Zika virus is transmitted to humans via the bite of an infected Aedes species mosquito, but maternal fetal and sexual transmissions have also been described.2 The most common symptoms are fever, maculopapular rash, arthralgia , and conjunctivitis, usually beginning 2 to 7 days after the infection. Other commonly reported symptoms are myalgia, headache, and asthenia. Treatment includes rest, fluids to avoid dehydration, and administration of acetaminophen to relieve pain and fever. Patients should be cautioned against the use of aspirin or nonsteroidal anti-inflammatory drugs to reduce the risk of hemorrhage.2In the past 20 years, arbovirus infections, caused by RNA viruses transmitted by arthropods, have become a major concern in the Western Hemisphere; examples include dengue, West Nile virus, and chikungunya.3 In April 2015, the first identified case in Brazil was reported. Since then, according to the Brazilian Ministry of Health, between 490,000 and 1,400,000 new cases may have occurred.4 Although approximately 80% of infected persons do not develop signs or symptoms, an explosive epidemic of microcephaly and Guillain-Barré syndrome has raised concerns about an etiologic association.5 On February 1, 2016, the World Health Organization announced that the cluster of neurologic disorders and congenital anomalies reported in Brazil constitutes a public health emergency of international concern.3 To the best of our knowledge, there are no data on Zika virus infection in patients with cancer. Here we report the first case.In the last 13 months, more than 30 countries have reported active Zika virus transmission in the Americas, Oceania, and Africa.A 58-year-old white woman, currently on treatment with anastrozol for a locally recurrent, hormone receptor–positive breast cancer, spent the New Year’s holiday in a coastal area of Sao Paulo State. In early January, she reported important tiredness, myalgia, headache, and retro-orbital pain. Two to 3 days later, she developed sudden episodes of high fever with intense sweating, which lasted 4 days. The patient sought medical assistance twice and was submitted to an extensive number of complementary examinations, the findings of which, besides a hemoglobin level of 10.0 g/dL (previously 13.5 g/dL), were normal. Approximately 10 days after the onset of the symptoms, the patient was referred to her oncologist in a tertiary center hospital, who noticed a mild abdominal maculopapular rash and ordered some serologic tests. Dengue virus immunoassays were negative, but indirect immunofluorescence assays for Zika virus demonstrated both immunoglobulin M (IgM) and IgG high titers. The patient is now recovered and had no neurologic symptoms.1This case illustrates some difficulties in diagnosing Zika virus infection in patients with cancer. As already highlighted in previous studies, cross-reactions with related flaviviruses are common and, unfortunately, Zika-specific tests are expensive and not widely available.Clinicians should be aware of the importance of including arboviruses as differential diagnoses of fever in patients with cancer, especially in epidemic areas. In doing so, patients would be less likely to be submitted to unnecessary and costly examinations. In addition, some patients with cancer who already have reduced platelet counts, secondary to the primary disease, chemotherapy, or other specific treatments, are at great danger of bleeding if not promptly advised not to take aspirin and nonsteroidal anti-inflammatory drugs if the infection is suspected. Also, a Guillain-Barré syndrome in a patient with cancer should be interpreted with caution in areas of the ongoing Zika virus transmission because it could easily be misdiagnosed as a paraneoplastic manifestation of the underlying disease.The Zika outbreak is evolving rapidly and the expected number of new cases in patients with cancer should increase. The course of the disease in immunosuppressed persons and in patients presenting with significant comorbidities is still uncertain. Therefore, close follow-up is required to identify late or chronic symptoms.


Spinous processes and posterior ligaments, such as inter- and supraspinous ligaments are often sacrificed either deliberately to harvest osseous material for final spondylodesis e.g. in deformity corrective surgery or accidentally after posterior spinal instrumentation. This biomechanical study evaluates the potential destabilizing effect of a progressive dissection of the posterior ligaments (PL) after instrumented spinal fusion as a potential risk factor for proximal junctional kyphosis (PJK).Twelve calf lumbar spines were instrumented from L3 to L6  and randomly assigned to one of the two study groups (dissection vs. control group). The specimens in the dissection group underwent progressive PL dissection, followed by cyclic flexion motion  to simulate physical activity and range of motion (ROM) testing of each segment with pure moments of ±15.0 Nm after each dissection step. The segmental ROM in flexion and extension was measured. The control group underwent the same loading and ROM testing protocol, but without PL dissection.P = 0.017) and a PL dissection in the instrumented segments showed a positive trend towards an increased ROM at UIV/UIV + 1.In the treatment group, the normalized mean ROM at L2-L3  increased to 104.7%, 107.3%, and 119.4% after dissection of the PL L4–L6, L3–L6, and L2–L6, respectively. In the control group the mean ROM increased only to 103.2%, 106.7%, and 108.7%. The ROM difference at L2-L3 with regard to the last dissection of the PL was statistically significant (A dissection of the PL at UIV/UIV + 1 leads to a significant increase in ROM at this level which can be considered to be a risk factor for PJK and should be definitely avoided during surgery. However, a dissection of the posterior ligaments within the instrumented segments while preserving the ligaments at UIV/UIV + 1 leads to a slight but not significant increase in ROM in the adjacent cranial segment UIV/UIV + 1 in the used experimental setup. Using this experimental setup we could not confirm our initial hypothesis that the posterior ligaments within a long posterior instrumentation should be preserved. According to a report by the Agency for Healthcare Research and Quality, approximately 488.000 spinal fusions were performed during U.S. hospital stays in 2011 (a rate of 15.7 stays per 10.000 population) –7. The rHowever, neither the precise pathophysiology of PJK nor clear strategies for preventing it have yet been established , 19.One of the keys to PJK is the quality and integrity of the posterior tension banding , 8, 12. The purpose of this biomechanical in vitro study was to evaluate the potential destabilizing effect of a progressive dissection of the PL, even within the fused segments caudal to UIV following posterior instrumentation on the segmental biomechanics at UIV/UIV + 1. It was hypothesized, that the PL even within the fused segments caudal to UIV are relevant for sagittal stability, that a dissection could therefore lead to an increase of range of motion (ROM) at UIV/UIV + 1 and could possibly represent a risk factor for the development of PJK and that the PL should therefore be preserved during surgery.Twelve L1–L6 calf lumbar spines (aged 12–18 months) obtained from a local slaughterhouse were used according to established biomechanical testing protocols , 20, 21.Posterior instrumentation at L3–L6 was performed by an experienced spine surgeon using a bilateral polyaxial pedicle screw rod system .The specimens were randomly assigned to one of the two study groups (treatment vs. control group). The tests were performed at room temperature, and the specimens were kept moist with physiological saline solution during testing.In the treatment group, six specimens underwent stepwise dissection of PL  within the fused segments and at last also at UIV/UIV + 1. After each level of ligament dissection , the specimens underwent cyclic flexion motion of 250 cycles in which a flexural moment of + 2.5 to + 20.0 Nm was applied  to simulate physical activity. The number of load cycles was limited to 250 cycles due to time constraints of in vitro testing with cadaveric specimens . The load magnitudes were chosen rather high to provoke an effect of stretching ligamentous structures beyond the elastic region to cause an increased motion in the segment to simulate the genesis of PJK. This was followed by a flexibility test with pure moments of ±15.0 Nm in flexion/extension for three load cycles measuring the ROM of each segment.In the control group, six specimens underwent the same test protocol with cyclic motion and flexibility tests, but without PL dissection Figs.  and 2.FiFlexibility tests and cyclic flexion motion were done in the same setup of a six degree of freedom spine tester. The specimens were loaded with pure moments, applied by a stepper motor. A six-component load cell with feedback control was connected to the stepper motor to control the loading of the specimens Fig. . No prelP values were corrected using the Greenhouse–Geisser method [p < 0.05.After instrumentation, a baseline flexibility test was performed. To facilitate data comparison, subsequent ROM measurements were normalized to the initial ROM. Statistical analysis was performed using IBM SPSS Statistics® for Windows, version 22.0 . For data comparison analysis, two-way analysis of variance (ANOVA) with repeated measures was performed  with post hoc analysis using Bonferroni correction. To account for possible sphericity violation among the states, r method . The sigThe ROM for each segment  was measured for each state   leads to a significant increase of ROM at the adjacent segment L2-L3 (UIV/UIV + 1) itself, whereas dissection of PL within the instrumented segments (keeping PL of UIV/UIV + 1 intact) results in a slight but not significant increment of ROM at the segment L2-L3 (UIV/UIV + 1) Fig. .Fig. 4Chp = 0.017). The interaction between treatment (dissection or control) and state  was also statistically significant (p = 0.002).In the treatment group, the normalized mean ROM in segment L2-L3  increased to 104.7%, 107.3% and 119.4% after dissection of the L4–L6, L3–L6 and L2–L6 PL, respectively . The interaction between treatment (dissection or control) and state  was also not significant (p = 0.171).The normalized mean ROM of segment L1-L2  increased to 105.5%, 108.6%, and 113.9% after dissection of the L4–L6, L3–L6, and L2–L6 PL, respectively  compared to the native state. The reason is the rigid posterior instrumentation spanning from L3-L6.The segments L4-L6 and L3-L4 (segments within the rigid posterior instrumentation) show only small absolute values of ROM Table  and theyThis study shows that a dissection of the posterior ligaments (the supraspinous and interspinous ligaments) at UIV/UIV + 1 does have a significant influence on the ROM in the adjacent segment and is therefore definitely a risk factor for PJK.PJK is defined as pathologic kyphosis in the first mobile proximal adjacent segment after instrumentation. Whereas some authors define PJK radiographically as kyphosis greater than 10° between the upper instrumented vertebra and the vertebral body two levels above it (UIV/UIV + 2) , others The incidence of PJK is reported to be in the range of 6–61.7% –6. In moVarious risk factors have been suggested in the literature as being responsible for PJK. Some of these cannot be controlled or influenced by the surgeon, but others can , 26–28. Cahill et al. have shown in a finite element modeling an increased angular displacement and nucleus pressure immediately after dissection of the posterior ligamentous complex at the level above the construct . DiffereHowever, there is no previous biomechanical study with an experimental setup evaluating the effect of a dissection of the posterior ligaments even within the instrumented levels, far away from the junction level, and not only at the adjacent level itself. Therefore, we conducted this biomechanical study which at least supports the theory that there is an important tension band effect of the PL preventing PJK in the adjacent segment. In our study a small, nonsignificant increase in ROM at UIV/UIV + 1 was detected after PL dissection even within the fused segments of the spine. Most notably, a PL dissection at UIV/UIV + 1 itself has a significant impact on the ROM in this segment (+ 18.9%) and therefore on the potential development of PJK.p < 0.02) at the final dissection. To simulate a physical activity a cyclic flexion motion was performed in both groups. Therefore, all ligamentous structures and other tissues are strained and might lose their tension over time. This can explain the increase of the ROM in the treatment group as well as the small increase in the control group over time.As an increase in ROM is only an indicator and not equal to an increased kyphosis, i.e. PJK, the hysteresis curves (applied moment vs resulting angular displacement) of the flexibility tests were further evaluated to emphasize the effect of posterior ligament dissection on segmental alignment. The ROM mainly increased in flexion, while the ROM in extension did not substantially change. With this the mean neutral zone as an indicator of the segmental alignment also shifted in the kyphotic direction. This became even more pronounced with an increasing dissection of the posterior ligaments Fig.  and the A limitation of the present study is the use of calf instead of human spines. These specimens do not represent the same sagittal profile as humans and therefore the absolute ROM values cannot be directly transferred to the situation in humans. However, the relative effects of the various states that were tested should be similar in humans. Previous studies have shown that the use of calf lumbar spines does allow accurate biomechanical studies comparable to human spines , 31. TheObviously, the biomechanical experiments were carried out with a limited number of specimens. However, due to the controlled laboratory environment the common confounding variables occurring in a clinical trial can be excluded. Therefore it can be assumed that if the biomechanical effect of an intervention cannot be shown in a controlled laboratory environment with a limited sample size, it is deemed to be unlikely to have a clinical impact.Furthermore a more extensive cyclic flexion motion after each PL dissection simulating a higher and longer physical activity could have been resulted in an even more increased ROM at UIV/UIV + 1 coming to statistical significance, especially for the ligament dissection within the instrumented segments and should be evaluated in the future.Dissection of posterior ligaments (supraspinous and interspinous ligaments) of the adjacent segment (UIV/UIV + 1) cranial to a posterior instrumentation leads to a significant increase in ROM in the adjacent segment itself. Therefore, an accidental injury to or an intentional dissection of the posterior ligaments at UIV/UIV + 1 could be a risk factor for PJK and should definitively be avoided.Dissection of the posterior ligaments within the instrumented segments while preserving the ligaments at UIV/UIV + 1 leads to a slight but not significant increase in ROM in the adjacent cranial segment UIV/UIV + 1 in the used experimental setup. Using this experimental setup we could not confirm our initial hypothesis that the posterior ligaments within a long posterior instrumentation should be preserved.Further clinical evaluation is needed to finally answer the question of the role of the posterior ligaments regarding sagittal stability after posterior instrumentation.


Vitis vinifera) cultivar ‘Kizil Sapak’ by high-throughput sequencing (HTS) and tentatively named “grapevine virus J” (GVJ). The full genome of GVJ is 7,390 nucleotides in length, which comprises five open reading frames (ORFs), including a 20K ORF (ORF 2) between the replicase (ORF 1) and the movement protein (ORF 3) genes. According to the level of sequence homology and phylogenetics, GVJ is proposed as a new member of the genus Vitivirus , with the closest characterized virus being grapevine virus D (GVD).This paper describes the nucleotide sequence and genome organization of a novel RNA virus detected in grapevine ( Vitis vinifera) is considered one of the oldest crops in the history of humanity [Vitivirus, subfamily Trivirinae, family Betaflexiviridae), whose type species is Grapevine virus A (GVA), which is associated with the etiology of rugose wood disease in grapevine [Vitivirus have a positive-sense single-stranded (ss)RNA genome with five open reading frames (ORFs), including a distinctive 20K ORF between the ORF1 (replicase) and the ORF3 (movement protein) [https://talk.ictvonline.org/), five of which are reported in grapevine: GVA, grapevine virus B, grapevine virus D (GVD), grapevine virus E and grapevine virus F (GVF) [Vitivirus genus. Nevertheless, the taxonomy of GVK should be further investigated after a phylogenetic study indicated that this virus is a variant of GVD [V. vinifera cv. ‘Kizil Sapak’ (KS) from Turkmenistan, a novel vitivirus was detected and tentatively named “grapevine virus J” (GVJ). A reverse transcription polymerase chain reaction (RT-PCR) assay was developed and used to confirm the presence of the new virus in the source plant.The grapevine (humanity  and is erapevine . Membersprotein) . To date F (GVF) , grapevi F (GVF) , grapevi F (GVF) , are prot of GVD . During de novo assembler and compared against the NCBI database of viruses using tBLASTx (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The previous analysis revealed nine contigs that ranged in size between 4,743 to 7,375 nucleotides (nt) and showed a distant relationship (average identity: 52%) to several viruses belonging to the genus Vitivirus. Consequently, the longest contig and near-complete genome sequence  of the putative virus was extended using a FirstChoice RLM-RACE kit (ThermoFisher Scientific) to obtain the 5’ and 3’ ends of the genome. The complete genome sequence (including a 12-nt polyA tail) of the new vitivirus, for which the name “grapevine virus J” (GVJ) is proposed, was 7,390 nt long  collection. The vine was grown in a screenhouse and assayed for known grapevine viruses as described by Al Rwahnih et al.  and subjhttps://www.ncbi.nlm.nih.gov/orffinder/), and these coding regions were then analyzed independently using the SmartBLAST algorithm and the NCBI Conserved Domain Search Tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Finally, Protein Information Resource (http://pir.georgetown.edu/pirwww/search/comp_mw.shtml) was employed to calculate the molecular mass of each of the predicted proteins. The genome of GVJ was found to contain five putative ORFs and 5’ and 3’ untranslated regions of 95 and 69 nt, respectively. ORF1 encoded a polypeptide of 1,702 amino acids (aa) (194.6 kDa), was similar (52% aa identity and 99% query coverage) to the replicase gene of GVA, and contained the following domains: a viral methyltransferase domain (G62-K340) found in a wide range of ssRNA viruses; an alkylation B (AlkB) domain (H620-R741); a viral RNA helicase domain (G931-A1147) belonging to the superfamily 1; and an RNA-dependent RNA polymerase domain (D1360-W1597). A 17.7-kDa protein of unknown function was encoded by the ORF2 (164 aa). ORF3  showed 52% aa sequence identity (95% query coverage) to the movement protein of GVA. A predicted coat protein (CP) with a molecular mass of 21.7 kDa was identified from ORF4 (197 aa), based on 86% aa sequence identity (81% query coverage) to the CP of GVD. Finally, the 103 aa (11.8 kDa) encoded by ORF5 showed similarity to a putative RNA-binding protein present in vitiviruses.ORFs were identified using ORF Finder  was included as an outgroup. In the phylogenetic tree, GVJ clustered with members of the genus Vitivirus  and GVJ-CP-R (5’-CTCTTGCAATGTGAGTTTGCTCAG-3’), which corresponds to sites that flank a conserved region of the viral CP gene, were used to generate a 396-bp amplicon. The RT-PCR reaction was performed using SuperScript II Reverse Transcriptase (Life Technologies) and GoTaq (Promega), and the thermocycling program consisted of 30 min at 52°C, 35 cycles of 30 s at 94°C, 45 s at 55°C, 1 min at 72°C, and a final elongation step of 5 min at 72°C. Subsequently, the KS selection tested positive for GVJ, and the PCR product was sequenced directly to confirm its identity.Vitivirus, subfamily Trivirinae, family Betaflexiviridae. Considering the current species demarcation criteria for members of the genus Vitivirus [Here, a new virus, grapevine virus J (GVJ) was identified in an asymptomatic KS grapevine by HTS. Based on its genomic arrangement, sequence similarity to other viruses and phylogenetic relationships, we propose that the identified virus should be placed within the genus itivirus , GVJ canFurther work is needed to identify the mechanisms by which GVJ is transmitted. Field surveys and biological studies are underway to determine the prevalence of GVJ in commercial vineyards and to assess its effect on vine performances. In addition, the incidence of GVJ in other selections introduced to FPS and the USDA National Clonal Germplasm Repository in Winters, CA, will be determined using the newly developed RT-PCR assay.


Surveillance is critical for the prevention and control of mosquito-borne arboviruses. Detection of elevated or emergent virus activity serves as a warning system to implement appropriate actions to reduce outbreaks. Traditionally, surveillance of arboviruses has relied on the detection of specific antibodies in sentinel animals and/or detection of viruses in pools of mosquitoes collected using a variety of sampling methods. These methods, although immensely useful, have limitations, including the need for a cold chain for sample transport, cross-reactivity between related viruses in serological assays, the requirement for specialized equipment or infrastructure, and overall expense. Advances have recently been made on developing new strategies for arbovirus surveillance. These strategies include sugar-based surveillance, whereby mosquitoes are collected in purpose-built traps and allowed to expectorate on nucleic acid preservation cards which are submitted for virus detection. New diagnostic approaches, such as next-generation sequencing, have the potential to expand the genetic information obtained from samples and aid in virus discovery. Here, we review the advancement of arbovirus surveillance systems over the past decade. Some of the novel approaches presented here have already been validated and are currently being integrated into surveillance programs. Other strategies are still at the experimental stage, and their feasibility in the field is yet to be evaluated. Arthropod-borne viruses (arboviruses) transmitted by mosquitoes are of public health and veterinary importance globally causing disease syndromes including encephalitis, viral haemorrhagic disease and arthritis. Dengue viruses (DENVs) alone cause an estimated 96 million clinical cases a year, especially in the tropics and sub-tropics . The flaWith the exception of YFV  and JEV Given the broadness of this subject, few attempts have been made to provide a synthesis of arbovirus surveillance methods. The objective of this review is to describe the development and implementation of mosquito-borne arbovirus surveillance strategies. First, we evaluate traditional methods that have been commonly used where arboviruses are a public health threat, then outline and assess recently developed methodologies, before identifying future research needs.Human or animal case surveillance relies on hospitals, laboratories and health practitioners notifying public health authorities of confirmed or suspected cases of arbovirus infection that occur in the population. Almost every state in the United States conducts surveillance of human WNV cases as a part of the national arbovirus surveillance system, ArboNET , whilst A major limitation of monitoring human and animal cases is that confirmatory laboratory testing is not available in many limited resource countries, so arboviral disease is diagnosed on clinical symptoms. However, symptoms can overlap between arboviruses, as well as with non-arbovirus pathogens, complicating their clinical diagnosis. Furthermore, most arbovirus infections are mild, or sub-clinical, which may lead to them being under-reported. Ultimately, using human and animal case data is not ideal, since it indicates that active transmission is already occurring.Sentinel animals provide evidence of virus activity and increased risk to the target animal or human population . For thiDifferent vertebrate species are used as sentinels Table  and choiEven though sentinel animal surveillance enables the timely detection of circulating arboviruses, it also comes with limitations. In many cases, the locations of enzootic arbovirus foci are unknown or difficult to access. Thus, animals are placed near towns, which may be too far from virus foci to detect elevated activity . FurtherAnother approach to vertebrate host surveillance relies on monitoring wild vertebrates or livestock, which are captured, sampled and released . HoweverMosquito-based arbovirus surveillance monitors vector populations and virus infection prevalence within them. Mosquitoes are collected, identified, pooled by species or other taxonomic grouping, and sent to the laboratory where they are tested for virus infection status. There are different strategies for mosquito collection. In areas with low-level mosquito infections or early in the transmission season, efforts should be directed towards performing targeted surveillance at “hotspots” where a high likelihood of arbovirus presence is suspected; as vector populations increase later in the season, the number of sampling sites should be expanded for broader monitoring . There aA variety of methods have been utilized for detection of arboviruses in captured mosquitoes. Historically, arbovirus isolations were conducted in animals, such as suckling mice and chickens. With the development and establishment of cell lines, virus isolation in cell culture became the gold standard for arbovirus detection from pools of mosquitoes. This method can only detect viable viruses, so a cold chain keeping samples at ultralow temperatures during transport needs to be maintained to preserve virus infectivity . Mainten.Nucleic acid detection using RT-PCR has become one of the most popular methods of virus detection and has potentially displaced virus isolation as the new gold standard. Real time quantitative RT-PCR (qRT-PCR) platforms, such as TaqMan®, are ideal for routine testing of mosquitoes, since they reduce processing time significantly (sometimes to less than an hour), allowing for high throughput screening , 36. SinRapid antigen detection assays were initially developed to test clinical samples but have proven to be a useful tool to test mosquito pools in the field . These a2, to increase collections. This comes in the form of dry ice or pressurized cylinders, which may not be readily available, or only allow overnight deployment of the trap. A cold chain of storage at < -50 °C is required to preserve the integrity of the virus for detection, which can be a challenge in remote locations. Finally, specialized laboratory equipment and infrastructure is required for diagnostics, which might not be available in developing countries.Traditional mosquito-based surveillance systems that target processing of pools of mosquitoes come with inherent limitations. Mosquito populations often have very low carriage rates, whereby only one in 1000 mosquitoes is actually infected . To incrAe. aegypti, which appears to obtain enough energy from blood and rarely feeds on sugar in domestic environments [2-baited box traps, which house nucleic acid preservation cards  soaked in honey and on which mosquitoes feed and expectorate onto [The majority of mosquito species feed on carbohydrates  which are the primary energy source of their diet . One excronments . The ingronments . It was ronments  and confronments , that inate onto . The FTA2-baited passive box trap (PBT) was developed by Ritchie et al. [Commonly used traps employed to collect mosquitoes  require batteries to operate which can be logistically challenging. To circumvent this limitation, a non-powered COe et al.  to collee et al. . Althoug2-baited traps [2 or electricity, so a number of stations can be deployed simultaneously, thus increasing geographical coverage. In a proof of concept, the sugar bait stations detected WNV before sentinel animals seroconverted in California. However, this method appears more efficacious in arid habitats, probably because of lack of competition with other sucrose sources, such as floral nectars. As sugar bait stations facilitate increased geographical coverage, they may have higher costs associated with analysing an increased number of samples, although this would be offset by savings by not having to use CO2 baited light traps.Free-standing sugar bait stations have the potential to be used instead of COed traps . These sKUN before sentinel animal seroconversions [KUNV, RRV, BFV, Edge Hill virus and Stratford virus [Sugar-based surveillance has several advantages over traditional methods. When mosquito populations are elevated, sorting becomes time consuming, and a high number of pools can overwhelm laboratory capacity. When combined, these issues can reduce the ability to provide results in a timely manner. Sugar-based methods potentially overcome these issues, since only 1-2 FTA® cards per trap are tested, compared to a variable number of mosquito pools. As only transmitting mosquitoes will yield positive results, the presence of virus in saliva expectorate is a better estimate of transmission risk. FTA® cards can preserve viral RNA for up to 28 days , making versions . Howeverrd virus –64.Ae. aegypti-borne arboviruses. The cards have been used in Biogents sentinel traps (BGS traps) and modified double sticky ovitraps for the detection of CHIKV in French Guiana [Ae. aegypti may be able to increase trap collections, thus increasing the likelihood of detecting virus. For instance, the Gravid Aedes Trap (GAT) [Ae. aegypti and significantly more gravid females than double sticky ovitraps [Ae. aegypti collections are usually small, and in many cases, it would be easier to pool the mosquitoes   collectsovitraps , which cA® cards ) and prot) values obtained by real time RT-PCR are high (> 30 cycles), reflecting the relatively small amount of saliva expectorated by mosquitoes [Like any system, sugar-based surveillance has some limitations. Perhaps its main limitation is that the cycle threshold . IntegrWolbachia, from a single mosquito in a single reaction [Traditionally, diagnostic assays utilised in arbovirus surveillance programs only screen for characterised endemic and enzootic viruses. Because virus specific primers and probes are used for molecular diagnostics, it is likely that many other viruses, whether pathogenic or not, remain undetected. Metagenomic analysis using next-generation sequencing (NGS), allows for the simultaneous identification of viruses, mosquito species, and endosymbionts, such as reaction  without reaction –81 and treaction  and mesoreaction  from fieSalmonella [At this stage, NGS methods have some disadvantages compared with other molecular methods of virus detection. NGS is less sensitive than qRT-PCR for the detection of samples with low virus titres . At preslmonella . It has lmonella , so it cWuchereria bancrofti after mass drug administration programmes [Anopheles stephensi mosquitoes 24 h after ingestion [Mosquitoes have the potential to act as environmental samplers  that feed on the blood of a variety of vertebrate hosts. Xenosurveillance offers an alternative to directly sampling hosts, a process that is time consuming and requires individual informed consent in the case of humans or animal ethics approval, in the case of veterinary pathogens. Mosquitoes can be used as a proxy for syringe sampling of small animals for virus titer determination . This apogrammes . Furtherngestion . In addingestion . Xenosurngestion  by perfongestion  on mosqungestion , Epsteinngestion , human hngestion .One of the main limitations of xenosurveillance is the difficulty in collecting sufficient blood engorged mosquitoes for analysis. Some of the methods to collect engorged mosquitoes (i.e. use of an aspirator) are labour intensive and can be intrusive, especially when sampling inside houses and villages . To circAe. aegypti females, collect field data, detect virus and create georeferenced infestation maps that are available in real time, providing information to optimize vector control. This system has been successful at reducing dengue in the municipalities that have adopted it.Integration of data acquisition, storage and sharing methodologies, such as cloud networks and geographic information systems, will form an integral component of surveillance and control programmes. An example of this is the Intelligent Dengue Monitoring technology (MI-Dengue) developed in Brazil . MI-DengAe. albopictus. To date, it has more than 30,000 registered participants [In the age of mobile phones, social media and internet, citizen science will undoubtedly play an important role in disease surveillance in general. In Spain, Mosquito Alert was implemented as a system to collect reports of invasive icipants . As a paicipants . Interesicipants . Mobile icipants . All theAnopheles species [Over the past 20 years, single device detection platforms for clinical and environmental analyses have been rapidly evolving. A promising technique for integration into surveillance programmes is the use of microfluidic devices  and bios species .2 in mosquito traps (such as fermentation using yeast) [2-free systems could provide an alternative in areas where dry ice or pressurized gas cylinders are not available. Deployment of in-field portable molecular laboratories or point of care assays could provide same-day assessment of arbovirus circulation and rapid response in these locations [Over the past decade, there have been key scientific advances in arbovirus surveillance, particularly with regard to sample collection, virus detection and data analysis. Table g yeast)  or CO2-focations . In the


We report a plasmonically enhanced virus inactivation, with greater than 3.7-log reduction measured by virus infectivity assays. Reliable virus inactivation was obtained for 10 s laser exposure with incident laser powers ≥0.3 W. Importantly, the fs-pulse induced inactivation was selective to the virus and did not induce any measurable damage to co-incubated antibodies. The loss in viral infection was associated with reduced viral fusion, linking the loss in infectivity with a perturbation of the viral envelope. Based on the observations that physical contact between nanorods and virus particles was not required for viral inactivation and that reactive oxygen species (ROS) did not participate in the detected viral inactivation, a model of virus inactivation based on plasmon enhanced shockwave generation is proposed.Femtosecond (fs) pulsed laser irradiation techniques have attracted interest as a photonic approach for the selective inactivation of virus contaminations in biological samples. Conventional pulsed laser approaches require, however, relatively long irradiation times to achieve a significant inactivation of virus. In this study, we investigate the enhancement of the photonic inactivation of Murine Leukemia Virus (MLV)  In particular, virus inactivation through exposure to ultrashort laser pulses is emerging as a potential alternative to existing biocides and ionizing radiation techniques7. The current interest in photonic virus inactivation derives from the need for new technologies that achieve a selective inactivation of the pathogens in the presence of other biomolecules or even living cells in food, feed stock in pharmaceutical bioreactors, therapeutic compounds and other sensitive areas with relevance for human and animal health. For many of these applications, harsh chemical or ionizing radiation techniques are not appropriate as they lack sufficient selectivity. Importantly, Kong-Hon Tsen and coworkers have demonstrated in a series of elegant studies that irradiation with femtosecond (fs) laser pulses with visible (425 nm)8 or near-Infrared  wavelengths results in an inactivation of both enveloped and non-enveloped viruses under conditions that do not impact mammalian cells13. Tsen and coworkers postulated that fs laser induced virus inactivation is a non-thermal effect that results from impulsive stimulated Raman scattering (ISRS) driven breaking of non-covalent bonds in the virus10. Although individual broken contacts can reform, the authors argued that an excessive bond-breaking results in an irreversible loss of structural integrity of virus particles6 and potentially in viral protein aggregation5. Theoretical analysis revealed that for ISRS to be effective, high laser intensities are required14. Under these conditions alternative mechanisms to drive structural and chemical transformations exist, including multiphoton absorption. It also deserves mentioning that a different group was unable to reproduce the inactivation of phages through fs laser irradiation15, highlighting the need for further research into the mechanism of inactivation.Pulsed lasers provide new opportunities for imaging or modulating cellular behavior in a diverse range of diagnostic10. The need for long irradiation times limits the scalability of the photonic virus inactivation process and impedes the practical implementation of photonic virus inactivation strategies in many cases.To date, photonic inactivation was successfully achieved with focused fs laser beams and relatively long irradiation times of ≥1 h16, they generate high local E-field enhancements in electromagnetic hot-spots17. The strong E-field generated by the plasmonic nanoparticles can enhance the previously discussed virus inactivation mechanisms and, as we show in this manuscript, facilitate new virus inactivation processes. We demonstrate that resonant nanoparticles whose LSPR overlap with the excitation pulse, enhance the virus inactivation but that non-resonant nanoparticles have no effect. The effect of resonant plasmonic nanoparticles on virus inactivation as function of laser power when irradiated for a short exposure time of 10 s is characterized in detail. Since both efficiency and selectivity are important figures of merit for photonic virus inactivation, we monitored the selectivity towards virus particles by measuring the functionality of IgG antibodies co-incubated with the virus particles during laser exposure. Our data indicate that the plasmonically enhanced photonic inactivation is highly selective towards virus particles and generates no detectable collateral damage to the antibodies, and that the mechanism of inactivation differs from those of previously established photocatalytic18 and photothermal19 inactivation methods.In this manuscript, we explore the plasmonic enhancement of photonic virus inactivation as a possible strategy to overcome some of the challenges associated with pulsed laser driven virus inactivation. Since noble metal nanoparticles convert incident electromagnetic waves into localized charge density oscillations, so called localized surface plasmon resonances (LSPRs)2 and the typical exposure time of the sample to the laser irradiation was 10 sec. All laser irradiation studies were carried out at 22 °C. After irradiation, the samples were immediately stored at 4 °C.The ultrashort pulsed (USP) excitation source used in this study is a femtosecond laser based upon a Legend Elite Duo (Coherent Inc.) Ti-sapphire regenerative amplifier. The laser produces a continuous train of 35 fs pulses at a repetition rate of 1 kHz centered at 805 nm with energies up to 7.5 mJ and spectral width of about 25 nm. The laser beam was incident on a quartz cuvette containing 250 µL of virus sample. Different laser powers on the sample were obtained using beam splitters and directing only a portion of the total laser beam to the sample area. The USP laser spot size was approximately 1 cm22. The nanorod sample was placed in a 1 mm quartz cuvette, which was located in a custom cell filled with water for ultrasound coupling. The cuvette was oriented at an angle of 45° relative to the 0.3 W fs laser beam. The photoacoustic signal (ultrasonic wave) was collected using a 2.25 MHz focused water immersion transducer.The acoustic signal generated by laser irradiated nanorods was collected in a homebuilt ultrasound detector. Figure et al. to synthesize gold nanorods23. 460 μL of a freshly prepared solution of 0.01 M sodium borohydride dissolved in 0.01 M sodium hydroxide was rapidly injected into 10 mL of 0.5 mM HAuCl4 solution containing 0.1 M CTAB under extensive stirring. A change of color from greenish to light brown indicated the successful formation of gold nanoparticle seeds. To synthesize gold nanorods with a longitudinal resonance at ~800 nm, 11.5 μL of 0.1 M silver nitrate solution was added to 10 mL of 0.5 mM HAuCl4 solution containing 0.1 M CTAB. Subsequently, 500 μL of 0.1 M aqueous hydroquinone solution was added, and the resulting mixture was hand-stirred until it became clear. Then, 160 μL of gold seed solution was added under thorough stirring. The resulting mix was allowed to age overnight. To obtain PEGylated nanorods, 2.5 µL of 10 mM PEG2 (HS—CH2CH2—(C2H4O)77—N3) were added together with 2.5 µL of 10 mM HS-(CH2)11-(CH2CH2O)6OCH2-COOH to 1 mL of gold nanorods in the presence of 3% v/v of Tween® 20. The samples were incubated overnight and washed by centrifugation and subsequent resuspension. Alkyne-functionalized annexinV was cross-linked to the azide functionalized nanorods in the presence of 500 μM ascorbic acid and 100 μM CuSO4via CuI catalyzed click-reaction24. The nanorods were cleaned again through repeated centrifugation and resuspension in DDI water or buffer.We used the seed-mediated growth technique described by Vigderman 25. For the virus fusion assay described below, HEK293T cells were co-transfected with pLNC-luc, pCL-Eco and S15-BlaM plasmids. To generate the S15-BlaM expression plasmid that expresses a Src-eptiope tagged β-lactamase fusion protein upon transfection, the N-terminal 15 amino acid sequence of c-Src (S15) was cloned in frame upstream of β-lactamase orf in pcDNA3.1/zeo + (Invitrogen) eukaryotic expression plasmid. S15-BlaM is incorporated into MLV particles upon virus particle budding from the plasma membrane of virus-producing cells27. Virus-containing supernatants were harvested 2 days post transfection, and passed through 0.45 µm filters. Virus was concentrated by ultracentrifugation on a 20% sucrose cushion . Virus pellets were resuspended in PBS, aliquoted, and stored at −80 °C until further use.HEK293T and Rat-2 cells were maintained in DMEM supplemented with heat-inactivated 10% fetal bovine serum (Invitrogen) and Pen/Strep (HyClone). Luciferase expressing, single cycle of replication competent MLV particles (MLV/luc) were derived via co-transfection of HEK293T cells with pLNC-luc  and pCL-Eco  plasmids, as previously describedgag monoclonal antibody; Clone 183-H12-5C; NIH AIDS Research and Reference Reagent Program, contributed by Dr. Bruce Chesebro) in RPMI medium was added to the mixture and the mixture was diluted by adding 1x Tris (pH 7) or PBS (pH 7.4) buffer to final volume of 1 ml. The nanorod to virus ratio was more than 10000:1. The concentration of the nanorods was quantified using a UV-vis spectrometer (Cary 5000 spectrophotometer).PEGylated nanorods or nanorods loaded with annexinV were centrifuged at 5000 rpm for 10 min. The supernatant was discarded and 17 µL of the pellet was added to 50 µL of MLV/luc, followed by overnight incubation at 4 °C. Then, 500 µL of IgG antibody (anti-p243) and Manitol were dissolved in 20 mM Tris (pH 7.0). Then Manganese (III) Tetrakis (4-benzoic acid) Porphyrin (MnTBAP) dissolved in 0.1 M NaOH was added. This solution was subsequently added to virus samples containing antibodies from a hybridoma preparation or to mixtures containing virus, PEGylated nanorods, and antibodies. All samples were diluted with Tris buffer. The final scavenger concentrations were 10 mM Manitol, 10 mM Sodium azide and 0.02 mM MnTBAP. The pH of the final solution was 7.A solution containing three different ROS scavengers were used. Sodium azide  was bound to HIV-Ig  coated wells and detected with unexposed control or laser exposed anti-p24gag monoclonal antibody, and HRP-conjugated goat anti-mouse secondary antibody (Sigma). ELISA was developed with a peroxidase substrate  and the standard curves were generated with each of the control (untreated) or fs laser-exposed anti-p24gag monoclonal antibodies.To test the efficacy of the fs laser inactivation protocol on functionality (epitope recognition) of a monoclonal antibody, an anti-p2429 was utilized with some modification. Briefly, untreated or laser-exposed MLV/luc particles containing S15-BlaM fusion protein was used to infect target cells. After incubating 2 hours at 37 °C, cells were washed with CO2-independent media (Invitrogen) and incubated in CCF2 (fluorogenic substrate of β-lactamase)-containing media at 18 °C over night, washed, fixed with 4% PFA. The number of β-lactamase positive cells was determined by FACS analysis using a LSRII flow cytometer (BD).To investigate MLV/luc fusion to Rat2 target cells, a FACS based assayprior to lysis. The lysates were then used for measurement of luciferase activity using a chemiluminescent reporter . The ratio of the intensity of the luminescence to a calibration standard is measured in RLU (relative light units), and is a linear function of the reporter concentration. If RLUc and RLUs are the measured RLU values of the control and sample respectively, the Log-Reduction Value (LRV) defined as LRV = log10(RLUc/RLUs) gives a quantitative measure of the extent of virus infection in the culture.Virus inactivation was determined by infecting cells (mouse fibroblast cell line) with treated (laser-inactivated) or untreated viruses. Cells were then incubated for 48 h In order to test for potential cytotoxicity of nanorods, laser irradiated and non-irradiated nanorods media were harvested and incubated with Rat-2 cells at 37 °C overnight. The Trypan blue exclusion test was then used to determine the percentage of viable cells.Nanorod samples were run on 2% agarose gels made from 0.5x TBE. The same buffer was used as running buffer. The samples were loaded with ficol and were run with a current of 200 mA, voltage of 140 V for 30 min.The variability in the data were analyzed by Anova with subsequent post-hoc test as implemented in Matlab. Differences between samples with a significance level of P < 0.05 are marked with one asterisk (*), and for P < 0.01 with two asterisks (**). NS was used to mark non-significant differences.The datasets generated during and/or analyzed in the current study are available from the corresponding authors upon request.31. All experiments were performed with a recombinant virus that expresses the luciferase reporter gene upon establishment of productive infection in target cells, providing a robust strategy for the quantification of virus infectivity. In a first set of experiments we tested the hypothesis that nanorods whose LSPR overlaps with the incident fs laser pulse enhances the efficacy of light-induced laser activation. To that end, we compared the viral infectivity obtained with virus particles exposed to fs laser irradiation with constant pulse duration but different average powers. Importantly, we kept the exposure time of the individual samples constant at 10 s, which is 3 orders of magnitude shorter than the exposure time in all previous photonic virus inactivation studies.We chose Murine Leukemia Virus (MLV) as target for our viral inactivation studies due to its similarity with endogenous mouse retroviruses that are known contaminants in the biopharmaceutical industry33. We used gold nanorods with an aspect ratio of around 4, with typical lengths of ~60 nm. We found that these nanorods sustain a longitudinal plasmon mode that peaks close to the wavelength of the fs laser emission (805 nm) as is shown in the UV-Vis spectra of the nanorod “control” (no laser exposure) in Fig. 34 using cetrimethylammonium bromide (CTAB) as ligand, which favors growth along the crystallographic [110] axis. Due to the functionalization of the nanorod surface with CTAB, the nanorods have a positive zeta-potential of approximately ζ =  + 23 mV. As CTAB is cytotoxic, we replaced it with polyethyleneglycol (PEG) ligands. In a first set of experiments we tested the stability of the nanorods when irradiated with 805 nm laser pulses with 35 fs duration for 10 s. We varied the average laser power between 0.3 W, 1.2 W and 3 W and recorded UV-Vis spectra after laser irradiation. These spectra and the spectrum of the non-irradiated control sample are summarized in Fig. 32. The peak wavelength of the longitudinal mode intensities and the associated full width at half maximum (FWHM), are plotted in Fig. The LSPRs of gold nanoparticles are strongly morphology dependent, and the aspect ratio of nanorods represents a rational control parameter to tune the plasmon resonance across the visible range of the electromagnetic spectrum to the Near-Infrared (NIR) and beyond36 of the nanorods leads to shorter and wider rods with increased oscillator strength for the transverse plasmon mode and a blue-shifted longitudinal plasmon resonance. Early studies experimentally showed that an average of ~60 femtojoule (fJ) energy, which is close to our experimental condition, is required to melt a single gold nanorod with an aspect ratio ~437. Fig. 38. The different spectral responses account for the broadening of the plasmon band centered at around 800 nm in the nanorod spectra. The observed agglomeration indicates a decomposition of the nanorod surface ligands under pulsed laser irradiation that results in a decrease of colloidal stability.The observed spectral changes can be attributed to two main effects. Surface meltingAlthough the nanorods show some morphological restructuring and agglomeration, we emphasize that both the UV-Vis data and the gel studies confirm that the sample still contain a substantial fraction of resonant nanorods under the chosen irradiation conditions.6 relative light units (RLU). We performed the inactivation studies with PEGylated nanorods as well as with rods functionalized with annexinV that binds to phosphatidylserine (PS) of the enveloped MLV. For the latter, Ca2+ was added to the binding buffer when incubating annexinV-functionalized nanorods with virus particles to achieve a final Ca2+ concentration of 2 mM.In a typical virus inactivation experiment, PEGylated nanorods were incubated with MLV particles in a ratio of more than 10000:1 in a total volume of 67 μL at 4 °C for up to 12 h. The samples were then diluted by adding 933 μL of PBS, pH 7.4 to a total volume of 1 mL. The final virus concentration was such that infections of Rat2 reporter cells with the luciferase encoding recombinant viruses resulted in approximately 1 × 10In many applications it is necessary to inactivate the virus in a complex biological matrix. For instance, virus inactivation plays a critical role in the pharmaceutical industry for the fabrication of monoclonal antibodies (mAbs). We emulated realistic process conditions by performing the virus inactivation in an IgG antibody  containing Tris solution as biological matrix. The proteins were added after overnight co-incubation of nanorod and virus.In a first set of experiments, we tested the hypothesis that resonant plasmonic nanorods enhance fs pulsed laser driven virus inactivation. To that end, virus samples were irradiated for 10 s with 805 nm laser pulses with a width of 35 fs in the presence and absence of pegylated nanorods. All samples contained IgG antibodies. Figure In a second set of experiments, we used the maximum average laser power of 3 W and measured viral inactivation in the presence of three different nanoparticles: virus + PEGylated nanorods; virus + annexinV-functionalized nanorods; virus + spherical nanoparticles. Unlike for the nanorods, the LSPR of the 40 nm diameter gold nanoparticles (530 nm) did not overlap with the exciting laser pulse. The nanorod and nanoparticle to virus ratios were both higher than 10000:1. Additional controls included laser irradiation of viruses without nanoparticles. After laser irradiation for 10 s, virus infectivity was quantified Fig. . The 805We tested for potential collateral damage to IgG antibodies through laser irradiation  of PEGylated and annexinV functionalized nanorods through ELISA Fig. . This tiOne unexpected finding from Fig. The fact that annexinV fails to enhance virus inactivation and the absence of a significant non-specific binding to MLV in the case of the PEGylated nanorods suggests that a direct binding between nanorods and virus particles is not required for the nanorod-enhanced photonic virus inactivation.The LRVs measured in Fig. 11 particles/mL, which corresponds to one rod in a cube of solvent with side length of approximately 2 μm, we can exclude that virus particles and nanorods co-localize close enough to generate a sufficient E-field enhancement to impact ISRS or multiphoton absorption of the virus. For similar reasons, it is unlikely that thermal effects are the underlying cause of the observed virus inactivation. Although the resonant fs laser excitation induces a temperature jump in the nanorod and its immediate environment, this effect is local , superoxide anion (O2−), or hydroxyl radical (•OH)39. Even if their formation is limited to the immediate vicinity of the nanorods, the ROS can diffuse in solution. Singlet oxygen, for instance, has a typical diffusion length in water of approximately 200 nm40. If these reactive species result in the disabling of important surface groups required for binding to and uptake into host cells, they could – in principle – account for the observed MLV inactivation. To test whether these ROS were involved in the virus inactivation, we repeated the fs laser irradiation of a mix of MLV and resonant nanorods in the presence of ROS scavengers. In particular, we used sodium azide (scavenger for 1O2), MnTBAP (scavenger for O2−), mannitol (scavenger for •OH)42. While the concentration of ROS scavengers used in these experiments did not show any cytotoxicity (data not shown), addition of ROS scavengers did not suppress nanorod mediated virus inactivation . It has been demonstrated in previous studies that plasmon excitation in gold nanocubes can generate singlet oxygen (ion Fig. , suggest44. Intense pressure wave generation is expected to dominate only for very short pulse durations as used in this work. We emphasize that although Tsen and coworkers dismissed the pressure wave argument in their studies due to insufficient laser power, the situation is fundamentally different in our case due to the presence of the plasmonic nanoparticles, which we expect to greatly reduce the power threshold for shockwave generation. Based on published analyses of threshold intensities in the presence of nanorods45, we estimate that average powers ≥0.3 W are sufficient to trigger shockwaves under our experimental conditions using pulse durations of 35 fs. We recorded the photoacoustic response of the nanorods, which represent a broad band acoustic emissions followed by a gradual fluctuation that decreased to near zero until another distinct oscillation arrives (Fig. Another potential virus inactivation mechanism that does not require direct contact between the virus and the nanorods is plasmonically-enhanced shockwave generation. The duration of the incident pulse is crucial in determining the feasibility of inactivationves Fig. . AlthougIndependent of the exact physical mechanism that causes the plasmonic enhancement of virus inactivation, it is key to establish which stage of the viral infection is affected by it. Since TEM inspections after laser exposure did not reveal obvious morphological changes in the virus samples, we hypothesized that the effect is primarily caused by molecular-level perturbations of the MLV membrane and/or surface groups, which prevents an effective binding to the host cell and subsequent fusion. To test this hypothesis, we repeated the fs laser irradiation of MLV/luc particles containing S15-BlaM fusion protein in presence and absence of resonant nanorod. Figure We have demonstrated the enhancement of fs-pulsed NIR radiation induced photonic virus inactivation through plasmonic nanoparticles. Irradiation in the presence of resonant nanorods significantly reduced viral fusion with the host cell, suggesting that the plasmonic enhancement acts on viral surface functionalities responsible for early stages of the viral infection rather than on the viral genome or enzymes. LRVs of ≥3.7 were achieved with MLV in as little as 10 s of irradiation. The inactivation was highly specific to the virus while co-incubated IgG antibodies did not show any loss in functionality. The ability to enhance the efficacy of selective photonic virus inactivation with NIR radiation through plasmonic nanoparticles paves the path to a broader applicability of the technique in research and technology.Supplementary Information


This study estimated the direct medical costs of osteoporotic fractures from a large claim database in Japan. We further identified several comorbidities which drove the treatment costs. The results would contribute to health economic analysis as well as understanding of individual financial burden in Japan.The purposes of this study were to estimate treatment costs of osteoporotic fractures and to investigate the cost drivers.Male and female patients aged 50 years and older with a hip, vertebral, or non-hip/non-vert (NHNV) fracture between April 2008 and December 2016 were analyzed from claim database. Two types of costs were estimated. The incremental yearly costs of fractures and comorbidity treatments  were calculated by subtracting pre-fracture costs from post-fracture costs. The costs exclusive for fracture treatments (fracture treatment costs) were estimated by summing up the costs of fracture treatments within 1 year after fracture. The associations between comorbidities and costs were examined with a generalized linear model.Total 12,898 patients were identified . The total medical costs of fractures were $14,592 for male-hip, $15,691 for female-hip, $4268 for male-vertebral, $3819 for female-vertebral, $3790 for male-NHNV, and $4259 for female-NHNV. The fracture treatment costs were $4506 for male-hip, $5427 for female-hip, $1022 for male-vertebral, $1044 for female-vertebral, $1035 for male-NHNV, and $1408 for female NHNV. Three comorbidities were associated with increasing fracture treatment costs whereas four comorbidities were associated with decreasing fracture treatment costs. Five comorbidities were associated with increasing total medical costs whereas one comorbidity was associated with decreasing total medical costs.Yearly treatment costs were increased considerably after fracture. Several comorbidities were considered to be cost drivers for osteoporotic fracture treatment. The cost estimates with different patient profile would support conducting health economic analysis in the future. Osteoporotic fractures often have serious health impacts. For example, in a previous research, those with osteoporotic fractures had a higher mortality rate and decreased individual quality of life , 2. It iUnder this situation, the health economic analysis in osteoporosis and osteoporotic fracture is featured more than ever. In recent years, new drugs, such as teriparatide and denosumab, have been launched in Japan with higher drug prices than conventional bisphosphonates. In line with the increasing varieties of osteoporosis drugs, attention by researchers has been paid to cost-effectiveness of these drugs. Ding et al. 2008) analyzed the cost-effectiveness of risedronate sodium compared with no-treatment . Moriwak analyzedTherefore, the primary objective of this study is to estimate the current yearly treatment costs due to osteoporotic fractures as well as treatment costs of osteoporosis patients in Japan using large-sized RWD. It will not only help estimate the financial burden of osteoporotic fracture but also help conduct various health economic analyses. The secondary objective of this study is to identify cost drivers among osteoporotic fracture patients. We defined cost drivers as the factors associated with increased or decreased treatment costs of osteoporotic fracture hereafter and explored them via the retrospective database analysis.In this retrospective observational study, the medical claim database provided by Medical Data Vision Co. Ltd.  was used. MDV accumulated anonymous patient-level medical claim data from more than 17.23 million unique patients in more than 240 acute-care hospitals, which is almost one-eighth of the total Japanese population .The MDV database includes patient demographics (age and gender), resource use, and diagnosis history including the date when it was first diagnosed at enrolled hospitals. The resource use history covers amounts used and prices for each resource . However, it does not contain socio-economic status (SES) records, patient behavioral records such as smoking history for outpatients, and treatment outcome records  in outpatient service.Patients aged 50 years and older with diagnosis of osteoporotic fracture, dating from April 2008 to January 2017, were identified from the database using International Classification of Diseases (10th revision) (ICD-10). Patients with osteoporotic fractures were defined as those with M80  or those with both M81 (osteoporosis without fracture) and ICD-10 code for fractures which can be assumed as osteoporotic fractures. These fractures were decided based on the guidelines and expert opinion, and categorized into three groups: hip, vertebral, and non-hip and non-vertebral (NHNV) fractures . The lisThe index date was defined as the date when the fracture was first diagnosed since they were enrolled in the database. If a patient develops a fracture of different ICD-10 code from the first one within 1 year, the fracture was defined as the second fracture. Patients who had a record on January 2017 were excluded as they may still continue their treatments thereafter. As a result, we used the patient data from April 2008 to December 2016. In addition, patients whose initial records or final records in the database were within 1 year from the index fracture were excluded to avoid including those who changed hospitals or died within 1 year.Two types of treatment costs were estimated in this study. One is the cost incurred by any medical resource use due to fracture development, and this was named as “total medical cost.” The other is the cost incurred by the medical resource use for osteoporosis and fracture treatments, and this was named as “fracture treatment cost.” Medical costs in Japan can be categorized into four: doctor consultation costs, drug costs, non-drug treatment costs (including diagnosis), and inpatient specific costs (such as room charges). The “total medical costs” includes all of these whereas the “fracture treatment cost” includes drugs and non-drug treatment costs related to fracture and general orthopedic treatments. The total medical cost was estimated by subtracting the treatment costs within 1 year before index fracture from the treatment costs within 1 year after index fracture. The fracture treatment cost was estimated by aggregating the costs for the specific medical resources used for fracture treatments within 1 year after fracture development. These resources were defined by using two reference books which describe the resources by disease indication , 12. TheCosts were calculated by multiplying the amount used by the unit price for each resource use record from public health care payer’s perspective. The prices used in this study were adjusted to the latest unit prices issued in 2017 by the government, which are applied to every hospital/clinics in Japan. All the costs were transformed into USD (assuming 1 USD = 110 JPY).t test was used for the total medical costs and Wilcoxon ranked sum test was used for the fracture treatment costs, as it was expected that the fracture treatment costs were skewed compared with the total medical costs.Both the total medical costs and the fracture treatment costs were estimated at patient level and reported as means with 95% confidence interval (CI). Before reporting the descriptive statistics, data with extreme values in the total medical costs were excluded as outliers. The data were stratified by gender and fracture category, then a box-and-whisker plot was made for each subgroup. The outliers were defined as those outside the whisker. When comparing the costs of those patients with the second fracture to that of single fracture patients, Comorbidity profiles of the patients were also gathered to use as explanatory variables. Both highly frequent comorbidities and diseases associated with osteoporosis risk were included. To extract highly frequent comorbidities, prevalence rates of comorbidity within 3 months before and after index fracture were calculated for each of three cost groups . The following two criterion were used to determine the comorbidities included in the later analysis: (1) If prevalence rates were more than 30% for all the three groups or (2) a prevalence rate in the group A was more than 20%. Medical conditions with M45–49 in ICD-10 code (spondylopathies) were excluded from the list, as it was assumed to be highly correlated with vertebral fractures. To extract the comorbidities associated with osteoporosis risk, those described as major risk factors in the guidelines for prevention and treatment of osteoporosis were also included.To identify the cost drivers for fracture treatment costs based on patient profiles , a generalized linear model (GLM) with gamma family with log link function was applied for patients with single fracture. The increased or decreased association was further examined by conducting a subgroup analysis of the patients with one type of fracture . In this subgroup analysis, the driving factors for the total medical costs were also tested by a GLM with Gaussian family and identity link function. In this regression analysis with the total medical cost, the associations between patient profiles  and change in the total medical costs were examined. Drivers for the treatment costs of NHNV fracture were not examined because NHNV fractures consist of fractures at different body parts , and its result would be difficult to interpret as we would not be able to identify which NHNV fracture was driven by a particular factor. All statistical analyses were performed using R version 3.3.3 .From April 2008 to December 2016, a total of 14,001 patients were identified as patients with a single osteoporotic fracture for the analysis. Of those patients, the fracture treatment costs were incurred among 12,898 patients. These 12,898 patients were included for the later analysis because the remaining 1103 patients were assumed to be those who did not receive fracture treatments at the enrolled hospitals but may have received at different hospitals. While there was little difference in gender proportion, age group, and fracture type between these two datasets, total costs were generally higher among dataset of those 12,898 patients. Their characteristics are shown in Table Only female patients with the second vertebral fractures were reported in this study, since male patients with the second fractures and female patients with the second fractures that were not vertebral fractures were very few. For this reason, in order to analyze the impact of the second fractures on individual treatment costs, 100 female patients with the second vertebral fractures alone were included in the comparative analysis of fracture costs between patients with single fracture and those with the second fracture (in the next section). Their mean age was 78.9, which was slightly higher than the mean age of female patients with single vertebral fracture (77.6 [not reported in the table]).Treatment costs incurred were stratified by gender and fracture site as shown in Table Similar result was observed in the fracture treatment costs. The significantly higher costs were observed in hip  and no large difference was observed between the vertebral and NHNV fracture patients’ costs. Among hip fracture patients, the fracture treatment costs were higher in female.p < 0.05. For the fracture treatment costs, mean was $2458 in the second vertebral fracture and $1044 in the single vertebral fracture; p < 0.01).When comparing the costs of the second vertebral fracture female patients with the costs of the single vertebral fracture female patients, the second fracture costs were significantly higher in both the total medical costs and the fracture treatment costs  were included. COPD was most prevalent among those 6 medical conditions but less common compared to the 12 medical conditions. Prevalence rates of 4 out of the 6 medical conditions  were very low among studied patients (less than 2%).The result of regression analysis seeking drivers for the fracture treatment cost is presented in Table Among comorbidities we identified, associations with the increased fracture treatment costs were suggested for hypertension and iron deficiency anemia (hypertension 1.13 [95% CI 1.06 to 1.20] and iron deficiency anemia 1.15 [95% CI 1.07 to 1.25]). On the contrary, associations with the decreased fracture treatment costs were observed for dorsalgia (0.90 [95% CI 0.85 to 0.96]), disorder of refraction and accommodation (0.86 [95% CI 0.80 to 0.93]), and COPD (0.91 [95% CI 0.84 to 1.00]).An association was further investigated after limiting the type of patients to hip fracture and vertebral fracture patients Tables . With reDrivers for the total medical costs were also explored in the hip and vertebral fracture patient groups Table . It was In terms of comorbidities, associations between the increased total medical costs and three medical conditions  were observed as well . In contrast to the previous analysis, an association between dorsalgia and the decreased total medical costs was observed only among the vertebral fracture patient group (−$426 [95% CI: −$724 to −$127]). In this analysis, associations between two new comorbidities  and the increased total medical costs were observed .This study estimated the total medical costs and the fracture treatment costs in osteoporotic patients in Japan. We also explored the common comorbidities among osteoporotic fracture patients, and examined the potential cost drivers for the treatment costs.Although a few studies have reported the treatment costs incurred for osteoporotic fracture in Japan, the number of patients was limited and the hospitals from which the patients were recruited was underrepresented. Our cost estimation was based on the latest claim data of 12,898 patients including both inpatients and outpatients, thus considered to be adequately representing Japanese population despite limitation of the database itself. Kondo et al. (2009) investigated the treatment costs of 41 hip fracture patients in three hospitals in Japan . They reThe treatment costs borne by the patients with the second fracture were significantly higher compared with single fracture patient, which is consistent with previous studies in USA and UK –18. In tThe results of cost estimation indicate that total medical costs would be preferable in economic evaluation because the fracture treatment costs were almost only one third of total medical costs. Other two third would consist from non-fracture treatment costs such as doctor consultation costs, comorbidity treatment costs, and inpatient specific costs. The amount of these costs are not negligible, and hence they should be considered when evaluating the economic value of fracture treatment. To our best knowledge, there has been no study in Japan comparing the total medical costs and the fracture treatment costs, and the results would support the official guideline for the economic evaluation of drugs/medical devices recommending the inclusion of related comorbidity costs for cost parameters .Interestingly, it was found that more fracture treatment costs were incurred among female hip fracture patients whereas less total medical cost was incurred female vertebral fracture patients, suggesting that more non-fracture (comorbidity) treatment costs were incurred among male than female. Male could be more likely to worsen their comorbidities. In fact, one study reported higher relative hazards of all-cause mortality after hip fracture in male during 0–1 year age interval . Male maAssociations between comorbidities and treatment costs were explored, and nine comorbidities were detected as cost drivers. The associations with three medical conditions  were consistent with previous research. Firstly, in our study, it was found that hypertension was associated with increased fracture treatment costs in hip fracture subgroups. This positive association was persistent when investigated in the total population. The association between hypertension and bone remodeling has been suggested , and oneSecondly, the result showed a strong positive association between iron deficiency anemia and treatment costs . It was reported that several potential interactions between iron deficiency and bone loss  . Thus, iLastly, it is interesting to find that diabetes had an association with the decreased fracture treatment costs among hip fracture patients. One possible explanation for this negative association is the influence of some antidiabetic drugs. Although it has not been concluded yet, drugs such as insulin and metformin have had positive effects such as improved bone bridging or osteogenic effect in some experimental studies . TherefoUnpredicted positive associations were also found with three medical conditions . Sleep disorders were positively associated with both the fracture treatment costs among hip fracture patients and the total medical costs among hip and vertebral fracture patients. Sleep disorders are common claims among the elderly population. In a previous study, it had potential associations not only with increasing risks of falls but also with osteoporosis , suggestLastly, other functional intestinal diseases had a positive association only with the total medical costs. Fracture patients would increase the risk of other functional intestinal disorder because of the physical inactivity during the hospitalization period. Vertebral compression fracture itself can cause constipation as complication . Thus, iOur study also showed unexpected negative associations between treatment costs and the other three medical conditions . Although the causal association between these medical conditions and treatment costs needs to be carefully interpreted with further research, potential causal pathways are hypothesized for each comorbidity as follows. The first one is dorsalgia. It had a negative association with the fracture treatment costs in hip fracture patients and the total medical costs in vertebral fracture patients, and the association between dorsalgia and fracture treatment costs remained in total population. The guidelines for prevention and treatment and osteoporosis suggest that dorsalgia is one of the factors for diagnosing osteoporosis . GeneralThe following limitations were recognized in this study. Firstly, a selection bias due to the nature of the database was possibly considered. The claim data used in this study were collected from acute care hospitals, allowing us to collect severer patients in Japan. On the other hand, these acute care hospitals may spend less treatment costs due to DRG-like payment system in Japan (DPC). In fact, it was shown that less treatment costs were incurred in DPC hospitals , which cIn conclusion, our study reported the treatment costs incurred to osteoporotic fracture patients in Japan and examined the cost drivers by analyzing the associations with comorbidities. To our knowledge, this is the first pragmatic study estimating the treatment costs of osteoporotic fracture patients through the analysis of large-sized RWD, and it showed financial burden of osteoporotic fracture in Japan including patients with the second fracture. This study also detected the novel associations between some comorbidities and the increase or decrease in treatment costs. The findings from this study would help clinicians, researchers, and policy makers understand the potential financial burden of osteoporotic fracture and would also support various health economic analyses in the future.


Science by Zhang et al. of Nanyang Technological University, Singapore. They resolved a 1.58 Å resolution structure of the NS2B-NS3 protease of ZIKV and demonstrated how peptide and non-peptide inhibitors interact with this structure, along with the different conformational states that were observed. This protease crystal structure offers new opportunities for the design and development of novel antiviral drugs used for the treatment and control of ZIKV.Zika virus (ZIKV) is the cause of a significant viral disease affecting humans, which has spread throughout many South American countries and has also become a threat to Southeastern Asia. This commentary discusses the article “Crystal structure of unlinked NS2B-NS3 protease from Zika virus” published recently in the journal  Aedes aegypti (Ae. aegypti) mosquito species serves as the principal vector for the virus. ZIKV is a member of the Flaviviridae family, which includes other highly pathogenic viruses such as dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus  imidazole-1-yl) methanol). Although the EN300 did not directly bind with NS2B, it made several interactions of pi-stacking and hydrogen bonding in one of the pockets of the NS3 part of the protein, and there were no major conformational changes in the protein.The structural dynamics of the NS2B-NS3 protease in solution form were also observed through nuclear magnetic resonance (NMR) spectroscopy, which showed a properly folded form of the protein. The different conformational states of the protease enzyme were explored by titrating it with a bipeptide of acetyl lysine-arginine (AcKR) . The AcKn 100 μM . The 1H- general . The 15Nprotease  when theThese results show that the ZIKV protease can be further explored for other active binding pockets, which will be targets for inhibitor molecules. Although Zhang et al. focused on the need to design peptide-based drugs , every option of drug design from small organic molecules to large peptide-based inhibitors should be evaluated for inhibitory interactions with such an influential protease enzyme . Secondl


Previous methods for predicting protein-protein interactions (PPIs) were mainly focused on PPIs within a single species, but PPIs across different species have recently emerged as an important issue in some areas such as viral infection. The primary focus of this study is to predict PPIs between virus and its targeted host, which are involved in viral infection. We developed a general method that predicts interactions between virus and host proteins using the repeat patterns and composition of amino acids. In independent testing of the method with PPIs of new viruses and hosts, it showed a high performance comparable to the best performance of other methods for single virus-host PPIs. In comparison of our method with others using same datasets, our method outperformed the others. The repeat patterns and composition of amino acids are simple, yet powerful features for predicting virus-host PPIs. The method developed in this study will help in finding new virus-host PPIs for which little information is available. Viral infection involves a large number of protein-protein interactions (PPIs) between virus and its targeted host. These interactions range from the initial binding of viral coat proteins to host membrane receptor to hijack the host transcription machinery by virus proteins. Various viral diseases are caused by infection with pathogenic viruses. For instance, Ebola virus disease is a highly contagious and fatal disease caused by infection with Ebola virus. During the 2014 Ebola epidemic, the world witnessed over 28,000 cases and over 11,000 deaths [H. sapiens and M. tuberculosis H37Rv. Support vector machine (SVM) models developed by Cui et al. [So far, many computational methods have been developed to predict PPIs. However, most of these methods predict PPIs within a single species and cannot be used to predict PPIs between different species because they do not distinguish interactions between proteins of the same species from those of different species. Recently, a few computational methods have been developed to predict virus-host PPIs using machine learning methods. For instance, a homology-based method  predictsi et al.  and Kim i et al.  predictei et al. –8 are ali et al. .In this paper, we present a new method for predicting virus-host PPIs, which is applicable to new viruses or hosts using amino acid repeat patterns and composition. Proteins in a variety of species contain significant amino acid repeats, with more abundance of repeats in eukaryotic proteins than in prokaryotic proteins , 11. It   F1: sum of squared length of single amino acid repeats (SARs) in the entire protein sequence  F2: maximum of the sum of squared length of SARs in a window of 6 residues  F3: composition of amino acids in 5 partitions of the protein sequenceProteins are of different lengths and have different amino acid compositions. Many features of proteins have been used to predict PPIs from protein sequences. In this study, we represent a virus-host PPI by three features :F1, which is the sum of squared length of SARs in the protein sequence, is defined by .We constructed several datasets of virus-host PPIs to examine the applicability of the prediction method to new viruses and hosts. The first training dataset consists of PPIs of human with positive-sense single-stranded RNA (+ssRNA) viruses except hepatitis C virus (HCV) and severe acute respiratory syndrome (SARS) virus. The SVM model trained with the training dataset was tested on PPIs of five new viruses: HCV, SARS virus, influenza A (H1N1) virus, human papillomavirus (HPV-16), and human immunodeficiency virus HIV-1. Both HCV and SARS are positive-sense single-stranded RNA (+ssRNA) viruses, but the remaining three viruses are of different type. H1N1 virus is a negative-sense single-stranded RNA (−ssRNA) virus, HPV-16 is a double-stranded DNA (dsDNA) virus, and HIV-1 is a retrovirus.Mus musculus, Bos taurus, Rattus norvegicus, Sus scrofa, and Escherichia coli K-12.The second training dataset is composed of PPIs between human and +ssRNA viruses, including HCV and SARS virus. The SVM model trained on the second training dataset was tested on PPIs of new hosts: Data of virus-host PPIs were collected from IntAct  and ViruThe training and test datasets constructed in our study can be summarized as follows.  TR1: 638 PPIs between 499 human proteins and 25 +ssRNA virus proteins  TS1: 515 PPIs between 423 human proteins and 11 HCV proteins  TS2: 30 PPIs between 27 human proteins and 12 SARS virus proteins  TS3: 377 PPIs between 307 human proteins and 10 H1N1 virus proteins  TS4: 319 PPIs between 298 human proteins and 11 HPV-16 proteins  TS5: 1,578 PPIs between 1,056 human proteins and 46 HIV-1 proteins1. Training (TR) and Test (TS) Datasets for Assessing the Applicability of the Prediction Model to New Viruses  TR2: 689 PPIs between 522 human proteins and 35 +ssRNA virus proteinsMus musculus proteins and 116 virus proteins  TS6: 191 PPIs between 141 Bos taurus proteins and 34 virus proteins  TS7: 125 PPIs between 87 Rattus norvegicus proteins and 24 virus proteins  TS8: 86 PPIs between 79 Sus scrofa proteins and 10 virus proteins  TS9: 57 PPIs between 38 Escherichia coli K-12 proteins and 27 virus proteins  TS10: 78 PPIs between 64 2. Training (TR) and Test (TS) Datasets for Assessing the Applicability of the Prediction Model to New HostsMachine learning-based approaches to PPI prediction require both positive and negative PPI data, but negative data are not available in databases. Constructing a negative dataset of PPIs is not straightforward because there is no experimentally verified noninteracting pair . Eid et γ were obtained by running the grid search of LIBSVM on training datasets. Unless specified otherwise, the results shown in this paper were obtained with C = 2 and γ = 0.5. The SVM models take a pair of virus and host protein sequences as input. As output, the SVM models classify whether or not the virus protein interacts with the host protein. The SVM models and supporting data are available at http://www.bclab.inha.ac.kr/VHPPI.We built several support vector machine (SVM) models using LIBSVM  to evaluThe performance of the SVM models was evaluated by several measures: sensitivity (Sn), specificity (Sp), accuracy (Acc), positive predictive value (PPV), negative predictive value (NPV), and Matthews correlation coefficient (MCC), which are defined by the following equations:In –8), tru, tru8), We performed 10-fold cross validation of the SVM model with several datasets which contain different ratios  of positive to negative PPIs between +ssRNA viruses and human. As shown in We also examined the contribution of the features to the prediction performance of the SVM model. For features F1 and F2, we tried both single amino acid repeats (SARs) and double amino acid repeats (DARs) along with different partitions of a protein sequence. As shown in For feature F3, we tried several different partitions of a protein sequence in several datasets. As discussed earlier, we trained the SVM model with the training dataset TR1 consisting of PPIs of human with +ssRNA viruses except hepatitis C virus (HCV) and SARS virus and tested it on PPIs of new viruses which were not used in training the model. The test datasets include PPIs of five viruses  with human. H1N1 virus is a negative-sense single-stranded RNA (-ssRNA) virus, and HPV-16 is a double-stranded RNA (dsDNA) virus. HIV-1 is a retrovirus, which is a +ssRNA virus with a DNA intermediate.In addition to species difference, we also analyzed the sequence similarity between the training datasets and test datasets using EMBOSS Needle tool  to assesMus musculus, Bos taurus, Rattus norvegicus, Sus scrofa, and Escherichia coli K-12) with the viruses. As shown earlier in In order to examine the applicability of the SVM model to new hosts, we tested it on PPIs of viruses with new hosts, which were not used in training the model. As described earlier, the model trained with PPIs of human with +ssRNA viruses was tested on PPIs of five new hosts . As shown in Amino acid repeats are prevalent in a variety of proteins but are rarely used in predicting PPIs. We developed a new method that predicts potential interactions between virus and host proteins using global and local compositions of amino acids as well as amino acid repeat patterns.We tested the prediction model on independent datasets of virus-host PPIs, which were not used in training the model and have a very low sequence similarity to any protein in training datasets of the model. Despite a low sequence similarity between proteins in training datasets and target proteins in test datasets, the prediction model showed a high performance comparable to the best performance of other methods for single virus-host PPIs. In comparison of our method with others using same datasets, our method outperformed the others. Experimental results demonstrate that the repeat patterns and composition of amino acids are simple, yet powerful features for predicting virus-host PPIs. The method can be used to find potential PPIs of new viruses or hosts, for which little information is known.


Now a day׳s SSRs occupy the dominant role in different areas of bio-informatics like new virus identification, DNA finger printing, paternity & maternity identification, disease identification, future disease expectations and possibilities etc., Due to their wide applications in various fields and their significance, SSRs have been the area of interest for many researchers. In the SSRs extraction, retrieval algorithms are used; if retrieval algorithms quality is improved then automatically SSRs extraction system will achieve the most relevant results. For this retrieval purpose in this paper a new retrieval mechanism is proposed which will extracted the MONO, DI and TRI patterns. To extract the MONO, DI and TRI patterns using proposed retrieval mechanism in this paper, DNA sequence of 1403 virus genome data sets are considered and different MONO, DI and TRI patterns are searched in the data genome sequence file. The proposed Next Generation Sequencing (NGS) retrieval mechanism extracted the MONO, DI and TRI patterns without missing anything. It is observed that the retrieval mechanism reduces the unnecessary comparisons. Finally the extracted SSRs provide the useful, single view and useful resource to researchers. Specifications TableValue of the data•Data sets obtained from genomes of VIRUSES with NGS retrieval process have shown the high specificity.•These data suggest that SSR extraction is an useful method for providing information for various applications related to studies in VIRUSES.•Access to the raw sequencing data in VIRUSES allows researchers to perform further bio-informatics analysis based on their own computational algorithms.1Database has been developed using MySQL. The information stored in the database includes virus names, genome id, A,C,G,T percentages, tract length, category, motif types , the sequences of the motifs and frequencies of occurrence in the entire genome. The actual process of database is shown in 1.11.virus_category2.virus_acgt_count3.virus_ssrsIn this paper, we consider three tables from database and changed the structure to our own format so that additional analysis can be done easily. They are1.1.1This table has the information related to virus categories from virus files. The structure is as shown in the 1.1.2This table has the information related to virus A,C,G and T count, its percentage, tract length. The structure is as shown in the 1.1.3This table has the information related to virus_name, genome_id, motif, frequency and its position. The structure is as shown in the 1.2In this section we give detailed description of the 1403 virus genomes1.2.1We used a total of 1403 virus genome sequences. We categorized these genomes as shown in the 1.2.2We extracted the overall frequency, MONO, DI and TRI frequencies from the virus_ssrs those are shown in 1.2.3In this section, we described SSRs by executing SQL queries on virus_category for category wise counts and the results are shown in the 1.2.4We did a preliminary study on the genome sizes of all viruses as shown in the 1.2.5We extract the total of 4,692,149 continues MONO, DI and TRI SSRs are extracted from 1403 genomes. 1.2.6We extract a total of 12853740 continues DI SSRs are extracted from 1403 genomes. 1.2.7We extract a total of 14469215 continues TRI SSRs are extracted from 1403 genomes. 22.1Availability of next-generation sequencing techniques leads to the accessibility of genome sequences including that of organelles like virus, fungi, bacteria etc. Studying the hyper-mutating SSRs This approach scans the input virus genome sequence file and pattern files for MONO, DI and TRI patterns to find all occurrences of these patterns within this file using next generation retrieval mechanisms ngs_search, check_for_tandem_repeat and ngs_database_insertion segments are called for entire length of input file. In search segment, the pattern is searched in the input file, if match occurs then increments the occurrence count. In tandem repeat checking segment, the different between the occurrence positions are measured, if they are equal to length of the pattern then it is considered one tandem repeat. In database insertion segment, virus name, genome id, pattern, count and position is stored in the database.SSR NGS retrieval algorithm has shown the detailed explanation about the Next Generation Sequencing(NGS) retrieval algorithm. It consists of five segments called I/O, Main, search, tandem repeat checking and database insertion. In input segment virus and pattern files are considered as input. In output segment, the extracted mechanism provides the number of occurrences, positions of MONO, DI and TRI patterns. In Main segment the length of file and pattern are read, for each pattern,