 10 minutes, after which the supernatant was collected. PEG8000 was added to the supernatant at a final concentration of 10% (w/v) followed by centrifugation at 20,000 x g for 2 hours. The pellet was resuspended in 2 mL 1× phosphate-buffered saline (PBS) for RNA extraction. Random RT-PCR was performed, and the products (500–1500 bps) were collected and ligated into the pGEM-T vector  by incubating overnight at 16°C. Bunyaviridae family. To further establish the relationships between the new virus and the members of the Phleboviruses genus, we included all sequences for phleboviruses available in GenBank. Branching orders of the phylograms were verified statistically by resampling the data 1,000 times in a bootstrap analysis with the branch-and-bound algorithm, as applied in MEGA.Phylogenetic analyses were performed using the neighbor-joining method in the MEGA software package, version 4.0.2 After successful isolation of the novel bunyavirus, we developed an indirect IFA to detect specific antibodies in patient serum specimens, as previously described All 285 patients with FTLS were from the Henan Province of China and were provisionally diagnosed as suspected HGA on the basis of similar clinical manifestations The median age of patients was 57.2 years  and the male-to-female ratio was 1 to 2.27; 219 patients (92.02%) were farmers and 19 (7.98%) were workers or students. Among patients, 52 (21.85%) reported a tick bite within 2 weeks (5–14 days) before the onset of clinical manifestations; the remaining patients did not recall receiving a tick bite.The main clinical features in confirmed patients included sudden onset of fever (>37.5°C −40°C) lasting up to 10 days, fatigue, anorexia, headache, myalgia, arthralgia, dizziness, enlarged lymph nodes, muscle aches, vomiting and diarrhea, upper abdominal pain, and relative bradycardia . A smallLaboratory tests showed that confirmed patients characteristically developed thrombocytopenia, leukopenia, proteinuria, and elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels . BiochemOne lane for each of the two sample pools , each consisting of 10 samples, was sequenced. The proportions of high-quality sequences in FTLS and control pools were 98.08%  and 97.88% , respectively. Unique, high-quality sequence reads were then classified into broad taxonomic groups based on the taxonomy of the most frequent top-scoring BLAST matches for each sequence. Virus sequences constituted 0.0065%  and 0.0048%  of the reads in FTLS and control libraries, respectively . After cAdenoviridae (human adenovirus), Herpesviridae (herpesvirus), Papillomaviridae (papillomavirus) and Retroviridae (human endogenous retrovirus) were detected in both FTLS patients and healthy subject samples. Some hepatitis B virus (HBV) and human bocavirus sequences were detected only in patient sera, indicating the presence of HBV and human bocavirus infections among these patients. To detect novel viruses, we examined sequence data against the GenBank protein database using BLASTx. An analysis of the deduced protein sequences revealed four different virus families in sera from FTLS patients  from Anelloviridae, which has been reported to be associated with certain inflammatory states Parvoviridae family, including human bocavirus, which could cause febrile illness and signs of FTLS. Although human bocaviruses are not known to be transmitted by arthropods, feline panleukopenia virus, a parvovirus, is strongly suspected to be transmitted by arthropods Parvoviridae sequences detected in FTLS samples were also assembled and their protein sequences deduced. Included among these samples were four fragments, all of which were found to be highly homologous to human bocavirus; one fragment showed the greatest similarity to human bocavirus 2 isolate 53044 (identity  = 86%) with the lowest E-value (8×10−17). Human bocavirus was further detected by PCR in the pool of 10 serum samples, but only one individual sample within the pool tested positive for bocavirus. The final virus family detected in sera from FTLS patients was the Bunyaviridae family, which contains viruses known to cause FTLS after tick bites Bunyaviridae sequences detected in FTLS samples, including 11 fragments , were assembled and their protein sequences were deduced. Among these 11 novel virus fragments was a 168-bp fragment  of the polymerase gene that showed the greatest similarity to Tehran virus (identity  = 36%) with the lowest E-value (3×10−7). This suggested the presence of a novel virus or a known virus whose genome had not yet been sequenced.For detection of known viruses, non-redundant reads were directly aligned with the GenBank database of nucleic acids using BLASTn software. Some sequences from patients . Among tPhlebovirus genus  to RNA polymerase of the Uukuniemi virus of the Phlebovirus genus, whereas the M segment had the highest homology  to glycoprotein genes of the Punta Toro virus in the Phlebovirus genus. Of the two proteins encoded by the ambisense S segment, one had the highest homology to nucleocapsid protein  of the Rift Valley Fever virus, and the other had the highest homology to nonstructural protein genes  of the Punique Virus, both of which belong to the Phlebovirus genus. Collectively, these findings confirm that this virus belongs to the Phlebovirus genus of Bunyaviridae.Genome sequencing of one isolate (HN01) revealed three segments of negative polarity, single-stranded RNA, including a large segment , a medium-sized segment , and a small segment . The deduced amino acid sequence of the L segment had the highest homology , the three genomic segments of the novel virus, along with the SFTSV sequences, were highly divergent  were released. A comparison of these new SFTSV sequences with the sequence of this novel virus showed that they were highly homologous (>99% identity). Using sequences of ivergent .IgG antibodies to the novel bunyavirus were detected in 80 of 285 acute-phase serum samples from patients with FTLS . Of 95 pA. phagocytophilum infection. However, only a few  such patients had evidence for A. phagocytophilum infection, and none of the 285 patients tested positive for the many other pathogens capable of causing similar clinical and laboratory manifestations that were also investigated. These findings suggested novel infectious agents, including viruses.Since 2007, there has been an increase in reported cases of FTLS in Xinyang City, Henan Province. These patients were tentatively diagnosed as having Parvoviridae and Bunyaviridae families reportedly can cause signs of FTLS and be transmitted by arthropods. However, only one sample from a pool of ten samples tested positive for bocavirus by PCR, suggesting that bocavirus from the Parvoviridae is not likely involved in FTLS. For viruses in the Bunyaviridae family, the incidence of infection is closely linked to vector activity. For example, tick-borne viruses are more common in the late spring and late summer when tick activity peaks. Human infections with certain Bunyaviridae, such as Crimean-Congo hemorrhagic fever virus, are associated with high levels of morbidity and mortality Bunyaviridae family viruses.Traditionally, virus culture is very important for identifying an unknown viral infection. Before performing the Illumina sequencing strategy, we attempted viral and rickettsial culture with DH82 and BHK cell lines, but the lack of an obvious CPE led us to initially abandon this approach. Here, mass sequence data obtained by Illumina sequencing revealed four virus families that appeared only in FTLS patient sera. Among these four virus families, viruses from the Bunyaviridae family contains more than 300 members arranged in four genera of arthropod-borne viruses  and one genus (Hantavirus) of rodent-borne viruses Phlebovirus genus currently comprises 68 antigenically distinct serotypes, only a few of which have been studied. The 68 known serotypes are divided into two groups: the Phlebotomus fever group  comprises 55 members, and the Uukuniemi group (transmitted by ticks) comprises the remaining 13 members. Of these 68 serotypes, eight are linked to disease in humans, including the Alenquer, Candiru, Chagres, Naples, Punta Toro, Rift Valley fever, Sicilian, and Toscana viruses The entire Bunyaviridae for our analysis. A 168-bp fragment of the polymerase gene with the lowest E-value and high sequence identity was used as the sequence of the unknown virus. This virus sequence was detected in all 10 pooled samples, indicating that the virus is involved in FTLS. After detecting a possible novel bunyavirus through high-throughput Illumina sequencing, we inoculated Vero cell lines, which are known to be sensitive to phleboviruses, with sera from six positive patients and were subsequently able to detect the virus by RT-PCR Phlebovirus branch, but was divergent from other known phleboviruses. These results confirm the novelty of this virus within the Phlebovirus genus of the family BunyaviridaeIn screening for unknown viruses, species hits alone likely carry little weight. Thus, we used all sequences in the family PCR and serological tests were performed to further test the causal link between the new virus and FTLS. Although we have not completely fulfilled Koch's postulates, evidence implicating this new bunyavirus in the outbreak of the disease among patients with FTLS is compelling.In view of the fact that the disease is caused by a novel bunyavirus, and taking into account that the disease was first discovered in Henan (HN), we propose the name "Henan Fever" for the FTLS disease cause by the novel virus (proposed name “Henan Fever Virus” [HNF virus]). Since the submission of this manuscript, a bunyavirus was identified as the cause of FTLS in Chinese patients from other regions of China, and the authors have named this virus “SFTSV” to indicate that it is the cause of severe fever with thrombocytopenia syndrome A. phagocytophilum, E. chaffeensis, Crimean-Congo hemorrhagic fever virus, Hantavirus, dengue virus, Japanese encephalitis virus, and Chikungunya virus. Furthermore, the availability of the new virus will facilitate the future development of new therapeutic interventions, such as vaccines and drugs.The discovery of this new virus will assist in the rapid diagnosis of this disease and help to distinguish it from other diseases caused by pathogens such as Figure S17 copies of control RNA per reaction) was detected in this assay.Sensitivity and dynamic range of real-time PCR in the detection of bunyavirus RNA. To evaluate sensitivity of our RT-PCR, a real-time PCR was performed. Serial dilutions of in vitrobtranscribed bunyavirus RNA sequences were tested. A wide linear range (from 5 copies to 5×10(TIF)Click here for additional data file.


Renilla luciferase-Aequorea GFP fusion protein, β-galactosidase, and β-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in miceWe have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts.we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice.These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals. At least 7 million people die from cancer worldwide every year, with an estimated 12 million deaths in 2030 /2) and reported in mm3. Thirty-three days after tumor cell implantation, mice were injected with a single intravenous dose of 5 × 106 plaque forming units (pfu) of individual virus strains in 100 μL of PBS. Animals were observed daily for any sign of virulence. Fourteen days post virus injection, four animals from each group were sacrificed for analysis of virus titers in tumors as described previously [All mice were cared for and maintained in accordance with animal welfare regulations under an approved protocol by the Institutional Animal Care and Use Committee of Explora Biolabs . GI-101A xenograft tumors were developed in 6- to 8-week-old male nude mice . Comparisons of treatment groups were made by either unpaired t test or two-way analysis of variance (ANOVA) with Bonferroni comparison post-tests as indicated in the figure legends. The post-test was only performed when ANOVA revealed significance. Statistical analysis of survival was assessed using the log-rank test. Values of F14.5L, J2R , and A56R  loci of the VACV LIVP genome disrupted the open-reading frames (ORFs) F14.5L, J2R, and A56R, resulting in the recombinant VACV GLV-1h68 with enhanced tumor colonization specificity as well as reduced virulence in mice [F145L, J2R, and A56R remain disrupted in all the derivatives. The RUC-GFP expression cassette at the F14.5L locus was replaced with the short DNA sequence "GGATCCTGCGAAGCTT" comprising BamH I and Hind III sites (underlined). The foreign inserts (human transferin receptor and lacZ) at the J2R locus were replaced with the non-coding DNA sequence "CTGTGAGCGTATGGCAAACGGTACCGAGCTCGGATCC" consisting of Kpn I, Sac I, and BamH I sites. The β-glucuronidase expression cassette at the A56R locus was replaced with the short DNA sequence "TAAGCTTCGCAGGATCCC" comprising Hind III and BamH I sites. The genotype of each derivative shown in Figure Previously, we have shown that insertion of the foreign marker gene expression cassettes into the  in mice ,11. To d-), GLV-1h73 (RUC-GFP-/gusA-), and GLV-1h74 (RUC-GFP-/lacZ-/gusA-) showed significantly enhanced virus replication at all time points examined, whereas GLV-1h70 (gusA-) and GLV-1h72 (lacZ-) demonstrated significantly better virus replication at 48 and 72 hpi, but not at 24 hpi, compared with the parent virus GLV-1h68 , GLV-1h73 (RUC-GFP-/gusA-), and GLV-1h74 (RUC-GFP-/lacZ-/gusA-) showed significantly higher replication efficiency at 48h, 72h, and all time points, respectively, than GLV-1h68 , GLV-1h73 (RUC-GFP-/gusA-), and GLV-1h74 (RUC-GFP-/lacZ-/gusA-) formed significantly larger plaques than GLV-1h68 in GI-101A cells  and GLV-1h72 (lacZ-) were consistently slightly larger than that formed by GLV-1h68. However, the differences in plaque size among GLV-1h68, GLV-1h70 (gusA-), and GLV-1h72 (lacZ-) were not statistically significant. Taken together, removal of any of the foreign expression cassettes or in combination, enhanced virus replication in each cell lines, irrespective of which foreign gene was removed. Among three foreign expression cassettes within the genome of GLV-1h68, removal of the RUC-GFP expression cassette had the greatest enhanced effect on virus replication. GLV-1h73 (RUC-GFP-/gusA-) with two of the foreign gene expression cassettes removed, replicated more rapidly than any of the single foreign gene expression cassette removal viruses, whereas GLV-1h74 (RUC-GFP-/lacZ-/gusA-) in which all three foreign expression cassettes were replaced, demonstrated the highest replication efficiency among all the virus strains tested. Thus, a series of recombinant VACV strains with fine-tuned replication efficiency were generated by replacing one, two, or three expression cassettes in the genome of GLV-1h68 with short non-coding DNA sequences.To examine whether removal of any of the originally introduced foreign gene expression cassettes from GLV-1h68 would affect virus replication, human breast tumor GI-101A cells in culture were infected with the parental virus GLV-1h68 and its derivatives at an MOI of 0.01 or 10. The infected cells were harvested at 24, 48, and 72 hpi and the viral titers at each time point determined in CV-1 cells using standard plaque assays. At the MOI of 0.01, GLV-1h71  showed the highest replication efficiency. It is interesting to note that the RUC-GFP expression cassette that was replaced in GLV-1h71 contains a synthetic early/late promoter (PE/L), the strongest promoter among the promoters introduced into the genome of GLV-1h68 [J2R locus was half of the index of the PE/L at the F14.5L locus (24.6) since the gene encoding human transferring receptor was inserted in the reverse orientation with respect to the promoter. Secondly, the promoter scores for GLV-1h68 and its derivatives were calculated by adding together the index of each inserted promoter remaining in each virus strain. The resulting promoter scores for GLV-1h68, GLV-1h70 (gusA-), GLV-1h71 (RUC-GFP-), GLV-1h72 (lacZ-), GLV-1h73 (RUC-GFP-/gusA-), and GLV-1h74 (RUC-GFP-/lacZ-/gusA-) were 99.3, 74.8, 50.1, 73.7, 25.6, and 0, respectively. Regression analysis indicated that there was an inverse correlation between the promoter scores and the virus replication efficiency in the GI-101A cell culture with a value of R2 being 0.9968 . All foreign gene expression cassette removal derivatives showed significantly more efficient tumor cell killing than their parent virus GLV-1h68 at certain time points after infection  and GLV-1h74 (RUC-GFP-/lacZ-/gusA-) with two and three of the foreign expression cassettes removed, respectively, gave significantly higher virus yields than their parental virus GLV-1h68. Although there was no statistically significant difference in virus yield between GLV-1h68 and single cassette-replaced virus strains, these virus strains consistently gave higher virus yields than GLV-1h68 in the GI-101A and other different tumor models tested  with each virus strain at 5 × 10d Figure . Regressd Figure .3 were injected i.v. with each virus strain at a single dose of 5 × 106 pfu/mouse. Tumor volume was monitored weekly for 84 days. As depicted in Figure -/lacZ-/gusA-) with all of the foreign expression cassettes excised, stopped growing three weeks after virus administration, which was two weeks earlier than the date when GLV-1h68-treated tumors ceased to grow. Overall, virus strains that replicated more efficiently in the GI-101A cell culture, also shank the GI-101A tumors faster.After demonstrating that removal of the marker gene expression cassettes enhanced virus replication and tumor cell killing in cell cultures as well as virus replication in tumors, we sought to determine if enhanced virus replication could be translated into improved therapeutic efficacy. Nude mice bearing the GI-101A tumors of 200-300 mm-) survived and looked healthy. Two mice each treated with GLV-1h68 or GLV-1h72 (lacZ-) died. Three, 5, and all 6 mice treated with GLV-1h70 (gusA-), GLV-1h73 (RUC-GFP-/gusA-), and GLV-1h74 (RUC-GFP-/lacZ-/gusA-) died, respectively. Statistically, there was a significant difference in survival between the GLV-1h68 treated group and the GLV-1h74-treated group, but no significant difference between GLV-1h68 treated group and any other virus-treated groups  treated group, we consistently observed higher virulence associated with GLV-1h73 (RUC-GFP-/gusA-) than GLV-1h68 in additional tumor models. Overall, virus virulence in tumorous mice directly reflected the replication efficiency of these virus strains in cell cultures.To evaluate if removal of the foreign gene expression cassettes from GLV-1h68 would affect virus virulence in mice, groups of 6 nude mice bearing the GI-101A tumors were treated with each virus strain individually as described above for the efficacy study (untreated group had 4 mice). Mouse survival was monitored daily. Due to excessive tumor burden, all untreated mice were sacrificed 102 days after tumor cell implantation . All treated groups were observed for 84 days after virus treatment. During this period, all mice treated with GLV-1h71  with two of the foreign gene expression cassettes removed replicated more efficiently than any of the virus strains with single foreign gene expression cassette removed. GLV-1h74 (RUC-GFP-/lacZ-/gusA-) in which all three foreign expression cassettes were replaced, demonstrated the highest replication efficiency among all the virus strains tested. Interestingly, we have noticed that replication efficiency of GLV-1h68 and its foreign gene removal derivatives was inversely proportionate to the added strength of the promoters present at the loci of F14.5L, J2R, and A56R in each virus strain. Thus, the expression of the foreign genes driven by the VACV promoters that adds an extra transcriptional and translational burden onto the virus and infected cells contributed to the attenuation of GLV-1h68 replication. Since the degree of attenuation of VACV strains is proportionate to the strength of promoters that drive foreign gene expression, therefore, it may be possible to fine tune VACV replication efficiency by choosing VACV promoters of appropriate strength to facilitate foreign gene expression in VACV as was demonstrated for the first time in this study.GLV-1h68, one of the most widely studied oncolytic VACVs, has been shown to be effective in treating large numbers of human and canine cancers in preclinical studies ,16-24. Iand A56R . Our preand A56R ,11. Inac in mice -27. In t in mice . GLV-1h7et al. reported that a VACV WR-derived oncolytic virus infected tumor-associated vascular endothelial cells, resulting in vascular collapse in infected tumors [et al. found that the immune response, but not direct viral oncolysis or replication, was a critical factor for reovirus-mediated tumor rejection or therapy [The mechanisms by which oncolytic viruses destroy tumors are currently under intense investigation. It is believed that an unarmed oncolytic virus destroys tumors through one, or most likely, a combination of the following mechanisms: 1) by direct viral oncolysis of tumor cells; 2) by destruction of the tumor vasculature ,28; and d tumors . In contd tumors . These d therapy . Previou therapy . However therapy . Instead therapy .Safety is a major concern for any cancer therapeutics. During the past years, strategies have been developed to enhance tumor selectivity and clinical safety of oncolytic viruses . These sTaken together, we have shown that virus promoter driven expression of foreign genes did in fact attenuate VACV replication. We found that virus replication efficiency was inversely proportionate to the added strength of promoters linked to foreign genes inserted into the viral genome. Thus, we propose that VACV replication can be fine tuned through carefully choosing the strength of promoters added to the VACV genome for the expression of foreign genes. Furthermore, VACV replication efficiency in cell cultures paralleled cytotoxicity in cell cultures, and importantly, replication efficiency in tumors as well as therapeutic efficacy in nude mice. However, enhanced virus replication efficiency in cell cultures resulted in increased virulence in mice.In conclusion, GLV-1h68 seems to have well balanced replication efficiency and minimum virulence in mice to achieve highly efficacious tumor rejection in nude mice with human xenografts. Virus strain attenuation by insertion of additional viral promoters of different strength into the VACV genome may result in rationally designed safe attenuated therapeutic strains, presumably due to competition for VACV RNA polymerase and cellular translation machinery required to facilitate transcription and translation of viral genes.Nanhai G. Chen, Yong A. Yu, Qian Zhang, and Aladar A. Szalay are affiliated with Genelux Corporation.F14.5L, A56R shuttle vectors, generation of the recombinant vaccinia viruses, virus replication assays, cytotoxicity assays, and virus titrations, performed the statistical analysis and interpretation of data, and drafted the manuscript. YAY was involved in the animal studies. QZ constructed the J2R shuttle vector. AAS conceived of the study, participated in its design and coordination, and prepared the final version of manuscript. All authors read and approved the final manuscript.NGC participated in the design of study, carried out the experiments including construction of the


Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic illness. There is little published data on the financial burden of this illness. The primary objective of this study was to determine the annual costs borne by families of a child with JIA living in Nova Scotia (NS).All families in NS with a child followed in the Pediatric Rheumatology Clinic at the Izaak Walton Killam Health Centre (IWK) in 2009 were mailed a self-report questionnaire. The questionnaire evaluated disease related costs, gross household income and perceived financial burden. Dillman's method was used to optimize return rates. Descriptive statistics were used to summarize results. Spearman’s correlation coefficient was used to assess the relationship of distance from the IWK and cost. The Mann–Whitney U test was used to compare median costs between groups.s = 0.18, P = 0.2). Families of a child with oligoarthritis had significantly lower costs than the families of a child with another subtype of JIA .Of 172 possible respondents, we received 54 completed questionnaires and 11 blank questionnaires . Approximately one third (35.9%) of parents rated the financial burden as moderate or large and 36% rated financial resources available as poor. The median annual total cost per patient was $619.50 CAD  which was a median 0.7%  of gross household incomes. The largest expense for families was visit related costs. There was not a significant relationship between total annual costs and distance from the IWK (rThe costs associated with having a child with JIA in NS are on average modest, but may be considerable for some families. Oligoarticular JIA is associated with smaller costs. Many families perceive the burden to be at least moderate and the availability of financial resources to be poor. Supports should be targeted to those families most in need. Juvenile idiopathic arthritis (JIA) is the most common rheumatic illness affecting children with approximately 1 in 1000 children having one of 7 categories [The Canadian health care system bears a significant portion of the medical costs, particularly physician and institution-based services. However, costs related to prescription medications, assistive devices and physiotherapy are often not included. Not all families have health insurance (whether private or government administered) to assist with these additional costs. There are also other non-medical costs which can add to the financial burden. These include costs associated with visits for medical care  and loss of paid work. The cost to families in order to provide their children with the recommended standard of care is unknown. However, anecdotal experience suggests that the burden for some families can be considerable.The goal of this study was to determine the annual out-of-pocket medical and related non-medical expenses borne by Nova Scotian families who have a child with JIA. We also assessed perceptions of financial burden and financial support for these same families.The Izaak Walton Killam Health Centre (IWK), located in Halifax, Nova Scotia, provides tertiary sub-specialist pediatric care to children in Nova Scotia (NS), New Brunswick and Prince Edward Island, Canada. This hospital is the only source of pediatric rheumatology care in these provinces, and most children with JIA are followed here.A questionnaire was created to estimate the annual out-of-pocket costs borne by families as a result of their child’s JIA , medication infusions and hospital admissions. Costs for these visits included transportation, parking, accommodations, childcare for other children, and communications related to the visit. Non-medical costs also included those related to home adaptations and driving their child to school.3. Finally, cost due to loss of paid work was defined as the loss of pay due to time away from work for reasons related to their child’s arthritis. This could be either or both parents.Gross annual household income was collected to allow calculation of costs related to their child’s JIA as a proportion of the family income. Perceptions of the financial burden associated with having a child with JIA and access to financial resources were also evaluated in the questionnaire using ordered response categories.Descriptive statistics were calculated for all parameters of the questionnaire. For consistency with previous literature, costs were summarized as medical, non-medical and those related to loss of paid work. The total cost was also compared to the household income per patient to determine the proportion of income lost. Spearman correlation coefficients were calculated to evaluate the relationship between costs and distance from the IWK. For this analysis, distance was estimated using travel time to the IWK, as the data was more complete than for actual distance.The Mann–Whitney U-test was used to compare costs for between groups of children. Given the small number of patients, these analyses were considered exploratory.Of 172 questionnaires mailed out, 5 were returned unopened due to incorrect addresses. Fifty-four (31.4%) were returned completed, and another 11 (6.6%) were returned blank or the family called to decline participation. Mothers completed 81.8% of the questionnaires.Participant characteristics are summarized in Table The median number of trips to the IWK for clinic appointments in 2009, excluding trips for joint injections or medication infusions, was 4 (range 0–27). Six children had joint injections at the IWK . Two children also came to the IWK for regular infusion (10 visits and 13 visits).Data on annual costs for 2009 are presented in Table The median cost per IWK clinic visit was $45.00 (range 0-$762.00). The median cost per clinic visit outside the IWK was $15.00 (range 0-$100.00). Due to small numbers, similar results were not calculated for emergency, infusion or joint injection visits.s = 0.18, P = 0.2). There was a moderate correlation between distance from the IWK and total annual visit costs to the IWK .There was no significant correlation between distance from the IWK and annual total costs  while those with all other categories combined had a median annual total cost of $877.00 . Children living more than 60 minutes from the IWK had a mean annual total cost of $893.50 (range $0-5535.00) while those living closer than 60 minutes away had a mean annual total cost of $460.38 .Families who rated the financial burden of having a child with JIA as being either large or moderate had a median annual total cost of $1205.00 (range $120-5036.00) compared to $474.88  for those who rated it as minimal or none. The proportion of gross family income represented by the total costs of JIA were 3.0% (large or moderate burden) and 0.5%  (P = 0.002).This study reports the financial costs borne by families of children with JIA living in Nova Scotia in 2009. We have shown that costs vary dramatically between households. The majority of costs were related to medical visits, followed by costs related to time away from paid work. Medical costs formed a relatively small component of the total costs. We found that disease category was associated with cost, with oligoarticular JIA resulting in smaller costs than other types. We also saw a trend towards increasing costs with increasing distance from the IWK.On average, total costs were relatively modest. However, our data show 2 important related findings. First, while costs were not large for most families, some families experienced total costs as high as 37% of their gross household income. Second, almost 36% of respondents reported their perception of the financial burden as being either moderate or large. It could be argued that having nearly 2/3 of families experiencing mild or no financial burden is a success of the health care system. However, we believe that we can do better, and our study highlights financial burden as an important issue for many families, and one that should be monitored in pediatric rheumatology clinics. Given that 36% of respondents also considered resources to assist with costs to be poor, further attention is needed to this issue.We expected that distance from the IWK Health Centre would be an important factor impacting cost. Given the relatively widespread, rural population of Nova Scotia, this is an important issue in our clinic. However, our data showed no significant correlation between distance and total cost and only a moderate correlation between distance and IWK specific visit costs. There was a non-significant trend for families living more than 60 minutes away to have higher costs. The explanation for this lack of a relationship is unclear. It may be that some costs are unrelated to distance . Additionally, it is possible that follow-up frequency is influenced by distance for children with similar JIA. It is important that all children receive optimal care and follow-up, regardless of residence. Our data was not adequate to investigate this question, but this is an issue which bears additional study. We did note that costs associated with IWK visits were higher than those associated with local visits ($45.00 vs $15.00), suggesting that distance had at least some impact.Comparing our results to previous work has some important challenges. The costs associated with health care and the financial responsibility for these costs varies considerably between countries and between regions within countries. This makes extrapolation of research conducted in one country or region to another country or region difficult. Health care costs can also vary considerably over time, as new medications become available or health care coverage changes. For example, in JIA the advent of biologic medications has the potential to markedly influence medical costs.Despite these limitations, some research in this area has been conducted. Allaire et al. reported costs for 70 families with a child with juvenile rheumatoid arthritis (JRA) from the New England area in 1989 . Total yMinden et al. reported on a group of 215 adults in Berlin with a history of JIA  . They foBernatsky et al. compared 155 children with JIA to 181 outpatient clinic controls without JIA in Montreal and Vancouver, Canada in 2005 [Minden et al. studied 369 children with JIA from a national German database, with costs estimated for 2003 [Thornton et al. studied 297 children newly diagnosed with JIA at 4 UK hospitals [Finally, Yucel et al. studied 100 children with JIA in Turkey in 2008 [In our study, we found that costs were higher than reported by most investigators, other than Allaire et al. . HoweverOnly 13% of patients in this study were treated with biologics. Over time, this proportion is increasing. However, the impact on costs to families is not easy to predict. Given the expense of these medications, it is unlikely that most families could afford them without insurance or other support. In our clinic, we have not prescribed biologics where families have not had some form of coverage. Even with insurance, families may have co-pay charges which could be considerable. Biologic therapy could also have an impact on visit costs, as some of the biologic treatments require infusions at the hospital, clinic or infusion centre. These evolving costs will require monitoring over time.As with all questionnaire-based research, this study needs to be interpreted with certain limitations in mind. First, we experienced a relatively modest response rate. This is not uncommon in this type of research, but does raise questions about how well respondents represent the overall population. The JIA category distribution of respondents is similar to our overall clinic population. If there is a bias introduced here, it is difficult to predict in which direction it may operate. It is possible that some families with significant financial concerns would be more likely to respond. However, it is also possible that those same families would be less likely to respond due to concerns about being stigmatized. A second limitation is recall bias. To minimize this, a chart review was completed to supplement and confirm data from the questionnaire, especially the number of visits to the IWK. Finally, costs are specific to location and health care system. This limits the direct applicability of our results to other jurisdictions. However, our overall findings are likely to be similar to other Multidisciplinary Rheumatology Clinics that provide services to large geographic areas.The impact of the financial burden on families of children with JIA is unknown. Beyond psychosocial stressors in the home, concern has been raised about the effect on outcomes. A prospective cohort study in a tertiary care center in the United States showed a significantly higher physical disability rate and lower health-related quality of life in children with JIA who had Medicaid or other similar state-programs for low-income families compared to those children with JIA who had insurance coverage . This stWe conclude that while the overall costs associated with having a child with JIA in Nova Scotia are modest for many families, the financial burden can be significant for some and may become a barrier to the provision of appropriate care. Many families perceive the financial burden to be high and the access to resources to be poor. Assistance in the form of targeted programs to aid families with the greatest costs should be considered. The impact of the financial burden on outcomes in children with JIA is unknown and warrants further evaluation.JIA: Juvenile idiopathic arthritis; NS: Nova Scotia; IWK: Izaak Walton Killam; JRA: Juvenile rheumatoid arthritis.The authors have conflicts to declare, whether financial or otherwise.The project was conceived, developed and conducted by AE under the supervision of AH. BL, SR and ES contributed to project development. AE and AH conducted the analysis. All authors contributed equally to manuscript preparation and revision. All authors read and approved the final manuscript.AE (at time of study) was a resident in pediatrics at the IWK Health Centre. She is currently a fellow in pediatric endocrinology at the IWK Health Centre. AH, BL, SR and ES are pediatric rheumatologists at the IWK Health Centre.Appendix. Cost of JIA Questionnaire.Click here for file


Also, in this study, we found that the identified mutations are in close proximity to the center of the repeat array rather than at the ends of the repeat array. Several studies have examined the mutational mechanisms of the minisatellites according to infinite allele model (IAM) and the one-step stepwise mutation model (SMM). In this study, we found that this locus fits into the one-step mutation model (SMM) mechanism in six out of seven instances similar to STR loci.Little is known about the general biology of minisatellites. The purpose of this study is to examine repeat mutations from the D1S80 minisatellite locus by sequence analysis to elucidate the mutational process at this locus. This is a highly polymorphic minisatellite locus, located in the subtelomeric region of chromosome 1. We have analyzed 90,000 human germline transmission events and found seven (7) mutations at this locus. The D1S80 alleles of the parentage trio, the child, mother, and the alleged father were sequenced and the origin of the mutation was determined. Using American Association of Blood Banks (AABB) guidelines, we found a male mutation rate of 1.04 × 10 The human genome can be grossly partitioned into three categories: nonrepetitive (single copy sequences), moderately repetitive (families of retroposon-like sequences), and highly repetitive  DNA . MinisatStudies have shown that minisatellites may mutate by unexpectedly complex conversion-like events . Higher Little is known about the general biology of minisatellites . The purThe D1S80 locus was previously used for forensic analysis due to its small size; however, this locus has recently been used as an associative tool in the elucidation of the chromosome 1p36 deletion syndrome which is one of the most common deletion syndromes located near a chromosomal terminus (one in 5000 births) , 25.Recent and past work with respect to this locus has included the use of two single nucleotide polymorphisms (SNPs). These included rs16824398, which is a SNP that involves a HinfI restriction site at a nucleotide 58 bases downstream from the forward primer and another SNP in the 3′ flanking region that involves an Fnu4HI restriction site, at the base next to the last repeat. Interestingly, all 18-repeat alleles to date have been found to be associated with HinfI(+) and Fnu4HI(−) restriction site polymorphisms at the 5′ and 3′ ends, respectively . On the Buccal epithelial cell samples from 45,000 trios for paternity analysis were collected in quadruplicate from each individual using cotton-tipped swabs and air dried at ambient temperature. Each paternity short tandem repeat (STR) assay was done using duplicate independently prepared DNA extractions and assayed using proprietary inhouse STR multiplexes. Each multiplex included a shared locus  with other multiplexes to confirm sample identity. D1S80 was assayed in combination with two to three other STR loci in multiplex reactions. The other STRs were selected to produce lower molecular weight fragments that would not interfere with analysis of the larger D1S80 amplified products. Amplified products were analyzed using nondenaturing high-resolution polyacrylamide gel electrophoresis with silver stain detection. Alleles were manually determined by comparison with allelic ladders containing all common D1S80 alleles. All STR-based parentage testing was performed with full knowledge and consent of the tested individuals or authorized parent/guardian. Appropriate institutional review board approval was obtained for sequence analysis of the samples.flSTR Identifiler human identification kit . This was essential to confirm that the mutation occurred in the D1S80 locus and was not the result of nonpaternity.To fully understand the implications and delineate the mutational mechanisms that the D1S80 locus undergoes, we examined the number of parent child allele transfers analyzed at this locus for the year 1996 from Laboratory Corporation of America, Burlington, NC. Observed mutations were confirmed as coming from concordant father or mother by analyzing short tandem repeat (STR) data from samples collected from alleged father, mother, and child using AmpSeveral factors were used in the elucidation of the origin of the mutation between the alleged father and mother of the child. The first factor was the phase of the SNPs at the 5′- and 3′-flanking sequence of the repeat array. Another factor was a match of the repeat units of the alleles most likely to be associated between father and mother and finally we assumed that the minimal size change was the most probable one.A mutation rate was then estimated by the number of samples sequenced that contain the mutation and compared to the number of trios sampled that year at Laboratory Corporation of America. Parentage calculations and mutation rate analysis were performed following guidelines put forth by the American Association of Blood Banks (AABB) .μL of Tris-EDTA (TE) buffer overnight. One to five μL of the gel extract was used for amplification of single alleles using the same primers described above. The alleles were sequenced using big dye terminator chemistry and ABI 310 genetic analyzer . Homozygous samples or samples that did not yield clean sequence were cloned into pBluescript plasmid vector. The DH5α competent cells were transformed using the recombinant vector and the cells were grown in LB/ampicillin agar plates. The resulting colonies were screened for the presence of the right size insert by directly sequencing the plasmid DNA isolated from minipreps of individual colonies. Sequencing of the cloned alleles was performed using big dye terminator chemistry and the sequence was analyzed using Applied Biosystems DNA sequencing analysis software version 5.2.Total genomic DNA was isolated from the trios that contained the mutation by standard organic extraction with phenol/chloroform/isoamyl alcohol followed by purification and concentration using Amicon Ultracel centrifugal filters . Quantitation of total genomic DNA was performed using Quantifiler human DNA quantitation kit . Paternity was confirmed by analyzing a set of fifteen short tandem repeat (STR) markers using the Identifiler human identification kit  as per manufacturer's recommendations. The samples were run on an automated 310 Genetic Analyzer and analyzed with GeneMapper ID software version 3.2 . The D1S80 locus was amplified using primers described by Kasai et al. . Allelesin vivo germline events rather than somatic mutational events.Paternity calculations were performed using AABB guidelines  and a reOnly two studies are reported that have dealt with the mutational events within this locus –37. The −3 to 1.53 × 10−3 [−4 [−5. The mutation rate of 7.77 × 10−5 represents the overall mutation rate rather than a rate specific to the male or female germline. Only one event could be narrowed down to the mother and two events attributable to the father. In the remaining four trios, we could not choose the progenitor because of the similarities of the maternal and paternal sequences. For the four indeterminate mutations, the AABB prorates them based on known mutation rates [−4  and 5.18 × 10−5 , respectively. Some mutations among the trios might not appear as Mendelian errors in the analysis since the scoring was based on a size difference between alleles (difference in the number of repeats between parent and child), and single base changes would not be detected, and the actual mutation rates could be slightly higher.Mutation rates for minisatellites have been estimated to range from 0.5% to greater than 20% per generation in some studies and in another study the rate was reported as 0.53 × 103 × 10−3 . Only on10−3 [−4 . We founon rates . In thisSeveral studies have examined mutational mechanisms according to infinite allele model (IAM) and the one-step stepwise mutation model (SMM). They have found that minisatellites sometimes fit both mechanisms; however, short tandem repeat loci (STR) were most similar to the simulation results under the SMM model of mutation. In this study, we found that this locus fit into the one-step stepwise mutation model (SMM) mechanism in six out of seven instances similar to STR loci , 37, 38.Jeffreys et al.  reportedIn addition to the differences in the repeat regions of the progenitor and mutated alleles, Jeffreys et al. have also found that the polymorphisms flanking the minisatellite repeat array have failed to reveal exchange of flanking markers . This isThe data we have reported highlights the importance of a number of questions. (1) Do repeats have functions and do they have an effect on genome structure? (2) Are the allelic clades the result of constraints on mutation? (3) Is selection acting at the D1S80 locus?, and (4) Why is the D1S80 locus conserved among primates (unpublished data) including humans?


Increased frequency of arbovirus outbreaks in East Africa necessitated the determination of distribution of risk by entomologic arbovirus surveillance. A systematic vector surveillance programme spanning 5 years and covering 11 sites representing seven of the eight provinces in Kenya and located in diverse ecological zones was carried out.Mosquitoes were sampled bi-annually during the wet seasons and screened for arboviruses. Mosquitoes were identified to species, pooled by species, collection date and site and screened for arboviruses by isolation in cell culture and/or RT-PCR screening and sequencing.Anopheles funestus sampled from Tana-delta and from Aedes mcintoshi from Garissa. Insect only flaviviruses previously undescribed in Kenya were also isolated in the coastal site of Rabai. A flavivirus most closely related to the Chaoyang virus, a new virus recently identified in China and two isolates closely related to Quang Binh virus previously unreported in Kenya were also detected.Over 450,000 mosquitoes in 15,890 pools were screened with 83 viruses being detected/isolated that include members of the alphavirus, flavivirus and orthobunyavirus genera many of which are known to be of significant public health importance in the East African region. These include West Nile, Ndumu, Sindbis, Bunyamwera, Pongola and Usutu viruses detected from diverse sites. Ngari virus, which was associated with hemorrhagic fever in northern Kenya in 1997/98 was isolated from a pool of Active transmission of arboviruses of public health significance continues in various parts of the country with possible undetermined human impact. Arbovirus activity was highest in the pastoralist dominated semi-arid to arid zones sites of the country where 49% of the viruses were isolated suggesting a role of animals as amplifiers and indicating the need for improved arbovirus disease diagnosis among pastoral communities. Arboviruses constitute a heterogeneous group of mostly zoonotic viruses transmitted by hematophagous arthropods, including mosquitoes, ticks and sand flies . Most ofArbovirus infection can be most effectively controlled by use of vaccines. However, this is limited by lack of registered vaccines for the majority of circulating arboviruses in the region except the Yellow Fever vaccine and Rift Valley Fever vaccine for livestock use only. Early detection of virus activity or detection of increased virus activity in the vector populations can be a key indicator or early warning for appropriate action to reduce outbreaks. Surveillance programs designed to monitor virus activity in vectors also provides a system for mapping disease distribution and information needed not only to assess risk but also to identify vector species for targeted control . To asseAedes aegypti screened by RT-PCR. The highest number of mosquitoes sampled, pooled and tested were from Garissa where the predominant mosquito species  were flood water Aedes species  followed by Marigat where the predominant species  were Mansonia africana, Mansonia uniformis and Culex pipiens. Naivasha  and Kisumu (514 pools) at the basins of Lakes Naivasha and Victoria respectively were in third place with a predominance of Cx. pipiens, Mansonia species and Culex zombaensis.A total of 450,680 mosquitoes were collected mostly by CDC Light trapping. 15,890 mosquito pools each containing up to 25 mosquitoes, were tested by cell culture screening and 158 pools of Overall 84 virus isolates giving reproducible CPE were obtained and characterized by RT-PCR and partial sequencing. They included Alphaviruses (53) among them Sindbis (SINV), Ndumu (NDUV), Babanki (BBKV) and uncharacterized alphaviruses; Flaviviruses (23) including West Nile (WNV), Usutu (USUV) and Insect Flaviviruses; Orthobunyaviruses (8) including Bunyamwera, Pongola and Ngari viruses.Aedes species; Ae. mcintoshi (18), Ae. ochraceous (8) and Ae. tricholabis (6). Viruses isolated were mostly Alphaviruses including Ndumu virus (NDUV) (94% identity) from Aedes mcintoshi (2), Ae. ochraceous (2) and Ae. tricholabis (3), Babanki virus (94% identity) (from Ae. mcintoshi (2) and Ae. ochraceous (2)) and unidentified Alphaviruses (7) from Ae. mcintoshi and Ae. tricholabis (2). There were also 4 orthobunyaviruses from this area consisting of one Ngari virus (96% identity), one Pongola and two Bunyamwera viruses (99% identity) isolated from Ae. mcintoshi. In addition, isolates of West Nile virus (97% identity) were obtained from 5 pools of Culex univittatus. The second highest number of isolates (11) were obtained from samples collected from Marigat with 6 of these being NDUV (94% identity) isolated from Culex rubinotus and the other 5 isolated from other Culex and Mansonia species , Sindbis-like viruses (94% identity) from Culex species (2) and one isolate of Usutu virus (97% identity) obtained from Cx. pipiens from Kisumu. Sindbis-like virus (2) isolates and one Babanki virus (98%) isolate were obtained from samples from Naivasha  with 77% identity. This virus was recently isolated from mosquitoes sampled in China. Three unidentified flaviviruses from Ae. aegypti (2) and Eretmapodite quinquevittatus (1) and one unidentified alphavirus from Ae. aegypti were also isolated in Rabai.Around the lake basins, five virus isolates were obtained from samples collected from Kisumu, 7 from Busia and 4 from Naivasha. They consisted of 7 Ndumu isolates (94% identity) from Busia; Sindbis virus from opennata , SindbisAn. funestus from Tana-delta while isolates of Bunyamwera virus (99% identity) were obtained from 2 pools of An. funestus from Magadi. A single isolate of WNV from the Cx. uinivittatus was obtained from Turkana. The phylogenetic analyses of the three groups of viruses are available in Figures Another isolate of Ngari virus was isolated from a pool of Culex naevei in South Africa in 1959 [The importance of mosquitoes in arbovirus disease transmission and maintenance in East Africa cannot be over-emphasized. Mosquito-borne arbovirus diseases have caused outbreaks afflicting both human and livestock with devastating public health and economic consequences in recent times ,5 Implem in 1959  and was  in 1959 ,12 and s in 1959 ,14. The  in 1959 . West Ni in 1959 . The finAedes mosquitoes. These species were also associated with Rift Valley Fever virus transmission during the two outbreaks in the area [Aedes, all the WNV isolates in Garissa and Turkana were obtained from Cx. univittatus. This suggests that the cycle of transmission of arboviruses is linked to specific mosquito species associated with flooding of breeding sites which results in hatching of hibernating mosquito eggs in abundance as happens during Rift Valley Fever outbreaks and that West Nile virus transmission is mainly driven by Cx. univittatus species in this region. Rift Valley Fever virus was detected in flood water Aedes mosquitoes only during the outbreak [The sampling in all sites targeted the rainy season when vector abundance is usually expected to be highest resulting in increased host/reservoir and vector contact hence increased virus activity. The sites with the highest mosquito abundance were also sites with the highest number of viruses isolated (Garissa and Marigat). These also happen to be the sites that were the hotspots for Rift Valley Fever outbreak in 1997/98 (Garissa) and 2006/07 (Garissa and Marigat). These two regions are also classified as semi-arid to arid and are inhabited by communities whose economic main stay is pastoralism. Most of the viruses were isolated from the most prevalent mosquito species in Garissa which were the flood water the area . While Noutbreak  but noneoutbreak  further Culex rubinotus. This indicates the importance of this marsh breeding mosquito that is found in abundance in estuaries and riverines lakes [Cx. rubinotus is also known to feed preferably on rodents [Only Ndumu virus (from 11 pools) was detected from mosquitoes tested from Marigat with over half of these (6) being recovered from es lakes  in the m rodents , an indiCulex species from Naivasha, Kisumu and Budalangi, sites neighboring lakes Naivasha and Victoria. These viruses are usually associated with migratory birds which nest around lakes during their stopover breeding seasons. Culex species preferably feed on such birds and may also feed opportunistically on humans. A single isolate of Usutu virus was recovered from a pool of Culex pipiens from Kisumu. This is another virus that is associated with birds in its natural cycle. It is known to be widely distributed in Africa and Europe and a recent occurrence of an outbreak among birds in Europe has been reported [Isolates of Sindbis and the closely related Babanki viruses were recovered from reported . These oAnopheles funestus pool sampled in Tana delta and another isolate from Garissa consisted of Ngari virus strains. All the Orthobunyavirus isolates were amplified with the primers targeting sections of the S, M and L segments of Bunyamweravirus and those targeting the M segment of Batai virus. The Ngari virus strains were amplified by all the above primers except the Bunyamwera M segment primers. The results were confirmed by sequencing giving 96% identity with the strain detected in the 1997/98 outbreak in Kenya (9). Bunyamwera virus was also detected in mosquitoes circulating in these same sites. The Bunyamwera viruses were amplified by all the above mentioned primers except the Batai M segment primers. This is an important observation which demonstrates that Ngari virus, first reported in Garissa in northern Kenya during the Rift Valley Fever virus outbreak [An. funestus is an important malaria vector known to be anthropophillic and therefore with a potential to transmit this virus to humans that could lead to NRI virus epidemics. Hence, unlike Rift Valley Fever virus, the risk of transmission of NRI virus to humans through mosquito bite may be greater. In West Africa, Ngari virus has been isolated from diverse mosquito species including Anopheles and Culex[Further analysis of the Orthobunyavirus isolates revealed that the isolates from and Culex. These fThe findings of this 5 year entomologic arbovirus surveillance have demonstrated the active circulation and distribution of a variety of arboviruses of public health importance in parts of Kenya, bringing to the attention of relevant authorities, the potential disease risks attributable to arboviruses in diverse ecosystems and provinces in Kenya. The concentration of the viruses in the pastoral arid to semi-arid eco-zones is an indication that livestock and surrounding wild animals may be important in the amplification of these viruses and the public health authorities need to improve detection of these diseases among the pastoral communities. It is important to note that Ngari virus, recently associated with life threatening hemorrhagic fever is more widely distributed in the country than previously thought. This underscores the need for continued surveillance, mapping of disease risk and creation of awareness concerning the risks among health care providers to improve detection, timely response and prevention. In-depth studies of these viruses including vector competence and molecular and evolutionary studies will shed more light on the dynamics of transmission, molecular epidemiology and the potential for future emergence of serious pathogens.The study was approved by the institutional review boards of the Kenya Medical Research Institute and Walter Reed Army Institute of Research.Mosquitoes were sampled in 11 sites across seven administrative provinces and geographically distinct regions of Kenya Figure  namely RAcacia type species and Prosopis juliflora.Garissa  is in North Eastern province and falls within arid to semi-arid zone and is predominantly flat with low lying plains. Garissa has an AMT of 28.8°C and AMR of 576 mm and is characterized by infrequent rainfall and are prone to occasional flooding. The vegetation is predominantly Prosopis juliflora and some acacia. Magadi, on the shores of Lake Magadi has an AMT of 29°C and AMR of 469 mm. Turkana comprises of both low-lying plains with an altitude range of 370 - 900 m and the mountainous regions ranging from 1,500 – 1,800 m. It lies in the arid and semi arid zone with an AMT temperature of 30°C and an AMR of 300 mm.Sukari ranch  is located on the outskirts of Nairobi city in Central province and is generally cooler, with an AMT of 19°C and AMR of 1,065 mm. In the Rift Valley province, sampling was conducted in Marigat , Magadi , and Naivasha . Marigat, which is located on the shores of Lakes Baringo and Bogoria, has an AMT of 24°C and AMR of 655 mm. Naivasha has an AMT of 25°C and AMR of 750 mm and is located on the shores of Lake Naivasha. Vegetation in Marigat is predominantly Kisumu  lies on the shores of Lake Victoria in Nyanza province and has an AMT of 23°C and AMR of 1,427 mm. It borders Western province where we have the low plains of Busia  and Budalangi  also on the shores of Lake Victoria. Kakamega  and Mt. Elgon  are humid with heavy rainfall all year round probably influenced by the Kakamega tropical rainforest. Busia district is the warmest with an AMT of 25°C and AMR of 1,300 mm.Eight of the sites were selected based on historical arbovirus outbreaks, prior cases or reports of human cases of fevers of unknown origin and an ecological environment suitable for arbovirus transmission (determined by availability of abundant vector habitats). The three remaining sites: Magadi, Turkana and Tana-Delta, were sampled in response to reports of increased flooding and reported upsurge of cases of febrile illnesses. All collection sites were mapped by determining the coordinates (latitude and longitude) on a GPS-12 geographical positioning system .Mosquitoes were sampled only during wet periods when high mosquito population density is expected. Carbon dioxide-baited CDC light traps and landing collection methods were used to collect mosquitoes. Consent to participate in human landing mosquito collection was obtained from willing volunteers. The CDC light traps were hung at least two meters from the ground and baited with carbon dioxide held in igloos next to the traps  and left3, supplemented with 15% heat-inactivated fetal bovine serum (FBS), (Sigma-Aldrich), 2% L-Glutamine (Sigma-Aldrich), and 2% antibiotic/antimycotic solution with 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per ml (Sigma-Aldrich) and shaken vigorously. The supernatant was then harvested by centrifugation in a refrigerated bench-top centrifuge at 12000 rpm for 10 minutes and stored in a 1.5 ml cryovial at -80°C for testing.Pools of mosquitoes were homogenized in a biosafety level 2 laboratory based at the Kenya Medical Research Institute’s (KEMRI) Centre for Virus Research. One, 4.5-mm-diameter, copper-clad steel bead  was placed in eppendorf tube with the pool of mosquitoes and 1 ml diluent Eagle’s Minimum Essential Media (MEM)  with Earle’s salts and NaHCOAll mosquito pool supernatants were screened for viruses by cell culture inoculation in Vero cells . Fifty microliters of each mosquito pool suspension was inoculated into a single well of a 24-well culture plates containing a monolayer of Vero cell cultures in growth medium  with Earles salts, with 10% Fetal Bovine Serum (FBS), 2% Glutamine, 100 units/ml of penicillin, 100 ug/ml streptomycin and 1 μl/ml of Fungizone). Inoculated cultures were incubated at 37°C for one hour to allow virus adsorption. 1 ml of Maintenance medium was then added . Cells were incubated at 37°C and observed daily for cytopathic changes. Cell culture supernatants were harvested when cytopathic effect (CPE) involving 50% of the cell monolayer was observed. Due to the costs involved in this screening, only 10% of the negative cultures from the first inoculation were subcultured (including those that were not clearly negative) to improve chances of virus recovery. A virus isolate was suspected when the CPE was reproducible in a passage of the initially harvested culture. The supernatants from all cultures showing reproducible CPE were analyzed further.RNA was extracted from a 250 μL of each cell culture supernatant using the Trizol®-LS - Chloroform extraction method . The finA Positive control cDNA and a no template negative control were included during the setting up of all PCR reactions. Electrophoresis of the amplified DNA products was done on a 1- 2% Agarose gel in Tris-borate EDTA buffer stained with ethidium bromide. The PCR product bands were visualized by a UV transilluminator and recorded using a gel photo imaging system.Amplified target DNA bands were either purified directly from the PCR reaction or from the gel using Wizard® SV Gel and PCR Clean-Up System kit (Promega). Sequencing was outsourced and performed using ABI-PRISM 3130 Genetic Analyzer . The sequences were compared with available sequences using Basic Local Alignment Search Tool  and the The authors hereby declare that they have no competing interests.CO contributed in the detection and identification of the virus isolates, data analysis, drafted manuscript, and final manuscript preparation. JL conducted fieldwork, contributed to field study design and contributed to drafting manuscript, and final manuscript preparation. AM, HK and EC performed virus isolation and contributed to the data analysis and review of the manuscript. SY and JM conducted fieldwork, LM, SK, JHR and JB contributed to field and lab study design and final manuscript preparation. DS and EW, the directors of the Global Emerging Infections surveillance and response System/Department of Emerging Infectious Diseases provided overall project management and guidance and ensured continued funding for the project. RS contributed to overall study design, overall supervision of implementation, data analysis, drafted manuscript and final manuscript preparation. All authors read and approved the final manuscript.Table showing the total number of mosquitoes collected for each species, the number of pools tested for each species and the viruses isolated from diverse mosquito species and sites.Click here for file


Here, we used sRNA deep-sequencing combined withtranscriptome profiling to determine the global impact of tobamovirus infectionon Arabidopsis sRNAs and their mRNA targets. We found thatinfection of Arabidopsis plants with Oilseed rapemosaic tobamovirus causes a global size-specific enrichment ofmiRNAs, ta-siRNAs, and other phased siRNAs. The observed patterns of sRNAenrichment suggest that in addition to a role of the viral silencing suppressor,the stabilization of sRNAs might also occur through association with unknownhost effector complexes induced upon infection. Indeed, sRNA enrichment concernsprimarily 21-nucleotide RNAs with a 5′-terminal guanine. Interestingly,ORMV infection also leads to accumulation of novel miRNA-like sRNAs from miRNAprecursors. Thus, in addition to canonical miRNAs and miRNA*s, miRNAprecursors can encode additional sRNAs that may be functional under specificconditions like pathogen infection. Virus-induced sRNA enrichment does notcorrelate with defects in miRNA-dependent ta-siRNA biogenesis nor with globalchanges in the levels of mRNA and ta-siRNA targets suggesting that the enrichedsRNAs may not be able to significantly contribute to the normal activity ofpre-loaded RISC complexes. We conclude that tobamovirus infection induces thestabilization of a specific sRNA pool by yet unknown effector complexes. Thesecomplexes may sequester viral and host sRNAs to engage them in yet unknownmechanisms involved in plant:virus interactions.Tobamoviruses encode a silencing suppressor that binds small RNA (sRNA) duplexes RNA silencing is a sequence-specific mechanism that coordinates the expression,protection, stability, and inheritance of eukaryotic genomes. It is involved intuning critical developmental, stress-responses, and bodyguard functions byregulating the expression of genes at the transcriptional and post-transcriptionallevels, or by triggering the formation of heterochromatic DNA regions Arabidopsis thaliana genome contains four DCL and ten AGOgenes. Several sRNA classes that depend on different pairs of DCL and AGO proteinshave been identified. For example, whereas DCL1-dependent miRNAs guide AGO1, AGO7,or AGO10 to corresponding target RNA transcripts, DCL3-dependent siRNAs guide AGO4,AGO6 or AGO9 to DNA targets Plants encode several members of these protein families. For instance, theTobacco mosaicvirus (TMV) and related tobamoviruses encode a VSR that resides in thesmall subunit of their replicase invitro and interferes with their methylation Tobacco etchvirus (TEV) or p19 of tombusviruses Oilseed rape mosaic virus (ORMV)in vitroCymbidium ringspot virus occurs through an activity independentof binding by the VSR p19 and that miR168 functions in translational repressionrather than in mRNA cleavage despite of the increased AGO1 mRNA levels RNA silencing is known to play a critical role in defense against viruses in plantsand insects So far our knowledge on changes of sRNA and transcriptome profiles induced by a virusin its host is limited. Our current knowledge is based on the analysis of a limitednumber of sRNAs and of their targets by Northern blots or on small-scale sRNAcloning We have used a combination of mRNA profiling and sRNA deep sequencing to understandthe impact of ORMV infection on the Arabidopsis sRNA and transcriptome profiles. Wedescribe here a global analysis of viral sRNAs and the changes in cellular sRNAprofiles during systemic infection. Our analysis shows that virus infection changesthe pattern of sRNAs that are processed from miRNA and ta-siRNA precursors. Thevirus-induced sRNA profile is inconsistent with the proposed stabilization of sRNAduplexes by the replicase as a sole mechanism and suggests that the observed changesin the sRNA profile involve additional mechanisms. In particular, we found that ORMVinfection leads to the specific enrichment of 21 nt sRNAs with a 5′ terminalguanine. This suggests that these sRNAs associate with size-specific and 5′nucleotide-specific effector complexes of yet-unknown nature. Our mRNA profilingdata demonstrate that increased levels of miRNAs and siRNAs do not correlate withsignificant changes in target transcript levels indicating that the virus-inducedsRNA fraction is sequestered or active at different levels. Virus infection alsoleads to the accumulation of novel miRNA-like siRNAs (ml-siRNAs) encoded by miRNAprecursors that might be part of a specific plant response to pathogens.Arabidopsis thaliana genomes  of the reads originated from the virus (To gain insights into the effects of ORMV infection on the Arabidopsis sRNA profilewe conducted Illumina sequencing of sRNA populations extracted from ORMV-infectedand mock control-inoculated Arabidopsis Col-0 plants at 7 days post-inoculation(dpi). Following removal of adapter sequences the reads were mapped to the virus and genomes , Aand ohe virus . Consisthe virus . The vsRhe virus . A similComparison of the profiles of the mapped Arabidopsis-encoded sRNA obtained formock and virus-treated samples reveals a significant impact of virus infection.The normalized, size-specific distribution of sRNAs shows a significant decreasein the proportion of 24 nt sRNAs, whereas the proportion of 21 nt sRNAs isincreased .ConsistTASgenes. The biogenesis of ta-siRNAs is initiated by AGO-mediated cleavage ofTAS transcripts guided by miR173(TAS1a/b/c and TAS2), miR390, or miR828 (TAS4) TAS1a,b,c and TAS2loci  siRNA strand is enriched in infected tissues, thecomplementary strand (TAS2 3′D6(-)) of the ta-siRNA duplex is not.Conversely, specific enrichment is observed for the TAS2 D7 (-) siRNA strand,whereas the level of the complementary TAS2 3′D7(+) strand remainsunchanged. Consistent with previous observations TAS1a,b,c and other loci, does not show anyincrease upon virus infection.Arabidopsis ta-siRNAs are derived from four families of AS2loci . InteresTAS RNAs from which they arederived. Specifically, enriched ta-siRNAs with a 5′G are predominantlyderived from TAS2 including the validated 3′D6(+)and 3′D7(-) species  areunderrepresented compared to ta-siRNAs starting with A or U  and AGO7(TAS3a) and subsequent processing of theTAS RNA duplexes by DCL4 are not affected in infectedcells. The distribution of the mapped ta-siRNA reads mapped to theTAS genes confirms that the enrichment is restricted tospecific ta-siRNAs and that in most cases only one of the two strands of theprocessed ta-siRNA duplexes is enriched. Collectively, these observationsindicate that the virus-induced enrichment of ta-siRNAs occurs at apost-processing step, presumably through stabilizing associations with specificeffector complexes, as is suggested by the strand, size and nucleotidespecificity of the ta-siRNAs.To test whether infection has an effect on the phased processing of ta-siRNAs, wemapped the frequency of unique ta-siRNA species with more than 5 reads to3a genes . A changTAS loci. A similar effect is indeed also observed for otherRDR6/DCL4-dependent, secondary siRNA-generating loci We note that the specific enrichment of 20–21 nt siRNAs is not restrictedto Next, we investigated the impact of virus infection on the profile of miRNAs.Previous studies using specific Northern blot probing or small-scale sequencingindicated an enrichment of miRNAs in tobamovirus infected tissues Although ORMV starts to spread systemically within 2–3 days afterinoculation of individual leaves , viralsymptoms are only observed after 10 days. From this time onward the newlyemerging leaves and progressively also the older leaves show curling andretarded growth, and the oldest leaves show signs of necrosis. Three weeks afterinoculation, the plants show a clear growth retardation phenotype .2 fold-change cut-off of 2 and a significancevalue of p<0.001. Of those, 175, 605, and 1119 genes displayed differentialexpression in virus-infected compared to mock-inoculated samples exclusively at7, 14 and 21 dpi, respectively. 179 genes were differentially expressed uponORMV infection at all the three time points. In addition, 75 genes wereregulated upon ORMV infection at the two earlier time points (7 dpi and 14 dpi),40 genes were differentially expressed upon ORMV infection only at 7 dpi and 21dpi, and 1023 genes exhibited differential expression at the two later timepoints (14 dpi and 21 dpi). Independent samples were used for qPCR analysis,which reproduced the infection-induced changes in the expression of the testedgenes (data not shown). The quality of the data was also validated by principalcomponent analysis  and a gradual increase in symptomseverity . Thetotsis PCA,  and hiersis PCA, , indicat2>2; p<0.001) revealsthat at 7 dpi there are responses of gene classes related to responses to bioticstimuli, other organisms, stress, defence, and immune system processes, whereasresponses at the later time points are focussed to gene classes ratherresponding to metabolic processes and abiotic stimuli , a public platform forexploring microRNA-target interaction maps from Argonaute CLIP-Seq (HITS-CLIP)and degradome sequencing  data To determine whether enriched ml-sRNAs could play a role in RISC-mediateddegradation of target transcripts, we used them as queries for a DegradomeSearchwith StarBase  or may be controlledby established RISCs that are stable and thus resistant against virus-inducedchanges in sRNA levels. Second, since some of the mRNA targets show changes inexpression levels, the virus-induced sRNA enrichment may occur predominantly inspecific tissues in which the majority of the corresponding targets is notexpressed. The enriched sRNAs may also function in sRNA-guided translationalrepression rather than in target transcript cleavage. Translational suppressionby sRNA-guided AGO complexes is a common phenomenon in plants and animals With regard to functional diversification it is also important to note that alarge proportion of the sRNAs that accumulate in tobamovirus-infected plants isnot methylated The enrichment of miRNA*s suggests that they might have a conditionalfunction, as may also be supported by their association with predicted targetsand the presence of potential target RNA cleavage products in degradomedatabases In addition to miRNA* sequences, virus infection also causes the accumulationof ml-sRNAs, which also may play important roles during infection. The detectionof ml-sRNAs indicates that miRNA precursors can produce additional sRNAs inArabidopsis upon virus infection. Processing of multiple miRNAs from oneprecursor is a common phenomenon in animals where miRNA precursors often foldinto complex secondary structures with multiple hairpins, each coding for one ormore miRNAs. In plants, miRNA precursors usually form one hairpin coding for onemiRNA. However, in rare cases, plant miRNA precursors can code for more than onemiRNA. In these cases, miRNAs were shown to be processed sequentially from thelonger hairpins by DCL1 from the hairpin base Pseudomonas syringaeThe ml-sRNAs that we have identified here are rare or absent in non-infectedtissues and accumulate to high levels upon ORMV infection. We identified 19ml-siRNAs derived from 14 precursors . The enrArabidopsis thaliana col-0 plants were grown inhumidity-controlled growth chambers at 21°C using a 12 h/12 h light/darkcycle. Nicotiana benthamiana plants were grown under greenhouseconditions at 24°C using a 16 h/8 h light/dark cycle. To generate theinoculum for ORMV infection, N. benthamiana plants wereinoculated with ORMV RNA in vitro transcribed from aninfectious clone. Crude, virus-containing extracts (sap) from these infectedplants as well as virus-free extracts from non-infected plants were used formechanical inoculation of young Arabidopsis plants (5 leaves stage). The rosetteleaves were harvested at 7 dpi, 14 dpi, and 21 dpi. The harvested leaves of16–20 mock- or ORMV-inoculated plants were pooled per sample. Threeindependent samples of each treatment and time point were prepared.Invitrogen, Switzerland) extraction protocols followingthe instructions of the producer. The quality of the samples was verified byhybridization with specific probes to detect viral RNA and viral/endogenoussRNAs. Affimetrix gene chip hybridization for RNA profiling was performed at theFunctional Genomics Center Zurich. The resulting Affimetrix microarray datasetwas analyzed by using the R environment (http://www.R-project.org)http://www.biocinductor.org). For sRNA analysis, the total RNAsamples were separated by gel electrophoresis. 18–30 nt sRNAs wereisolated form the gel, ligated with adapters and sequenced using Solexatechnology .Total RNA extracts were prepared by using classical Trizol were generated. Following removal of adapter sequencesthe reads were grouped and counted according to sequence identity using acustomized Python script (available upon request). The reads were mapped againstthe ORMV and sRNAs were detected by Northern blot hybridization using radiolabeledoligonucleotide probes as previously described sRNA targets were identified by searching degradome databases with the StarBaseon-line tool http://www.ncbi.nlm.nih.gov/geo/) datasets used in the assaywere GSM253622 (AGO1), GSM253623 and GSM304285 (AGO2), GSM253624 (AGO4),GSM253625 (AGO5), and GSM304283 (AGO7).Potential associations of sRNAs with AGO proteins were identified by searchingdatabases of AGO-associated sRNAs. The GEO  Number of normalizedsRNA reads (RPM) mapped to the Arabidopsis thaliana (A.t.)and viral genomes. (B) Proportion of virus- and plant-derivedsRNA reads in the population of sequenced and mapped sRNAs of ORMV-infectedplants. (C) Size distribution of vsRNAs. Virus infectionincreases the number of 21 nt sRNAs whereas the number of 24 nt sRNAs isreduced. (D) The normalized frequency (RPM) of vsRNAs accordingto their specific 5′ nucleotide. (E) vsRNAs mapped to theplus strand (black) and minus strand (grey) of the ORMV genome.(TIF)Click here for additional data file.Table S1Size-specific profile of ta-siRNAs in mock- and ORMV-treated plants (7dpi).(DOC)Click here for additional data file.Table S25′nucleotide-specific accumulation of TAS2-derived21nt siRNAs in ORMV-infected tissue (7 dpi).(DOC)Click here for additional data file.Table S3Size-specific profile of siRNAs encoded by RDR6-dependent loci in mock-and ORMV-treated plants.(DOC)Click here for additional data file.Table S4Size-specific profile of siRNAs encoded by IR71 in mock- andORMV-treated plants (7 dpi).(DOC)Click here for additional data file.Table S5miRNA and miRNA* reads in mock and ORMV-infected plants.(DOC)Click here for additional data file.Table S62-fold change, p<0.001) upon ORMVinfection.Functional annotations for genes with modulated expression(log(DOC)Click here for additional data file.