Zika virus (ZIKV) has spread widely in the Pacific and recently throughout the Americas. Unless detected by RT-PCR, confirming an acute ZIKV infection can be challenging. We developed and validated a multiplexed flavivirus immunoglobulin M (IgM) microsphere immunoassay (flaviMIA) which can differentiate ZIKV-specific IgM from that due to other flavivirus infections in humans. The flaviMIA bound 12 inactivated flavivirus antigens, including those from ZIKV and yellow fever virus (YFV), to distinct anti-flavivirus antibody coupled beads. These beads were used to interrogate sera from patients with suspected ZIKV infection following travel to relevant countries. FlaviMIA results were validated by comparison to the ZIKV plaque reduction neutralization test (PRNT). The results highlight the complexity of serological ZIKV diagnosis, particularly in patients previously exposed to, or vaccinated against, other flaviviruses. We confirmed 99 patients with ZIKV infection by a combination of RT-PCR and serology. Importantly, ZIKV antibodies could be discriminated from those ascribed to other flavivirus infections. Serological results were sometimes confounded by the presence of pre-existing antibodies attributed to previous flavivirus infection or vaccination. Where RT-PCR results were negative, testing of appropriately timed paired sera was necessary to demonstrate seroconversion or differentiation of recent from past infection with or exposure to ZIKV. Flavivirus of the family Flaviviridae. The virus was identified in 1947 in Uganda [Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) assigned to the genus n Uganda . When prn Uganda . A suspen Uganda ,5. From n Uganda ,7,8. WheWe previously described an IgM capture ELISA (MAC-ELISA) which could sensitively and specifically differentiate between infections due to any of the four DENV serotypes, Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), Kunjin virus (KUNV), Alfuy virus (ALFV), Kokobera (KOKV) and Stratford virus (STRV) . We subsWe describe the development, validation, challenges and application of the flaviMIA consisting of a panel of 12 flaviviruses which are either endemic or relevant to travelers visiting or returning to Australia. The flaviMIA allows identification of plaque reduction neutralization test (PRNT)-confirmed ZIKV-specific IgM among sera sent to a public health virology laboratory for characterization.6 beads were coupled to approximately 10–12 µg of antibody. The antibody was either purified in-house from stocks of murine ascitic fluid, or obtained commercially . Coupled microspheres were stored at 4 °C at approximately 107 beads per mL.Bio-Plex Pro Magnetic carboxylated beads  were coupled to the 6B6C-1 flavivirus monoclonal antibody  using a ZIKV strain MR766 was sourced from the American Type Culture Collection . This was the only strain available at short notice permitting us to rapidly develop tests in response to a public health need. The literature suggests there is only a single serotype of ZIKV and there is no evidence that different phylogenetic lineages react differently to IgG and IgM . The vacEach inactivated virus preparation was pre-titrated to determine optimal working dilution by flaviMIA testing of serial two-fold dilutions against known positive human sera and selecting the dilution giving maximum reactivity. Each virus preparation was diluted to 4 mL in MIA buffer  + ProClin300  as a preservative) then mixed with its corresponding 6B6C-1-coupled beadset at 1:20. The virus–bead mixes were stored in light-resistant 4 mL bottles  which were incubated at room temperature on a shaker for several hours after which they were stored at 4 °C.Because infections by various viruses included in the flaviMIA occur infrequently , characterized clinical reference sera are rare and often unavailable commercially.Where possible, reference sera were sourced from IgM positive patients with RT-PCR-confirmed recent flavivirus infections who were willing to donate a large volume of sera for laboratory quality assurance purposes. When this was not possible, control sera were constructed by pooling reactive patient samples.Patient serum samples referred to our laboratory from public hospitals or private diagnostic laboratories specifically for flavivirus serology were first screened for flavivirus IgG and IgM by in-house ELISAs . These E® Universal System and QIAamp One-For-All Nucleic Acid Kit,  or EZ1 and Virus Mini Kit v2.0,  were added to 15 µL RT-PCR reactions  removal reagent  to remove potentially interfering IgG and rheumatoid factor. This was performed in a microtiter plate with 5 µL serum diluted 1:20 in 95 µL MIA buffer. RF removal reagent was added (100 µL) to each well, followed by incubation at room temperature for 15 min and a final dilution of 1:800 in MIA buffer.The flaviMIA antibody binding reaction was then performed in 96-well filter plates , in wells pre-wetted with PBS-Tween. Each flavivirus and control beadset were further diluted 1:20 in MIA buffer in sufficient volume for the number of samples being tested. The bead mixture was added to the wells of the plate at 100 µL per well followed by washing in PBS-Tween using a Bio-Plex Pro II washer. Diluted sera were added to the plate, along with a specific positive control serum for each virus, and a negative serum in duplicate. The plate was incubated at room temperature with shaking for 45 min after which the washing was repeated. Phycoerythrin-labelled donkey anti-human IgM antibody  was diluted to previously-determined optimal concentration in MIA buffer and added to each well of the plate. Incubation and wash steps were repeated and the beads were resuspended in each well by addition of 150 µL MIA buffer.® software . Results were exported to Microsoft Excel® and presented graphically for easier determination of specificity and analysis. A mean fluorescent intensity (MFI) cut-off of 2000 units was assigned based on comparison to RT-PCR-confirmed dengue sera; values above 2000 were considered a positive result. Specificity determinations were based on the MFI result of one viral beadset preparation being 1.5 times higher than the MFI of any other beadset in the panel. Where a single virus-specific IgM could not be determined, i.e., if there was reactivity to two or more flaviviruses in the panel (excluding between dengue serotypes) the result was reported as “cross-reactive” , with 100 beads per well read in 100 µL of buffer using Bio-Plex Managereactive” . This cr® CCL-81TM). Following virus absorption for 60 min, the cells were overlaid with a medium containing Gibco Opti-MEM , 3% foetal bovine serum  and 1.5% carboxymethylcellulose . The Vero cell plates were then incubated for seven days, after which the supernatant was removed and the cells washed with PBS and stained with naphthalene black stain (0.1% (w/v) naphthol Blue Black, 6% (v/v) glacial acetic acid, 1.36% sodium acetate). Plaques were counted and sera (at 1:10) demonstrating >90% plaque neutralization by comparison of plaque counts in duplicate “no antibody” control wells were reported as positive.Because PRNT is considered a gold standard assay for confirmation of flavivirus antibodies, a ZIKV PRNT was conducted to interrogate ZIKV results from the ZIKV component of the flaviMIA. Sera were screened at a dilution of 1:10 after incubation at 37 °C for 90 min with an equivalent volume of diluted ZIKV MR766 whole virus antigen preparation. Serum from a confirmed ZIKV Asian lineage infected patient was used as a positive control and a pool of flavivirus negative sera was used as the negative control. ZIKV was pre-titrated to give approximately 75 plaques per well of a 6-well plate. After incubation, 100 µL of each serum/virus mix was inoculated in duplicate into 6-well plates seeded one day previously with Vero cells  sera from patients positive for ZIKV (RT-PCR or PRNT confirmed cases) but who have no serological evidence of previous flavivirus infection (primary ZIKV cases), 834 acute and convalescent-phase sera known to be positive for one of nine other flaviviruses and 70 flavivirus IgG and IgM screening ELISA negative sera were analyzed by flaviMIA . The sen90 titer).A strain from the ZIKV Asian lineage  was isolIn responding to clinical and public health need, our laboratory has identified a total of 101 patients with flaviMIA results indicating specific IgM to ZIKV . Of thesOf the 43 patients determined as having recent primary ZIKV infection, 23 (53.5%) had acute samples which were negative by flaviMIA while 20 (46.5%) were positive. All but one of the positive sera had specific ZIKV IgM. Twenty-eight (65.1%) of the 43 patients had a follow-up sample collected. In 27 patients, the flaviMIA was specific for ZIKV, while the other patient exhibited cross-reactivity to other flaviviruses .Our laboratory also tested samples from 21 patients whose results indicated that infection with ZIKV was secondary to a previous flavivirus infection. Previous infection was determined by the presence of flavivirus IgG (detected by ELISA) concurrent with the detection of ZIKV RNA or in one case (Reference No. 24) Flavivirus IgG in the absence of IgM within five days of return from overseas travel. Of the 21 acute sera, nine (42.9%) were negative and the remainder were positive by flaviMIA. Six of the flaviMIA positive sera were ZIKV IgM specific, two were cross-reactive between ZIKV and at least one other virus, while the remainder had IgM that specifically reacted to either DENV-2 or DENV-4 (both suggesting previous infection with these dengue serotypes) and two to YFV (consistent with previous vaccination with YFV associated with travel to South America). Unfortunately, these prior infections could not be confirmed (by PRNT) owing to routinely insufficient sample volume, however pre-existing specific antibodies were confirmed by indirect fluorescent antibody test.A convalescent serum sample was collected from 12 of the patients and each was tested in parallel with its paired acute serum to demonstrate change in antibody levels. Eight of the follow-up sera were positive and specific for ZIKV IgM including the acute sample with initial DENV-4 IgM (Reference No. 8), three from initially negative patients, three from the initially ZIKV-specific patients, and from one patient whose acute sample was YFV IgM reactive (Reference No. 83). Three further patients had cross-reactive IgM in the convalescent sample, two from initially negative patients (Reference No. 23 and 36) and one from the DENV-2 specific patient (Reference No. 11). One convalescent specimen (Reference No. 10) was DENV-2 specific by flaviMIA with ZIKV infection clearly demonstrated by both RNA detection and PRNT. Co-infection of ZIKV and DENV in this patient cannot be excluded, although DENV RNA was not detected.Past ZIKV infection was demonstrated in 14 patients by parallel testing demonstrating stable antibody levels between the samples. One of the 14 acute samples had cross-reactive IgM (Reference No. 90) while the remainder were ZIKV IgM specific. All convalescent sera were ZIKV-IgM specific and all were confirmed by PRNT.Patient Reference No. 17 had a single sample which was DENV-1 RNA positive, but the flaviMIA result was ZIKV IgM specific. The sample was also positive by ZIKV PRNT, suggesting previous infection or exposure to ZIKV. Balmaseda et al. reported DENV-1 to be the most common DENV to cross-react with ZIKV .Patient Reference No. 22 was found to have ZIKV-specific IgM and ZIKV neutralizing antibodies in the acute sample, while the flaviMIA result on the convalescent sample showed cross-reactive IgM between ZIKV and DENV. This result is suggestive of dengue infection secondary to an earlier infection or exposure to ZIKV.Huge numbers of suspected ZIKV infections were recorded across the Americas in 2015 and 2016 but most were not confirmed by laboratory methods. This and other regions have co-circulating viruses which can produce a clinically indistinguishable initial picture to that from ZIKV infection. Laboratory methods are the only way to ensure accurate diagnoses. With the discovery of congenital Zika virus syndrome, robust diagnosis to support patient prognosis has become more urgent however, definitive results require a complex and resource intensive process. Molecular methods used on serum samples rapidly, sensitively and accurately identify ZIKV RNA but suffer from the short window of detection using sera. Whole blood and urine may extend this window over testing of sera alone ,18.Other researchers have described new serological tests incorporating panels of flaviviruses but these usually include fewer virus targets than we have, are region-specific, or use recombinant rather than whole-virus antigens ,20,21,22Paired sera, collected two weeks apart, are essential for antibody-based identification of virus infection and serology is important to support the diagnosis of ZIKV infection. Despite only 60.6% of our sera being paired, the addition of a convalescent sample frequently permitted clarification of an initially unclear result as when IgM persisted from a past flavivirus infection or vaccination. This has been reported by others . Hence, In our study, a combination of RT-PCR and flavivirus serology identified and confirmed the presence of ZIKV infection in 99 suspected cases. By combining molecular and serological techniques, we could differentiate primary from secondary infections and recent from past infections in most cases as described by others . The difPatients originating from some areas of the world may have been exposed to multiple flaviviruses over a lifetime. Among primary ZIKV infections, we found antibody reactivity by flaviMIA was usually specific when follow-up samples were available and collected within a relevant time-frame from onset of disease. In the case of secondary infections however, results were often less clear, with the earliest detected antibody often directed towards a presumed earlier virus, or to prior YFV vaccination.The flaviMIA is a specialized technique requiring an experienced laboratory. The use of a multi-target antibody testing protocol such as this for screening samples from patients with suspected flavivirus infections benefits from including viral targets most appropriate to a patient’s exposure history .The results of our study suggest that all patients with suspected ZIKV infection should have a submitted specimen tested initially by RT-PCR see . Where ROur in-house flaviMIA protocol affords broad concurrent flavivirus serodiagnostic capacity. In our professional laboratory setting, it proved easy to use, was amenable to high throughput testing, had a short turnaround time and generated reproducible results. The flaviMIA was rapid in both analysis time and in interpretation of results. However, our results highlight the complexities of flavivirus diagnostic serology, which can be confounded by several factors including the presence of cross-reactive antibodies and high titers of specific antibody to antigenically different viruses that have resulted from recent or concurrent infection, vaccination or because of an anamnestic response triggered by a new ZIKV infection.


Betaflexiviridae and a novel virus belonging to the family Tymoviridae as well as two viroids. The number of identified viruses and viroids in each transcriptome ranged from one to six. We obtained 18 complete or nearly complete genomes for six viruses and two viroids using transcriptome data. Furthermore, we analyzed single nucleotide variations for individual viral genomes. In addition, we analyzed the amount of viral RNA and copy number for identified viruses and viroids. Some viruses or viroids were commonly present in different cultivars; however, the list of infected viruses and viroids in each cultivar was different. Taken together, our study reveals the viral population in a single peach tree and a comprehensive overview for the diversities of viral communities in different peach cultivars.Many recent studies have demonstrated that several known and unknown viruses infect many horticultural plants. However, the elucidation of a viral population and the understanding of the genetic complexity of viral genomes in a single plant are rarely reported. Here, we conducted metatranscriptome analyses using six different peach trees representing six individual peach cultivars. We identified six viruses including five viruses in the family  Prunus persica (L.) Batch] belongs the genus Prunus which includes almond, apricot, cherry, and plum1. In Korea, the peach is the third fruit tree, which is widely cultivated after apple and mandarin. Diverse peach cultivars with different harvest times are being cultivated due to their short storage times. Peach cultivars are clonally propagated to maintain fruit traits via grafting. Viruses and viroids infecting peach trees can be transmitted by grafting using virus-infected materials. To date, more than 20 different viruses infecting peach trees have been identified. Of known viruses infecting peach trees, Apple chlorotic leaf spot virus , Plum pox virus , Prunus necrotic ringspot virus , and Plum bark necrosis stem pitting-associated virus 5. Furthermore, the peach is susceptible to two different viroids including Hop stunt viroid  and Peach latent mosaic viroid 7. Although most peach trees infected by HSVd and PLMVd are asymptomatic, some PLMVd isolates cause severe disease symptoms such as albinism, leaf mosaics and blotches8.The peach is a kind of popular stone fruits in the world. Peach [10. However, such approaches have only led to the identification of known viruses and viroids, rather than novel viral pathogens. Several next-generation sequencing (NGS)-based approaches have been conducted to overcome traditional approaches, which has resulted in the identification of known and novel viruses13. For example, two recent studies using NGS identified novel viruses in nectarines that might be responsible for stem-pitting disease symptoms15. Moreover, a recent study using NGS reported a novel virus referred as Peach leaf pitting-associated virus (PLPaV) in the genus Fabavirus, which is responsible for a leaf symptom16. Furthermore, peach-assoicated luteovirus and Peach virus D (PeVD) are also newly identified using NGS18.Thus far, the identification of the viruses and viroids that infect plants relies on collecting samples displaying viral disease symptoms followed by direct detection using RT-PCR and ELISA19. In fact, many horticultural plants that are usually clonally propagated are reservoirs of a large variety of viruses and viroids. However, although studies associated with a single virus infection in a specific plant are numerous, viromes or viral populations in a particular plant have not been intensively examined. Thus far, only a limited number of studies have revealed viromes in specific plants such as grapevines22, sweet potato23, and pepper24. Furthermore, NGS-based approaches enable the assembly of viral genomes and reveal single nucleotide variations of present viruses26.The term “virome” is defined as the genomes of all the viruses inhabiting a specific organism or environmentAlthough peach is an important fruit tree; however, the studies associated with viruses and viroids infecting peach trees in Kore are limited. In this study, we conducted a peach RNA virome study in six different peach cultivars, which are commercially important in Korea. We identified both known viruses and viroids and a novel virus. In addition, the peach RNA virome reveals host specific viral communities in different peach cultivars, but unique viral communities of each peach plant associated with variety of factors.Apple chlorotic leaf spot virus  and two viroids (HSVd and PLMVd) in various peach trees by RT-PCR to study the peach virome in different peach cultivars. The RT-PCR results demonstrated that most peach trees were infected by at minimum either ACLSV or PLMVd. Most peach trees used in this study grew vigorously  to 146,623 (BC)  Table . The valApple green crinkle associated virus  were partial sequences for Asian prunus virus , which can be further divided into APV1 and APV2. Moreover, contigs associated with Maize rayado fino virus (MRFV) and Anagyris vein yellowing virus (AVYV), belonging to the family Tymoviridae were identified as partial sequences or nearly complete genome sequences for a novel virus.The assembled contigs from each library were blasted against a house-made viral genome database to determine virus-associated contigs. Ninety-two virus-associated contigs were identified after removing endogenous virus-associated sequences Table . Althougde novo assembled partial or nearly complete genomes for identified RNA viruses and viroids. We examined the portion of virus-associated contigs in individual cultivars  (two contigs) in MB cultivar. APV-associated contigs (16 contigs) derived from APV1 and APV2 were dominant in JH and similarly, 11 contigs derived from APV2 were enriched in BC. By combining all virus-contigs, APV (27 contigs), ACLSV (20 contigs), PLMVd (20 contigs), and HSVd (11 contigs) were highly present. As a result, we identified six viruses: APV1, APV2, ACLSV, APCLSV, CNRMV, and one novel virus, and two viroids: HSVd and PLMVd, from six peach transcriptomes.JT cultivar was only infected by PLMVd, while JH and BC cultivars were infected by four viruses and two viroids Fig. . The mosars Fig. . For exade novo transcriptome assembly using the Velvet program28. Using contigs obtained by the Trinity and Velvet programs, we could assemble the nearly complete genomes for several viruses and viroids  in NCBI, and the BLASTN results showed that a viral genome from a BC cultivar shared a 95% nucleotide identity with the recently identified PeVD18, while a viral genome from the JH cultivar shared a 88% nucleotide identity with the PeVD. Since the viral genome from BC shared a strong sequence similarity to the PeVD, we have named it “PeVD isolate BC.” However, the novel viral genome from the JH cultivar was different from PeVD. Thus, we have tentatively named it “Peach virus T (PcVT) isolate JH” , peptidase (Pep), helicase, RNA-dependent RNA polymerase (RdRp), and coat protein (CP), while ORF2 encodes movement protein (MP). We constructed two different phylogenetic trees using sequences from regions A and B to reveal the possible taxonomical classification of PeVD and PcVT , Grapevine asteroid mosaic associated virus (GAMaV), Nectarine virus M (NeVM), and Oat blue dwarf virus (OBDV) belonging to the genus Marafivirus in the family Tymoviridae. Furthermore, a phylogenetic tree using the polyprotein of ORF1 clearly showed that PcVT isolate JH is different from PeVD   Table . Therefos) Table . A phylots Table . Thus, wts Table . As expeMB Table  was clos32 were subjected to phylogenetic tree construction. The phylogenetic tree using 387 HSVd genomes displayed five HSVd isolates in this study were grouped with Group C containing HSVd variants from plum; however, the five HSVd isolates from peach were grouped in a different clade . In general, the HSVd genome sequences were highly conserved. The five HSVd genomes and 382 HSVd variants from a recent studyade Fig. .We amplified 40 PLMVd genomes from each cultivar by RT-PCR using two different primer-pairs to reveal genetic diversity of PLMVd in different peach cultivars. In total, 240 PLMVd genomes were subjected to phylogenetic tree construction. The phylogenetic tree identified at least ten different groups of PLMVd genomes Fig. . The numIn general, viruses and viroids with RNA genomes replicate with high mutation rates, which results in significant genetic diversity. In this study, we analyzed SNPs for the viruses and viroids in which we could assemble genomes from transcriptome data. We aligned raw sequence reads from each transcriptome on the each assembled viral genome and subjected to SNP identification. All detailed information associated with identified SNPs was described Table . In the 33. We subjected two transcriptomes, BC and JH, to FPKM analysis. Based on the number of the expected read count (expected count), PeVD (46%) was the dominant in BC  and CJ  transcriptomes contained relatively low numbers of virus-associated reads, whereas MB  and JH  possessed a high number of virus-associated reads Fig. . Next, w BC Fig.  while AP BC Fig. . In JH,  BC Fig. . HoweverWe conducted RT-PCR using each virus- and viroid-specific primer to confirm the presence of the viruses and viroids identified by NGS Table . In factBetaflexiviridae, which includes two genera, Foveavirus (APV1 and APV2) and Trichovirus (ACLSV and APCLSV)  Fig. . MoreovePeach trees are usually propagated by grafting. Therefore, many viruses and viroids may be transmitted through scions and rootstocks. In Korea, peach seedlings are normally used for the rootstocks of peach trees. In this study, all rootstocks were peach seedlings derived from a single wild peach tree grown on a mountain without human contact. Furthermore, we have confirmed that this wild peach tree had not been infected by any viruses and viroids by using NGS and RT-PCR. Therefore, we could be certain that viruses and viroids were not transmitted from rootstocks in our study.35. In general, the first step of virus identification is to observe the disease symptoms caused by viruses12. However, viral infection in plants does not always cause disease symptoms. The disease symptoms might depend on the virus, host, and environmental conditions. The peach trees used in this study did not always show virus disease symptoms, which indicates the importance of environmental conditions such as cultivation method, climate, pesticides, and fertilizers. In addition, disease symptoms were highly dependent on the host. For example, only BC and JH out of the six cultivars displayed disease symptoms.The emergence of several NGS systems is an innovation in many research areas including virology36. However, the complex of the virome in a single tree made it difficult to determine the major virus or viroid that might be associated with disease symptoms. Several studies have shown that the coinfection of several viruses could create competition of mutually beneficial cooperation38. Furthermore, the coinfection of PNRSV and PLMVd resulted in synergistic effects at the transcriptomic level of peach fruits39. We supposed that at least closely related viruses can be beneficial for replication, movement, and pathogenesis due to their common characteristics. However, the impact of coinfection among entirely different viruses and viroids in the viral lifecycle and the host should be studied. Moreover, we found that the coinfection of different viruses in the host did not always correlate with disease symptoms, as shown in the high quality fruits obtained from the six cultivars.A recent study has suggested possible effects of the virome with regard to host health and disease13, small RNAs40, and extracted double-stranded (ds) RNAs34. Theoretically, NGS using a ribosome-deleted library possibly identifies both poly-A tailed viruses and dsRNA without poly-A tail and viroids with circular RNA genomes. However, our recent studies have demonstrated that the mRNA library prepared by oligo dT is also adaptable enough to identify different types of viral genomes such as DNA viruses, double stranded RNA viruses, and viroids regardless of the presence of poly-A in the genome26. A recent study found that a wide range of RNA viruses contain nontemplated oligo(U) or (U)-rich tails, suggesting widespread 3′ uridylation in eukaryotic viruses41. Thus, we carefully supposed that some (U)-rich regions in viruses without poly-A tail can be amplified by oligo dT, which is used for mRNA preparation. Of course, the mRNA based library might be not useful to amplify viruses without poly-A tail; however, the mRNA libraries in this study were successfully applied in de novo assembly of 12 virus genomes with poly-A tail. In addition, we confirmed the absence of Little cherry virus 1 and Peach mosaic virus in the family Closteroviridae which do not possess poly-A tail in the six peach cultivars by RT-PCR. Therefore, we carefully suggested that the library type should be dependent on the purpose of the study.The preparation of a library for NGS is a very important factor for virome study. Many recent studies have preferentially prepared libraries from ribosome-deletedThe pooling of samples might increase the number of identified viruses, but this would make obtaining the detailed information of the virome in a single plant impossible. Prior to conducting RNA-Seq, we checked the infection of known viruses and viroids by RT-PCR. This approach enabled us to compare different viromes from individual single peach cultivars. Sometimes, NGS did not detect viruses or viroids in low titer, even though RT-PCR could detect them. In addition, in the preparation of the six libraries, we added barcode sequences to each library and the pooled libraries were sequenced using a HiSeq. 2000 system. Technically, the individual barcoded sequences should be sorted during data analysis, but we found that some identified viruses with small number of reads should be carefully analyzed. Sometimes, they might contaminate sequences derived from other libraries. Thus, a combination of at least two different approaches was necessary for virus identification and diagnosis.42, for determining closely related genomes. The assembled contigs associated with identified viruses in our study were sufficient to cover most regions of the target virus. In addition, the information of available reference genome sequences was beneficial for virus genome assembly.A complex mixture of viruses in a single plant was an obstacle to distinguishing closely related viruses. For example, NGS results showed the coinfection of APV1 and APV2 in JH, and the coinfection of ACLSV and APCLSV in BC. Complete or nearly complete genome sequences are very useful, as described previouslyCompared to previous studies, our study precisely revealed the virome in a single tree and elucidated the abundance of individual viruses or viroids in a single tree by viral reads, copy number, and FPKM values. In fact, it was difficult to determine the abundance of viral RNAs due to the natural mutation of RNA viruses and viroids. We used assembled viral genome sequences generated from the corresponding transcriptome for the calculation of viral abundance and SNPs to minimize possible mismatches caused by mutations. Furthermore, we carefully suggested that FPKM values might be a good tool for the calculation of viral abundance using transcriptome data.43. In particular, the advance of NGS techniques facilitates the identification of virus SNPs using sequenced data. The analyses of SNPs revealed the mutation frequency for each identified virus or viroid. As previously reported44, the 240 PLMVd genomes in this study showed strong genetic variations, whereas the HSVd genomes were relatively conserved. Out of the examined RNA viruses, the sequences of five ACLSV isolates showed a low rate of SNP, whereas APV1, APV2, CNRMV, and PeVD showed relatively higher rates of SNPs. Two closely related PeVD and PcVT showed that the SNP frequency of PeVD was very high in BC as compared to that of PcVT with low SNP frequency. This result might indicate a possible host-specific mutation of viral RNA genome.Viral quasispecies, which are a collection of diverse variants, contribute to the characteristics of the viral populationTaken together, we provide a comprehensive overview of the peach RNA virome in a single tree and different viral communities in different cultivars. We successfully applied intensive bioinformatics analyses in genome assembly, SNPs, abundance of viral RNAs, and phylogenetic analyses. We suppose that several unknown factors including environment and genetic background contributed to the formation of unique features for individual peach viral communities in different cultivars. The elucidation of the possible impacts of individual peach viral communities associated with plant health might be very challenging and complicated due to the complexity of viral communities.The peach cultivars used in this study were grown in an orchard in Hoengseong, Korea. In total, we used six different peach cultivars, which are widely grown in Korea: Jangtaek (JT), Mibaek (MB), Cheongjung (CJ), Cheonghong (CH), Janghowon (JH), and Baekcheon (BC) for the peach virome study. We used leaf samples from a single plant for individual peach cultivar. In general, the peach cultivars did not show any visible viral disease symptoms. We examined viral disease symptoms in the selected peach plants for the years 2013–2016. JH and BC cultivars showed visible viral disease symptoms in leaves in years 2013 and 2014; however, we could not observe any visible disease symptoms in 2015 or 2016.Five leaf samples from a single plant were rapidly frozen in the presence of liquid nitrogen and kept at −70 °C for further experiments. Frozen leaf tissues were ground in liquid nitrogen with a mortar and pestle. We extracted total RNA using two kits: Fruit-mate for RNA Purification  and the RNeasy Plant Mini Kit  according to manufacturer’s instructions. The quality and quantity of extracted total RNA were checked. Only high quality total RNA was used for the library preparation for RNA-Seq.Extracted total RNAs were used for the library construction using the TruSeq RNA Library Preparation Kit v2  according to the manufacturer’s instructions. In this study, we generated six mRNA libraries derived from six different plants. In brief, mRNAs with poly-A tail were extracted using poly-T oligo-attached magnetic beads. The purified mRNAs were subjected to the first strand of cDNA followed by a second strand of cDNA, and the adenylation of 3′ ends was carried out. Adapters were ligated, and PCR amplification was performed to selectively enrich DNA fragments with adapters and amplify the amount of DNA in the library, respectively. We conducted quality control for the generated libraries using a 2100 Bioanalyzer . The libraries were paired-end sequenced by Macrogen Co.  using the HiSeq 2000 platform.de novo transcriptome assembly. We subjected two paired-end sequenced FASTQ files for each individual library to de novo transcriptome assembly using the Trinity program  with default parameters27. We used a workstation with two six-core CPUs and 256 GB RAM operated using the Ubuntu 12.04.5 LTS operation system for most bioinformatics analyses including de novo transcriptome assembly and BLAST search. In addition, we used the Velvet/Oases assembler (version 0.2.08)28 for the de novo transcriptome assembly to complement results from the Trinity program.We used the raw data obtained from six different libraries for 45 with a cut-off E-value of 1e−5 to identify viruses and viroids in assembled peach transcriptomes. We combined sequences for plant viruses and viroids from the complete reference sequences of viruses and viroids (http://www.ncbi.nlm.nih.gov/genome/viruses/) and descriptions of plant viruses (http://www.dpvweb.net/) to establish a specific database for plant viruses and viroids46. We blasted the assembled contigs from each peach transcriptome against a database composed of plant viruses and viroids. The identified contigs associated with viruses and viroids were extracted and used for a tBLASTx search against a NCBI NR (non-redundant proteins) database. After filtering endogenous virus-like sequences, only clean contigs associated with viruses and viroids were obtained.We used the MEGABLAST algorithmde novo transcriptome assembly for individual peach cultivar by two different programs to assemble complete or nearly complete viral genome for the identified viruses and viroids: Trinity and Velvet. Based on our previous study26, both programs have advantages and disadvantages for the transcriptome assembly. The best way is to combine the contigs associated with viruses and viroids for viral genome assembly. Viral contigs were aligned on the corresponding virus genome using ClustalW program implemented in the MEGA7 program for de novo assembly followed by BLAST search47. In many cases, we could fill gaps in a viral genome with contigs obtained from Velvet. Moreover, we again mapped raw sequence reads on the assembled viral genome to confirm consensus sequences using a Burrows-Wheeler Aligner (BWA) program with default parameters48. The open reading frames (ORFs) for known viruses were analyzed based on the respective reference viral genome. In the case of PeVD and PcVT, ORFs were predicted by ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) and conserved domains were analyzed by SMART program (http://smart.embl-heidelberg.de/)49.We conducted http://www.ncbi.nlm.nih.gov/genbank/) to reveal phylogenetic relationships with other known viruses. After filtering partial sequences, we only retrieved complete genome sequences homologous to each virus from the nucleotide database. We aligned the genome sequences for each virus using the ClustalW program with default parameters. After sequence alignment, unnecessary sequences and poly-A tails at the 5′ and 3′ regions, respectively, were removed. Aligned sequences were manually edited and used for the construction of a phylogenetic tree using the MEGA7 program47. The phylogenetic tree was constructed via the neighbor-joining method with 1,000 bootstrap replicates and Kimura 2-parameter distance.We generated phylogenetic trees for viruses in which the genome sequences were complete or nearly complete in this study. Sequence similarity between ACLSV and APCLSV genomes meant that we could use five ACLSV genomes  and an APCLSV isolate BC for the construction of phylogenetic tree. In a similar manner, a genome for APV1 isolate JH and two genomes for APV2 isolates BC and JH were used for the construction of a phylogenetic tree. In total, 18 ACLSV genomes, two APCLSV genomes, five APV1 genomes, six APV2 genomes, three APV3 genomes, and six CNRMV genomes were used for phylogenetic analyses. Three CTLV genomes were used as outgroup in the CNRMV phylogenetic tree. The accession number for each viral genome was indicated in the phylogenetic tree. We conducted BLAST using the identified virus as a query against GenBank  and sequenced by the Sanger sequencing method. The accession numbers for HSVd genomes were KX371905–KX371909. We combined five HSVd genomes in this study with 382 HSVd variants from a recent study to construct a phylogenetic tree. In total, we aligned 387 HSVd genome sequences, and converted the aligned sequences file into a nexus file. The high degree of sequence similarity of HSVd genomes meant that we used the Splitstree programIn the case of PLMVd, we conducted RT-PCR using two different primer-pairs. The amplified PCR products were cloned and sequenced as described for HSVd. Each primer-pair amplified 20 clones. Thus, we sequenced 40 clones for each cultivar. We used 240 PLMVd derived from six different cultivars for PLMVd phylogenetic analyses; again, we used the Splitstree program. We identified ten different groups, and calculated the number of PLMVd isolates in each group.25. In brief, we mapped the raw sequence reads from each transcriptome on the identified individual viral genome using the BWA program with default parameters. We used the assembled viral genome from each transcriptome for reference to increase SNP specificity. In general, the application of known viral reference genomes for the analysis of SNPs led to the identification of unexpected SNPs. We converted the SAM files generated by the BWA program into BAM files via SAMtools51. The sorted BAM files were used to generate the VCF file format using the mpileup function of SAMtools for SNP calling. Finally, we used BCFtools implemented in SAMtools to call SNPs. The positions of identified SNPs on each viral genome were visualized by the Tablet program52.We analyzed SNPs for only viruses and viroids, in which complete or nearly complete genomes were obtained in this study. We used the same method to analyze SNPs for each viral genome as described previouslyhttp://hannonlab.cshl.edu/fastx_toolkit/) for this, and we subjected the converted FASTA files to MEGABLAST. The second was the estimation of transcript abundance based on alignment-based abundance estimation methods used for RNA-Seq33. We aligned the raw sequence reads on the identified viral contigs using the Bowtie 2 program53 and subjected this to the FPKM method to calculate the normalized expression value “fragments per kilobase transcript length per million fragments mapped” (FPKM). In the case of BC and JH cultivars, we combined the FPKM values for each identified virus or viroid to estimate the copy number.We used two different approaches to quantify viral RNAs associated with identified viruses and viroids. The first was to calculate the sequenced reads associated with respective viruses or viroids via MEGABLAST search. We converted the FASTQ files into FASTA files using the FASTX-Toolkit  repository with the following accession numbers SRR2290949, SRR2290951, SRR5074727, SRR5074715, SRR5074728, and SRR5074714. The viral genome sequences obtained from this study were also deposited in GenBank, NCBI with respective accession numbers.Supporting informationSupplementary File S1-11Supplementary File S10


Gracula religiosa), demonstrating close homology with Usutu viruses circulating in Europe.The complete and annotated coding sequence and partial noncoding sequence of an Usutu virus genome were sequenced from RNA extracted from a clinical brain tissue sample obtained from a common hill myna ( Turdus merula) and great grey owls (Strix nebulosa) in Austria  that died 24 h after the first demonstration of clinical symptoms tested positive using an in-house Usutu virus real-time reverse transcription-PCR (RT-PCR) (data not shown). During autopsy, a swollen liver and spleen were detected as sole macroscopic lesions. The brain tissue was homogenized in phosphate-buffered saline (10% [wt/vol]), pretreated by 0.45-µM-pore-size selective filtration and nuclease, and RNA was extracted as previously described . Stretches containing unidentified nucleotides (N) were trimmed using Cutadapt version 1.3    . De novoge.net/) . The proge.net/) .The nearly complete genome of Usutu virus strain Gracula religiosa/U1609393/Belgium/2016 was obtained with an average coverage of 493× and contains a single 10,305-nucleotide (nt) open reading frame (ORF) encoding a polypeptide precursor protein, sharing a high nucleotide homology with Usutu viruses circulating in wild birds and mosquitoes in Germany in 2011 to 2013 and Usutu viruses circulating in 2011 to 2015 in central and western Europe in general.KY315178.The complete coding sequence of Usutu virus strain Gracula religiosa/U1609393/Belgium/2016 was assigned DDBJ/EMBL/GenBank accession no.


In the past two decades, methods have been developed to measure the mechanical properties of single biomolecules. One of these methods, Magnetic tweezers, is amenable to aquisition of data on many single molecules simultaneously, but to take full advantage of this "multiplexing" ability, it is necessary to simultaneously incorprorate many capabilities that ahve been only demonstrated separately.Our custom built magnetic tweezer combines high multiplexing, precision bead tracking, and bi-directional force control into a flexible and stable platform for examining single molecule behavior. This was accomplished using electromagnets, which provide high temporal control of force while achieving force levels similar to permanent magnets via large paramagnetic beads.Eschericia coli (E. coli) bacteria, and see similar results to previous studies.Here we describe the instrument and its ability to apply 2–260 pN of force on up to 120 beads simultaneously, with a maximum spatial precision of 12 nm using a variety of bead sizes and experimental techniques. We also demonstrate a novel method for increasing the precision of force estimations on heterogeneous paramagnetic beads using a combination of density separation and bi-directional force correlation which reduces the coefficient of variation of force from 27% to 6%. We then use the instrument to examine the force dependence of uncoiling and recoiling velocity of type 1 fimbriae from This platform provides a simple, effective, and flexible method for efficiently gathering single molecule force spectroscopy measurements. Single molecule force spectroscopy (SMFS) has become a powerful tool for investigating the force dependence of biological phenomenon including, but not limited to, biological bonds –3,viscoeThe magnetic tweezer (MT) is a relatively recently developed instrument that allows the examination of hundreds of single molecule measurements simultaneously –11. ThisThe smallest detectable change in the height of a bead is referred to as the spatial resolution and determines the lower limit of biological length changes that can be detected. The spatial resolution in basic MT’s is estimated using the depth of field and is on the order of several microns . HoweverE. coli fimbriae under a wide range of forces. Finally, we discuss the tradeoffs when optimizing a MT for multiplexing versus spatial precision.Here we describe a multiplexed MT with precision bead tracking and bi-directional force control (MMTB) and characterize its capabilities. Because high forces of up to 100 pN are desired for many studies, and pose unique challenges for multiplexing with electromagnets, we describe use of the MMTB to obtain this force range. To achieve these high forces, large paramagnetic beads with high magnetic content were used. These beads showed significant bead to bead force variation, which can prohibit the accurate estimation of force dependent properties. To address this challenge, we developed a novel technique for precisely determining forces on beads with heterogeneous magnetic content, which reduced the coefficient of variation of force from 27% to 6%. We then used the MMTB to examine the uncoiling and recoiling velocities of Information about the beads used in the experiments in this publication are shown in Table Chamber slides  were placed in Acetone for 3 min before being rinsed with ethanol and water. A 100 μl droplet of fimbria in 0.02% bicarbonate buffer (1.5 μg/ml) was added to center of the bottom slides and incubated for 2 h at room temperature. A 100 μl droplet of 0.2% PBS-BSA was added to the center of the top slides and incubated at room temperature for 1 h. Bottom slides were rinsed with 0.2% PBS-BSA three times. Chambers were then assembled with double sided tape and 40 μl of 0.2% PBS-BSA was injected into the chamber via a 100 μl pipet. Chambers were covered and stored overnight at 4 °C.To separate beads by density, differential centrifugation was used similarly to a method used to isolate cellular organelles of different density. Briefly, 5 ml of 1.5 M NaCl  was added to a Nalgene 50 ml polypropylene centrifugation tube . 45 ml of a mixture of Percoll  and water was added to the tube to obtain a density of 1.09 g/ml. Paramagnetic beads were washed 3 times in PBS, and added to the Percoll solution. This solution was then spun at 30,000 x g for 30 min. A thumbtack was used to create a hole in the bottom of the centrifugation tube, and the solution was drained into 1 ml aliquots. Because Percoll forms a gradient during spinning, both the percent Percoll and the spinning conditions can be adjusted to spread magnetic beads as broadly as possible for maximum separation.In order to bind mannose-BSA to beads, 0.1 mL of 7.8 μm paramagnetic beads were washed two times in 1 mL of MES hydrate  with a pH of 4.5–7.5. After the second wash, the pellet was resuspended in 1 mL of MES hydrate and vortexed for 10 s. 10 mg of N-Cyclohexyl-N′-(2-morpholinoethyl) carbodiimide methyl-p-toluenesulfonate  was added to the beads. The beads were then vortexed for 10 s, and rotated at room temperature for 15 min. Beads were then washed two times in PBS, and resuspended in 1 mL of the same. The PBS was replaced with 1 mL of 100 mg/ml mannose-BSA  in PBS and the solution was rotated at room temperature for 2 h. Beads were then washed in 1 mL of 35 mM glycine  in water with 0.2% BSA  and rotated at room temperature for 30 min. Beads were then washed and resuspended in 0.2% PBS-BSA and stored at 4 °C.E. coli using magnesium precipitation as described previously [Fimbria were purified from eviously .V(f) is the uncoiling velocity as a function of force f, and using ∆L = 5.0 nm, and kbT= 4.114 pN-nm. The estimates of kbal, fbal, and ∆xu are shown in Table The models in Fig. The MMTB has four electromagnetic poles: two above and two below a chamber containing paramagnetic beads suspended in buffer Fig. . The desBy applying a voltage potential across the coils using two identical power supplies , a magnetic field gradient acts to pull the beads in the upward (upper magnet) or downward (lower magnet) direction. In practice, the lower magnet is first used to pull the beads to the bottom, functionalized surface. The current to the lower magnet is then turned off and the current to the upper magnet turned on, and the beads are pulled away from the bottom surface at a force controlled by the magnetic field. This cycle of pulling the beads toward and away from the bottom surface is known as a “pull”. A high speed camera  is mounted to an inverted microscope  and used to acquire images of the beads at rates of up to 100 Hz, using either a 0.45 NA 20× or 0.55 NA 40× objective. The use of objectives with longer working distances and low NA’s is due to spatial constraints below the chamber caused by the lower magnet. With the 20× objective, 40 7.8 μm diameter beads or 120 2.8 μm diameter beads can fit in the field of view (528 × 297 μm), with enough space between beads to allow bead tracking i.e. diffraction patterns do not overlap (see Bead Tracking). Multiplexing can be further increased using non-random tether techniques [A custom Labview program  is used to provide synchronous control of the microscope, power supplies, and camera. The Labview program allows the camera and power supply settings to be adjusted in real time, or a time-based script can be used to ensure repeatability between pulls. This flexibility is beneficial when measuring bond lifetimes: one may use a high frame rate to examine the rupture of short-lived bonds, and then switch to a lower frame rate to examine the rupture of long-lived bonds, thus avoiding collecting superfluous amounts of data. Using the Labview program, we found that it takes ~40 ms to switch from one current level to another (data not shown), similar to the findings by Snook and Guilford .Experimental images are analyzed using a custom Matlab script  that implements a variation of previously published tracking algorithms , 14. In To estimate the tracking accuracy of the MMTB over long distances, we tracked several beads under force as the beads traversed the axial distance of the chamber(~78 μm) under 0.6 amps of current. We then compared this to the chamber height, as measured by focusing on beads that were stuck to the top and bottom of the chamber and noting the microscope objective position. The difference in objective position was then multiplied by 1.33 to account for the refractive index mismatch. The tracked bead displacement was within a few percent of the chamber height with an error of 2.9 ± 0.5% (SEM) for 7.8 μm beads. This demonstrates that our system is capable of accurately tracking beads over long distances, an important ability when estimating the force on beads .To test the spatial resolution, we non-specifically bound beads to the surface, and determined their z-positions over time under no force (0 amps of current). The resolution was computed as the standard deviation of the bead displacement. We determined this resolution at 40× magnification under various conditions for 2.8 μm diameter beads Fig. . ExaminiTo examine the effect of objective properties and bead size on spatial resolution we determined the spatial resolution for beads of different sizes using a 0.45 NA 20× and a 0.55 NA 40× objective while using a single reference bead Fig. . At 20×,F is the drag force on the bead, μ is the dynamic viscosity of the fluid, r is the radius of the bead, v is the velocity of the bead, and λ is a correction factor due to the chamber surface effects [r > 15), or one surface is much closer (b/(b + c) < 0.2), the effect of the distal surface is insignificant [The force on the beads was estimated by determining the z-position of the beads as a function of time and then applying a modified version of Stokes’ law, effects  that depnificant , so lambnificant :3\documeλ was estimated using the tables and figures created by Ganatos, Pfeffer, and Weinbaum [λ in the middle third of the chamber ranged from 1.04 for 2.8 μm beads to 1.35 for 11 μm beads. It should be noted that taller chambers would minimize the need to compensate for surface effects when calibrating force even with larger beads.Considering that our average chamber height was approximately 78 μm, this estimation of lambda was used for estimating forces on 2.8 μm and 5.3 μm beads, where the position of the bead was assumed to be in the middle of the chamber (b = 39 μm). Because bead velocities were typically measured in the middle third of the chamber, we estimate that this approximation yielded an error in force of less than 5%. For the larger 7.8 μm and 11 μm beads, b/r was less than <15, and therefore the second surface had an effect on the bead velocity. In this case, Weinbaum . EstimatTo assess the magnetic field gradient variation in the chamber along the z-dimension, we examined the velocity of beads at two different heights of the chamber. Because the velocity of beads near the chamber walls are highly nonlinear due to the effect of the chamber walls, we examined the change in bead velocities at positions in the chamber where the velocities (and thus forces) were more stable: when the beads were at z-positions 25% and 75% the height of the chamber. Examining the velocity of fifteen 7.8 um beads measured on two separate days, we found that the velocity of the beads at a z-position of 75% of the chamber height is 1.11 ± .02 (SEM) times that of the beads at 25% of the chamber height. This increase is unsurprising as the beads are slightly closer to the magnets at the 75% height. Since this change in height is the same as when beads are on the surface compared to when they are halfway across the chamber , we expect the forces measured at the halfway point to be within 11% of the forces at the surface. This potential 11% force error is very similar to the force accuracy of other SMFS instruments .We determined the force on 7.8 μm beads at different currents, for both the upper and lower magnet Fig. . For theC95 represents the difference in contact time of 95% of the bead population, Fdown is the force applied with the lower magnet, COV is the coefficient of variation in force for the beads, and h is the height of the chamber. Using our maximum downward force of 3.5 pN and COV of 0.14 (see Force Variation), we estimate that 95% of beads will contact the surface within 1 s of each other. Eq. h or COV, or by increasing Fdown.The maximum downward force of 3.5 pN at 1 amps for the 7.8 μm beads allows an estimate of the contact time resolution of the beads with the bottom surface using Eq. βB is the bond temporal resolution, and η is the spatial resolution. For the 7.8 μm beads, the spatial resolution without reference beads or time averaging was about 75 nm. At the lowest force of 10 pN, the bond temporal resolution is estimated to be 0.5 ms using Eq. Similarly, the combination of the upward force and spatial resolution controls the bond temporal resolution, or the shortest bond lifetime that can be estimated.Expanding on this force data, the smallest upward force that can be applied using the MMTB is about 2 pN with 2.8 μm beads (data not shown), and is limited by the lowest applicable current of our power supply of 0.02 Amps. This force is about five times smaller than the smallest applicable force using an atomic force microscope , and coup-value of .00625, in accordance with Dunn [To assess the variation in force across the field of view, we parsed the field of view into nine sections of 176 × 99 μm per section Fig. , inset, ith Dunn . This raTo test the bead to bead force variation, we determined the coefficient of variation (COV) in force of 7.8 μm beads at 0.1 amps. This resulted in a COV of 27% for “Stock” beads: beads directly from the manufacturer’s container Fig. , Stock. m and y in Eq. Fupper, α is the estimated force on the bead using the upper magnet at current α, and Flower, α is the measured force on the bead using the lower magnet at current α. Proceeding pulls, where the beads were initially pulled down to the bottom surface, were used to estimate the upward force on beads that had adhered to the bottom surface using the initial downward force, and parameters m and b in Eq. To further increase the precision of the bead force estimations, we developed a method that correlated the downward force (toward the bottom surface with the lower magnet) and the upward force (away from the bottom surface with the upper magnet). Due to the difference in the amount of magnetic material across the bead population, there was a correlation between the downward force and upward force of individual beads, which could be used to increase the precision of the upward force estimations of adhered beads. This process began with a calibration step, where the downward and upward force on tens of beads was acquired. This data was used to fit parameters E. coli  adhesion on average, while the average for beads with Mannose-BSA had 23% (SEM = 5%) of beads adhered to the surface. This demonstrates that the vast majority of adhesive events were bound specifically to the fimbrial tips. Based on the net 22% adhesion rate, a Poisson distribution predicts that 12% of attached beads formed multiple attachments .N = 39 samples). We then disregarded any measurements that had bead displacements of less than 1.5 μm during the 5 s uncoiling pulls, or during the 2 s 100 pN force for the recoiling pulls. This eliminated any measurements in which fimbriae changed orientation to an upright position without uncoiling, since it is very unlikely that any single fimbria had a length greater than 1.5 um in the native state. Because uncoiling typically extends fimbria to ten times their native length [To ensure that measured velocities were due to fimbrial uncoiling and not reorientation of long fimbriae, we used atomic force microscope imaging to measure the lengths of the fimbriae, which were found to have an average native length of 0.43 ± 0.15 μm with no fimbriae longer than 0.8 μm  is about 10 pN, or <10% of the force applied. This discrepancy can thus be explained by the 10% error in force accuracy commonly seen using SMFS instruments [Figure truments . More deOur MMTB with its unique combination of multiplexing, precision bead tracking, and bi-directional force control was able to efficiently gather single-molecule uncoiling and recoiling velocities over a wide range of forces. Without any of the three aforementioned capabilities, this study would have been significantly more difficult.The multiplexing ability of the MMTB decreased the experimental time needed to acquire the data. In this case, the decrease was not large over traditional methods. Most of our pulls lasted only 6 s for the actual pull plus 60 s to replace the beads in the chamber, and provided an average of more than 30 measurements per pull, or 0.45 measurements per second. To run a similar experiment with traditional SMFS instruments, there would only be 1 measurement for every 6 s pull, or 0.17 measurements per second. We therefore estimate the MMTB acquired data ~2.7 times faster than traditional SMFS instruments for this experiment. The major limitation on efficiency in this experimental was that detached beads aggregate under the influence of the magnetic field, so beads cannot be reused for multiple pulls, requiring the minute-long bead replacement after each pull cycle. For long pulls where efficiency is most critical, this additional time is well worth it to provide multiplexing. However, for short pulls of less than 1 s, high efficiency would require improvements such as creation of regular arrays with microcontact printing , a microMultiplexed SMFS systems have been demonstrated with other MT’s , 12, andFor MT’s without precision bead tracking, the spatial resolution is estimated as the depth of field and is on the order of several microns . Since tThe ability to quickly change the bead force was imperative in estimating recoiling velocities. Because the recoiling velocities were quite rapid, there was a limited time at which the force-dependent velocity could be measured. Since we wanted to measure the dependence of this recoiling velocity on force, the force on the beads had to be set quite quickly, otherwise the fimbria would have completely recoiled before the new force level was attained. This was achievable with electromagnets where a new steady-state force was reached in ~40 ms, but would be more difficult with permanent magnets because a precise and fast shift in position would be required.Finally, our novel force correlation method, which required the use of the lower magnet, allowed force estimations of tethered beads with greatly improved precision. An alternative method for estimating bead forces uses the Brownian motion of the tethered bead and requires a known tether length and a significant amount of bead position data . This meHere we specifically designed and optimized the MMTB for efficiently gathering mechanical measurements of single molecules or molecular complexes. This necessitated both multiplexing and precision bead tracking, but prevents either of these capabilities from being maximized. However, others in the field may have need of instruments that emphasize other applications of SMFS, and we offer the following guidance for those considering building their own MT.To maximize the multiplexing ability of the MT, as many beads as possible should be fit into the field of view. This can be achieved by using a camera with a large field of view in combination with a low magnification objective and small beads. Such a setup does come with drawbacks. First, cameras with large field of views often have low frame rates. This frame rate reduction combined with the low magnification objective decreases the maximum spatial precision . Also, oConversely, maximum spatial precision requires a high magnification objective and a camera with a high frame rate and small pixel area. Such cameras tend to have smaller fields of view, which together with the higher magnification reduces the multiplexing factor. For very high speed cameras, a stronger light source may be required for proper illumination, of which superluminescent diodes are an option . Due to The preceding paragraphs have shown the spectrum of capabilities of the MT: maximizing multiplexing and throughput on one end, and maximizing spatial precision and bead force on the other. Our MMTB is a hybrid of these two extremes: capable of enough spatial precision to detect many biological phenomena , 47, 48,


Severe fever with thrombocytopenia syndrome (SFTS) caused by SFTS virus (SFTSV) usually have a high fatality. At present no effective therapy or vaccine are available, so early diagnosis of SFTS is crucial to prevent and control SFTSV infection. This study aimed to establish a national reference for these diagnostic kits of SFTSV genome and make the diagnosis of the disease effective.Six SFTSV strains isolated from different regions, and five relative viruses with similar clinical manifestations were selected as positive and negative references and assessed using real time quantitative PCR (q-PCR) using specific primers and probe and two commercial kits. The stability of the references was also assessed at 37 °C, room temperature or -70 °C for 8 days, 14 days or 8 months respectively, or following several cycles of freezing-thawing. Collaborative calibration of the references was performed by three labs.2 U/mL. The accuracy was coefficient of variation less than 5%. The references were highly stable at high temperatures and after long storing and freezing-thawing treatment.The references indicated good accuracy and specificity. The lowest detection limit was 10We successfully established a national reference with good accuracy, high specificity, sensitivity and stability, which can be applied for quality control of commercial SFTSV diagnostic kits, thus preventing and controlling SFTS.The references have been finished and it was retrospectively registered in the following article.The online version of this article (doi:10.1186/s12985-017-0682-z) contains supplementary material, which is available to authorized users. Phlebovirus (family Bunyaviridae) named Sever Fever Thrombocytopenia Syndrome virus (SFTSV), was isolated from a sample collected from a patient in Henan Province in China at 2009 [In 2007, some patients with severe fever, gastrointestinal bleeding and thrombocytopenia syndrome were first discovered in central and eastern China , 2, but  at 2009 . Then th at 2009 –6.Phlebovirus, is an enveloped, segmented minus-strand RNA virus. The genome consists of three segments: L (large), M (middle) and S , which respectively encode the RNA-dependent RNA polymerase, Gn-Gc envelope glycoproteins, nucleocapsid and non-structural proteins [SFTSV, like other viruses in genus proteins .SFTSV infection is clinically characterized by fever, thrombocytopenia and leukopenia, gastrointestinal symptoms, and multiorgan dysfunction. The initial case fatality of SFTSV infection was 30%  in ChinaEarly clinical diagnosis of SFTSV infection is crucial for controlling the spread of SFTS. Generally, infection is confirmed via detection of viral genome or virus-specific antibodies in the patient’s blood or serum . SeveralSix SFTSV strains  were provided by the National Vaccine & Serum Institute . Japanese encephalitis virus (JEV) SA14-14-2, dengue virus (DENV) Ban18 and yellow fever virus (YFV) 17D were provided by Arbovirus Vaccine Group, National Institutes for Food and Drug Control (NIFDC). Inactivated hemorrhagic fever with renal syndrome virus (HFRSV) and tick-borne encephalitis virus (TBEV) Senzhang strain was provided by Changchun Institute of Biological Products Co., Ltd.Nucleic acid detection kits for SFTSV (PCR-fluorescence probe techniques) were obtained from SinoMD Gene  and DaAn Gene . An RNA extraction QIAamp Viral RNA Mini Kit (cat 52904) was obtained from Qiagen . Reverse transcriptase was purchased from Promega , and La Taq was purchased from Takara .Four kinds of references  were investigated.References should be broad-spectrum and representative. So the phylogenetic tree of SFTSV, based on the S segment, was first draw using MEGA 6.0 Fig. . To reprFive arthropod-borne or blood-borne viruses , which cause similar clinical manifestations to SFTSV infection, including fever, headache, disturbance of consciousness, convulsions, bleeding and kidney damage, meningeal irritation and paralysis, were selected as negative references to evaluate the kit specify .The earliest isolated and extensively investigated SFTSV strain HB29 was used as references for sensitivity and accuracy.Six SFTSV strains AH12, HN1, JS3, LN3, SD4, and HB29 were amplified. Virus titers were determined before inactivation using the cytopathogenic effect assay (CPE). JEV A14-14-2 strain, DENV Ban18 strain and YFV 17D strain was amplified in BHK 21 cells, and the titers were determined using a CPE method. HFRSV L99 strain and TBEV Senzhang strain were provided by Changchun Institute of Biological Products. All viruses were inactivated using formaldehyde (1:1000) at 37 °C for 24 h and stored in −80 °C for use.The S gene represents a conserved region of the SFTSV genome . The who2O 37 μL) using the following procedures: denaturation at 94 °C for 2 min; 30 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and finally extension at 72 °C for 5 min. The PCR products were confirmed by sequencing .Virus RNA genome was extracted using QIAamp Viral RNA Mini Kit and then reverse transcribed into cDNA. The cDNA was used as template for PCR in a total volume of 50 μL  from SinoMD Gene, and DaAn Gene. The reaction system for specific primers and probes were: 0.1 μL LA Taq, 2.5 μL reaction buffer, 2 μL dNTP, 5 μL template, 2 μL primes, 1 μL probe, and 12.4 μL ddH6copies/mL of SFTSV HB29 was defined as 106 U/mL. Ten-fold serial dilutions of SFTSV HB29  were used as sensitivity references and labeled S1-S5. The lowest detection limit of the reference was assessed using SinoMD kit and DaAn kit for 4 times.The extensively investigated SFTSV strain HB29 was used as a reference for sensitivity testing. First, the recombinant plasmid containing the conserved S gene pMD18-TS was constructed, serial diluted 1:10 and then used to establish a standard curve. 4.83 × 104 U/mL (S3) SFTSV HB29) was detected using the SinoMD and DaAn kits for 10 times and the coefficient of variation (CV) of each Ct was calculated and defined as the accuracy of the reference.The reference , and means are presented. 50/mL, the titers of the JEV A14-14-2 strain, DV Ban18 strain and YFV 17D strain were 5.56 lg PFU/mL, 5.40 lg PFU/mL and 6.09 l g PFU/mL, respectively. The titers of the provided HFRSV L99 strain and TBEV Senzhang strain were 7.3 lg CCID50/mL and 8.0 lg LD50/mL, respectively.Using the CPE method, the titer of SFTSV ranged from 6.8 to 8.3 lg CCIDSequencing of these 6 positive and 5 negative references indicated that all conserved virus genes or genomes  shared 99% homology with genes from the same or similar virus strains.Next these positive and negative references were applied for q-PCR using SFTSV specific primers and probes, and kits from SinoMD and DaAn gene. The positive Ct value was defined as less than 35 for specific primers and probes, and less than 37 for the two kits according to the manufacturers’ protocol. The Ct values of 6 positive references were all less than 35, with typical S curves for specific primers and probes as well as two commercial kits Table , Fig. 2a12 copies/mL to 2.01 × 104 copies/mL in ten-fold dilutions. The sensitivity reference SFTSV HB29 strain was used at 4.83 × 106 copies/mL, serially diluted 1:10 five types (S1-S5), and detected using two commercial kits. As shown in Table 3 U/mL virus, while the detection limit of the DaAn kit was S4 with 102 U/mL virus. Although S5 with 10 U/mL virus can be detected using the DaAn kit in 3 of 4 times, the Ct value and dilution did not have a linear relationship. The detection limit was still S4 with 102 U/mL virus. The q-PCR reaction curves of sensitivity reference are shown in  SFTSV HB29 was detected using the SinoMD and DaAn kits in five independent experiments including two duplicates each. As shown in Table To evaluate the repeatability of the accuracy reference, a sample containing 104 U/mL S SFTSV HB2 U/mL virus and the CV values of Ct for all treated accuracy references were less than 5% . All these data suggest that all references exhibit good stability to temperature.The references were subjected to 3 cycles of freeze-thaw treatment and after each cycle tested using q-PCR. The accuracy reference CV values of Ct did not differ between the three cycles (Tables 2 U/mL. The CV of the accuracy reference ranged from 0.45 to 4.55%, which were all less than 5%.Calibration of the references is described in Table SFTS caused by SFTSV infection was first emerged in China in 2009 , followeWhen establishing reference standards, the references should be represent a broad-spectrum of relevant viruses . In 20072 U/mL, as SFTSV infection is reported to cause titers of 102-108copies/mL in the serum [When selecting sensitive and accurate references, the earliest identified and extensively studied HB29 strain was chosen. The serial dilution method was used to determine the detection limit of the reference. Through collaborative calibration by three labs, the detection limit was set as 10he serum –29. The We also investigated the capacity of the references to withstand freeze-thaw and conditions typical of storage, transportation and application, as previously reported . StorageThe national reference had been established successfully and proved good accuracy, high specificity, sensitivity and stability. It means that the reference will be widely used in the quality control of the similar commercial SFTSV diagnostic kits and guarantee their effectiveness, thus more SFTSV infection can be diagnosed rapidly and the targeted prevention and control measures will be developed.


We report here the complete genome of a Dezidougou virus (DEZV) isolated from a passaged culture of the Zika virus strain DAK AR 41524. The consensus DEZV sequence we recovered shows 99% nucleotide similarity using BLASTN to a previously reported DEZV (accession no. JQ675604.1). The current sequence has additional repeat regions as well as a deleted repeat region, which we confirmed by Sanger sequencing, that were not present in the originally published sequence, JQ675604.1. Negevirus taxon. Here, we report the most complete sequence for a Dezidougou virus available to date, which is missing only the start of the coding region. This virus was obtained from a passage of Zika virus (ZIKV) strain DAK AR 41524, which was isolated from a mosquito (Aedes africanus) in Kédougou, Senegal, on 17 November 1984. This isolate was passaged twice in mosquito cell lines, first in AP61 (Aedes pseudocutellaris) and then in C6/36  cells at the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). The isolate was then passaged twice in C6/36 cells, where it showed good cytopathic effect but failed subsequent molecular authentication for Zika virus. In parallel, the seed Zika virus from WRCEVA that was passaged in the Vero cell line (African green monkey) grew to high titers. Viral RNA was extracted from the supernatant of mosquito cell cultures and subjected to sequence-independent single-primer amplification, as described previously (Dezidougou virus (DEZV) is a single-stranded, monopartite, positive-sense RNA virus of approximately 9.3 kb in length, and it is a member of the recently proposed de novo assembled. BLASTN searches of these contigs were then performed against the GenBank nonredundant nucleotide database , we observed one 396-base insertion beginning at position 6714 in JQ675604.1. The 201-base duplication found in JQ675604.1 at position 8780 was not detected in the J. Craig Venter Institute (JCVI) consensus sequence. These duplications were then verified with Sanger sequencing technology. Sequences of additional DEZV strains will confirm the true consensus and the genomic structure of Dezidougou virus.KY968698 and BioProject ID PRJNA350806.The consensus sequence for DEZV was manually annotated and deposited in GenBank with the accession number


In Indonesia, 96% of children (< 24mo) are breastfed. However, only 42% of children (< 6mo) are exclusively breastfed, as per World Health Organization recommendations. Breastfeeding provides protective benefits such as reducing the risk of morbidity and mortality associated with diarrhea and pneumonia/respiratory disease (PRD). This study estimates the potential economic impact of not breastfeeding according to recommendation in Indonesia based on infants suffering from attributable diarrhea and PRD.n = 615) collected data related to out of pocket costs such transportation and opportunity costs such as wage loss. These figures were combined with the national disease prevalence rates from Indonesia Demographic and Health Survey 2012, and the relative risk of disease of not breastfeeding according to recommendation from literatures to extrapolate the financial burden of treatment.A cost analysis examined both the healthcare system costs and non-medical costs for children (< 24mo) with diarrhea and PRD. Data collection took place between 2015 and 2016 and healthcare expenditures were assessed in 13 facilities, in five sites including Bandung and Tomohon City. Costs from a provider perspective were estimated using healthcare records and 26 interviews with healthcare workers. A discount rate of 3% was used. A cross-sectional survey with caregiver-child pairs  contains supplementary material, which is available to authorized users. Breastfeeding has been shown to provide many benefits to children, mothers, health systems and economies. Breastfeeding is associated with decreasing maternal risk of breast cancer, ovarian cancer and Type 2 diabetes, and higher intelligence of the child and decreased risks of infections, malocclusion of the teeth, overweight and diabetes , 2. BreaNot breastfeeding according to recommendation is associated with diarrhea and pneumonia/respiratory disease (PRD) in children < 24mo) mo 4–8]..8]. Stud..8]. StuIn Indonesia, 96% of children (< 24mo) are breastfed at least once, but only 42% of children (< 6mo) are exclusively breastfed , 11.Even though Indonesia has policy level laws and regulations supporting exclusive breastfeeding , 13, theThe lack of accredited, “Baby-Friendly” breastfeeding promoting hospitals  and the Breastfeeding also has the potential to save costs for parents, insurance companies, employers, and society in general  –32. In sThe costs estimate attributed to not breastfeeding according to recommendation by Walters et al.   in accessing care?We aim to present the economic impact of not breastfeeding according to recommendation, extrapolated by treatment costs of diarrhea and PRD among children (< 24mo) from the healthcare provider perspective and patient perspective. To our knowledge, such study in Indonesia is limited to one study . Knowingposyandu), primary health centres (puskesmas), and “others” being other healthcare facilities. For hospitals, cost data was collected separately for inpatient and outpatient visits. The “others” category consists of facilities not grouped within the other five types of facilities. For these, we used the treatment cost of the most similar of the five types of institutions from our survey data. The list of other facilities, is provided in an Additional file The methods used were based on the Walters et al. and Bagriansky study , 34. ProData collection was performed in five separate sites, two cities and three non-urban districts, ranging from West to near East Indonesia. The five selected sites where; Bandung city (West Java), Serdang Bedagai district (North Sumatra), Tomohon city (North Sulawesi), Gianyar district , and Kupang district (East Nusa Tenggara). The site selection was based on discussions with stakeholders and experts from the Ministry of Health, the Ministry of Children and Women Empowerment, UNICEF Indonesia, and Alive & Thrive. The treatment costs obtained from each site were used to represent similar provinces. The list of provinces is provided in Additional file The Gates Reference Case principles  was follCosts from the providers’ perspective consisted of medical expenditures for treatment. To calculate these expenditures, the following information related to treating diarrhea and PRD for children (< 24mo) was collected from records and interviews with healthcare workers. The labour costs, equipment costs, number of equipment and the recurrent costs of upkeep and supplies as well as service utilization (i.e. number of visits/cases) were observed. Overhead costs such as utilities, rent and administration were not included. Interviews were conducted with 26 healthcare workers, two per facility in each region, to estimate the percentage of utilization of resources to treat patients. Annualized capital costs were calculated using a 3% discount rate, in which the total cost for capital items is spread over the useable life time of the equipment and is standard for economic evaluations of healthcare programs . The cosIn total, 615 caregiver/child pairs were interviewed to estimate out of pocket costs from patient perspective . Caregivers were interviewed regarding medical costs (e.g. lab and drug costs), and non-medical costs such as transportation and productivity loss. Productivity loss was measured based on the work time lost due to accessing the facilities. The mean total time for roundtrip transport, receiving treatment in facilities (including wait time) was assessed and multiplied by the estimated wage per minute of the accompanying family member(s) to estimate productivity loss. A monetary value was not allotted for those providing unpaid childcare such as housewives or stay-at-home fathers. Also, the productivity loss or extra expenses stemming from providing outpatient homecare was not calculated. A sampling of convenience was used and 20 to 70 caregiver/child pairs from each type of facility studied were interviewed. In locations without certain types of facilities, for example Tomohon city does not have a public hospital, more participants were interviewed in the other facilities. Prior to the interviews, respondents were required to give informed signed consent.puskesmas, posyandu, or others) from IDHS 2012 data [The Indonesia Demographic Health Survey (IDHS) 2012 data  was used012 data . The tot012 data  as it haThe total healthcare system costs consisted of tallying the total costs from provider and patient perspectives in treating diarrhea and PRD for children (< 24mo) due to not breastfeeding according to recommendation. Patient medical costs were excluded from the healthcare system costs analysis to avoid duplication of expenditures as these were also counted as expenditures by healthcare providers.To calculate the total healthcare system costs, the annual number of cases were multiplied by the treatment costs throughout the respective facilities and regions , resulting in the total treatment cost implications of these diseases due to not breastfeeding according to recommendation.In order to present the high and low estimates of our calculation, as sensitivity analysis was preformed where the main parameters were varied by up to ±25%. The treatment cost for each facility, prevalence rate of diarrhea and PRD and the rate of accessing care by was modify by ±25%, ± 25% and ±5%, respectively.Participants caregiver/child pairs for those treated for diarrhea and PRD were surveyed in the different types of healthcare facilities. Participant characteristics are presented below in Table puskesmas and posyandu had an average monthly income of US$31 and US$56, respectively. They spent about 13 min round-trip travel time to the facilities and approximately 50 min at the facilities. Most participants surveyed at the puskesmas or posyandu suffered from PRD.Participants visiting the The annual costs of outpatient and inpatient care for diarrhea and PRD treatment due to not breastfeeding according to recommendation in the five types of facilities are provided in Table posyandu, US$41.33 in public hospitals and US$146.11 in a private hospital. The costs for inpatient treatment for diarrhea can reach US$362.80 in a public hospital. The cost per case treated as outpatient PRD treatment ranged from US$2.34 in posyandu, US$45.62 in public hospitals. PRD inpatient treatment was US$366.82 in public hospitals and US$433.44 in private hospitals. The treatment costs in each facility was higher in Bandung city, except for outpatient treatment in the public hospital in which the treatment cost in Serdang Bedagai district is around one and a half to two times higher (unpublished data available from authors). The total annual healthcare system cost was around US$119 million . From the patient perspective, the non-medical cost share out of the total healthcare system cost differs between diseases, in which for PRD the non-medical cost share can reach around 55% of the total healthcare system cost, while for diarrhea the non-medical cost share is 20%. Our sensitivity analysis shows that the highest estimate may reach US$163.5 million, while the lowest estimate is US$80.3 million , which is 0.01% of Indonesian Gross National Income (GNI) in 2012 . Of thisSecondly, the costs of treating diarrhea and PRD for children (< 24mo) amounted to approximately US$88 million from provider perspective (i.e. excluding patient perspective costs), and accounts for 75% of the total healthcare system costs. This leaves around 25% of the healthcare system costs to come from non-medical costs borne by patients , amounting to around US$30 million per year (US$3.85 per case). Differentiating between diarrhea and PRD, we found that this percentage increased substantially for PRD , while the percentage is at 20% for diarrhea (US$3.33 per case). This shows that although overall the costs of diarrhea are higher than that of PRD due to higher number of cases, the latter cause much more burden to the patients through non-medical costs. Furthermore, PRD has a higher mortality rate in Indonesia . Note thThirdly, the patient out of pocket costs  of accessing both outpatient and inpatient care for diarrhea and PRD treatment are a potential problem for families as they account for more than 10% of average monthly income , and mucJaminan Kesehatan Nasional”, can be directed towards covering the costs of counselling on infant and young child feeding, including breastfeeding, information which is accessible to all mothers, therefore, potentially decreasing the treatment costs of diseases associated with not breastfeeding according to recommendation. Currently, the NHI is focused mostly on the curative side, and not so much on the preventive aspect. Therefore, there might be a need to revise NHI to cover more preventive and public health programs and interventions such as the counselling on breastfeeding to all mothers. The NHI may become one of the solutions to provide the appropriate funding to cover the costs of public health programs and interventions to ensure the success of breastfeeding. Thus, eliminating the incidence of diseases caused by not breastfeeding according to recommendation. Prioritizing resources to support breastfeeding according to recommendation is therefore crucial, with a strong return on investment for the entire nation.To reduce the incidence of not breastfeeding according to recommendation the following recommendations are proposed. The National Health Insurance policy (NHI) or “In addition, a comprehensive breastfeeding strategy should be implemented to ensure breastfeeding is promoted, protected, and supported. The role of workplaces, healthcare workers, and social support from families cannot be underestimated. Creating an enabling environment for breastfeeding is crucial, and all stakeholders play significant roles. Providing longer maternity leave and supporting women to continue breastfeeding after returning to work are just two examples of how environments can be designed to support breastfeeding according to recommendation –51. MoreIn terms of study limitations, Indonesia is an extremely diverse nation, and a broader sampling of treatment costs is needed for more accurate results. Data regarding treatment costs were assessed from two cities and three districts to represented the treatment cost of 98 cities, 416 districts and 34 provinces in Indonesia. Furthermore, our study sample to calculate patient costs is relatively small (615 respondents) in the context of the number of people in Indonesia (more than 250 million people). However, we found that patients per type of facility in each of the city/districts have similar trends in terms of distance/time travelled, and monthly income/expenditure. Therefore, as our selection of cities/districts is spread in five different parts of Indonesia, we believe that our sample, to some extent, may represent the trend in Indonesia. Future studies can explore treatment and patient costs from additional regions, so more islands or regions in Indonesia will be represented by the extrapolations of data gathered here.Another limitation was the assumption that the relative risk of diarrhea and PRD due to not breastfeeding according to recommendation was evenly distributed across Indonesia . FurtherLastly, the productivity value of housewives and stay-at-home dads were not estimated for the productivity loss and extra expenses due to providing outpatient care. Therefore, to some extent, this undervalues our calculation of non-medical cost. Future study may include these non-medical costs, and may go even further to estimate the costs of palliative care and future productivity loss of children and maternal breast cancer deaths attributable to not breastfeeding according to recommendation and provide a more complete picture of the severe impact of not breastfeeding according to recommendation in Indonesia.Although our study is based on the methodology used by Walters et al. , our stuThe 2016 annual economic impact of not breastfeeding according to recommendation in Indonesia based on the high cost of treating diarrhea and PRD warrants urgent attention. The healthcare system costs and non-medical out of pocket cost were US$11.37 and US$3.85/treatment respectively. The total healthcare system costs were US$118.62 million annually. Non-medical costs from patient perspective accounted for 25% of treatment costs. Out of pocket costs represented a significant burden and a financial barrier to seeking treatment as it accounts for more than 10% of the caregiver’s average monthly income.Importantly, these figures only include immediate costs stemming from not breastfeeding according to recommendation and do not take into account the cost of future cognitive losses, which could reach US$1.54 billion per year for Indonesia .Additional file 1.Classification of health facilities and proxy for treatment cost (based on IDHS 2012). This table shows the proxy of treatment cost used for facilities included within “others” category. (DOCX 15 kb)Additional file 2.Proxy for treatment costs in different provinces. This table shows the proxy of treatment cost used to represent each of the provinces in Indonesia. (DOCX 15 kb)Additional file 3.Summary of type of costs/parameters used. This table shows the summary of explanation, values, and sources of various costs and parameters used in the study. (DOCX 20 kb)


In the last decades, a group of viruses has received great attention due to itsrelationship with cancer development and its wide distribution throughout thevertebrates: the papillomaviruses. In this article, we aim to review some of the mostrelevant reports concerning the use of bovines as an experimental model for studiesrelated to papillomaviruses. Moreover, the obtained data contributes to thedevelopment of strategies against the clinical consequences of bovinepapillomaviruses (BPV) that have led to drastic hazards to the herds. To overcome theproblem, the vaccines that we have been developing involve recombinant DNAtechnology, aiming at prophylactic and therapeutic procedures. It is important topoint out that these strategies can be used as models for innovative proceduresagainst HPV, as this virus is the main causal agent of cervical cancer, the secondmost fatal cancer in women. In the last decades, novel diagnostic methods and therapies have been implemented in anattempt to combat cancer. However, the number of patients that succumb to the diseasehas increased globally , 85% of cervicalcancer cases occurs in less developed countries.Current studies estimate that 23% of all human malignancies are associated withinfectious agents . Among tPapillomaviruses (PVs) are not only associated to human cancers, but also with animalmalignancies. Although there is no epidemiological data about the number ofPV-associated incident animal cancers, this association is recognized since 1932 . MoreoveIn this review, we summarize relevant data and advances in papillomaviruses biology,including viral evolution, pathogenic mechanism of viral proteins and oncoproteins, waysof transmission, pathogenesis and oncogenesis. We also discuss the importance of BPV asa study model for HPV-associated oncogenic process.Papillomaviridae family (Chelonia mydas papillomavirus type 1) to8,607 bp (CRPV1 - Cotton rabbit papillomavirus type 1), divided inthree regions: early (E), late (L) and long codon region (LCR), as showed in ori)  . Current in ori) , which a in ori) .Although the virus origin is still uncertain ,studiesStudies based on molecular phylogeny suggest that these viruses originated in Africa,from where they disseminated to all continents . It was in situ hybridization probe for Shope papilloma virus (CRPV),which presents homology with the rabbit genome sequences  present in LCR  C-terminal and (2) transactivationN-terminal . The E24CGGT-3′ palindromic sequence present in four E2binding sites (E2BSs) in E6 and E7 promoter (P97)  . This inn levels .The E4 ORF codifies a family of proteins produced by splicing followed bypost-translational modifications .The E4 The L1 ORF is the most conserved among PVs . The L1The L2 protein has 64-78 kDa . The molA third structural protein (L3) has been described as present exclusively in BPV-4 . PDGFβR E6 is a small oncoprotein with 151-158 (HPV) . Later, In 1986 in an intriguing paper by BPV is a cosmopolitan virus, worldwide distributed, independently of the level ofexpertise on livestock exploration ,Epsilonpapillomavirus (BPV-5 and 8),Xipapillomavirus  andDyoxipapillomavirus (BPV-7)   allows to determine the number of viral copies, thismethod has the lowest reproducibility . Therefore, CISHrepresents and additional method used to demonstrate the physical state of these viruses or indirect contact withcontaminated surfaces . StudiesStudies of PV infection are based on the prototype BPV-1 , which a4 viral copies  , it seems that the virus can survive to pasteurization process  as adjuvant‥ Both vaccines areconsidered safe and well tolerated (Two prophylactic vaccines against HPV are available in the market since 2006: (1)Cervarix, produced by Glaxo-Smith Klein (GSK) and (2) Gardasil, produced by Merck . These vadjuvant . Gardasiolerated . For theEscherichia coli have demonstrated to be a useful alternative,because they have a lower cost, and not requiring the L1 VLPs, they are more stable.Studies have demonstrated that BPV early proteins (E6 and E7) show a therapeutic action,while later proteins (L1 and L2) have a prophylactic action . VaccinePapillomaviridae comprises the most extensive known family of viruses,able to infect all vertebrates including humans, in which it is responsible for 27-30%of all infectious agent-associated incident cancer cases. Moreover, PVs represent animportant problem in the veterinary field, inducing papillomas in dogs, felines andcattle. Also, BPV infects equines, resulting in sarcoids. Although novel discoveriescontributed to the understanding of the PVs oncogenic role, some carcinogenic mechanismsremain unknown, especially those following cancer initiation (itiation . Howeveritiation . In addithbirthday. She and her husband were the founders of the Laboratory of Genetics of theInstituto Butantan, in 1961. They were Brazil's pioneers in introducing human andvertebrate cytogenetics and the first to use cytogenetics as an important tool in humangenetic counseling. Dr. Beçak's contribution in classic and recent papers in the geneticliterature is very important and remarkable.This paper is dedicated to Dr. Maria Luiza Beçak on occasion of her 80