Jatropha curcus is a good candidate plant for biodiesel production in tropical and subtropical regions. However, J. curcus is susceptible to the geminivirus Indian cassava mosaic virus (ICMV), and frequent viral disease outbreaks severely limit productivity. Therefore the development of J. curcus to carry on durable virus resistance remains crucial and poses a major biotechnological challenge.J. curcus plants expressing a hairpin, double-stranded (ds) RNA with sequences homologous to five key genes of ICMV-Dha strain DNA-A, which silences sequence-related viral genes thereby conferring ICMV resistance. Two rounds of virus inoculation were conducted via vacuum infiltration of ICMV-Dha. The durability and heritability of resistance conferred by the dsRNA was further tested to ascertain that T1 progeny transgenic plants were resistant to the ICMV-SG strain, which shared 94.5% nucleotides identity with the ICMV-Dha strain. Quantitative PCR analysis showed that resistant transgenic lines had no detectable virus.We generated transgenic J. curcus plants to include a resistance to prevailing geminiviruses in Asia. These virus-resistant transgenic J. curcus plants can be used in various Jatropha breeding programs.In this study we developed transgenic The online version of this article (doi:10.1186/s13068-014-0149-z) contains supplementary material, which is available to authorized users. Jatropha curcas, a small woody plant belonging to the Euphorbiaceae family, is a non-food oil seed crop mainly grown in the tropical and subtropical regions. This plant possesses several traits render this woody plant suitable for biodiesel feedstock production. It is easy to propagate and grows rapidly. It has a short gestation period, low seed cost and high oil content. Moreover, the ability of J. curcus to thrive on degraded soil and its wide adaptability to different growth conditions allows the use of marginal or non-arable wasteland for the application of this plant on an industrial scale. However, the productivity of J. curcus in the field is limited by the occurrence of Jatropha curcas mosaic disease (JcMD) [e (JcMD) -3. The de (JcMD)  and up te (JcMD) .Jatropha plants in Africa and South Asia [Jatropha plantations prompted us to investigate the biology of the virus in more detail.We have previously reported the first full-length genome sequence of one geminivirus, a strain of Indian cassava mosaic virus (ICMV-Dha), as the causative pathogen of JcMD found in Southern India . Followiuth Asia -6. Recenuth Asia . The recGeminiviridae can be classified into four genera: Begomovirus, Mastrevirus, Curtovirus and Topocuvirus. So far, all five Jatropha viral pathogens belong to one genera: Begomovirus. Most of these viruses contain two genomic components termed DNA-A and DNA-B (approximately 2.7 to 3.0 kb) and they are all exclusively transmitted via the whitefly, Bemisia tabaci. The virus DNA-A-positive strand encodes the coat protein (CP/AV1) involved in the encapsidation of viral DNA, virus movement and viral transmission by B. tabaci). Among other encoded proteins, the replication associated-protein (Rep) AC1 is absolutely required for the replication of both genomic components. The transcriptional activator protein (TrAP) AC2 is needed for transcriptional activation of viral gene transcription and plant host gene expression. The replication enhancer protein (Ren) AC3 greatly enhances viral DNA accumulation by interacting with Rep/AC1. Another viral protein, AC4, acts as a gene-silencing suppressor to compromise the host defense system. All these five genes are essential for the virus life cycle and pathogenesis [Geminiviruses, which are single-stranded DNA viruses infecting a range of economically important crop species  in tropical and subtropical regions, have become a major threat to world agriculture in the past decade . Based oogenesis .Jatropha germplasms. This strategy has the advantage that segregation patterns can be clearly observed between resistant and susceptible lines [J. curcus. Recently, we and other groups have established transformation platforms which facilitate the transfer of foreign genes into the J. curcus genome [J. curcus.Because of the capacity of geminiviruses to evolve rapidly by mutation, recombination and pseudo-recombination, the development of plants with durable virus resistance continues to be a major challenge. One strategy involves genetic crossing of resistant and susceptible le lines . Howeverle lines . Therefos genome -13, and J. curcus lines by expressing a hairpin double-stranded (ds) RNA targeting five key geminivirus DNA-A genes. Some of the transgenic lines displayed broad resistance to related geminiviruses, with 94.5% nucleotide identity in the transgene sequences.A major strategy to produce transgenic plants with virus resistance is based on the concept of pathogen-derived resistance (PDR) in which the transgene is derived from viral sequences. The mechanism of PDR includes protein-mediated resistance and RNA interference (RNAi). Both mechanisms have been shown to confer geminivirus resistance in transgenic tomato, common bean, cassava and others -18. HereAC5 gene, fragment 2 (250 bp) targets genes for TrAP/AC2 and Ren/AC3 and fragment 3 (609 bp) targets genes for Rep/AC1 and AC4  gene  35S promoter with double enhancers and named it pX9-hpICMV RNAi. Upon chemical induction with 17β-estradiol, promoter . The PCRAgrobacterium and a total of 133 T0 transgenic plants were generated. We performed PCR analysis on genomic DNA using two pairs of primers  value difference between symptomless resistant transgenic plants and uninfected control plants. In total, 15 independent transgenic lines presented with the symptomless phenotype and no virus could be detected in these resistant transgenic plants. However, we detected very high virus titers in susceptible transgenic plants, which were similar to those found in infected WT plants Figure . Six monNext, we performed Southern blotting to examine the transgene copy number in resistant lines. As there are two EcoRV sites around the two loxP sites, the genomic DNAs were digested with EcoRV Figure A. SoutheJ. curcus plants with between three and four true leaves were inoculated with ICMV-Dha. Two months after the virus challenge, WT plants and transgenic line 82–3 showed very obvious virus disease symptoms, whereas transgenic plants 82–1 and 82–2 grew normally (like uninfected control plants) -PCR sequencing) showed a consistent virus resistance trait Figure C. T1 see) Figure A. qPCR a) Figure B. To ana) Figure C. Southe) Figure D. OveralJatropha tree , which shares 94.5% nt homology with the ICMV-Dha strain [We also tested whether the ICMV resistance conferred by hpRNAi has broad virus resistance. We recently reported a second ICMV isolate from the Singaporean a strain . Before J. curcus.We reasoned these ICMV-resistant plants should be also resistant to multiple ICMV strains within a similar evolution clade. Multiple sequence alignment of full-length DNA-A was carried out using the ClustalV program Vector NTI®  with default parameters. Phylogenetic trees were constructed from multiple alignments using the neighbor-joining method in the MEGA4 program , and a bAC1 gene , the coat protein gene and AC4 gene have been shown to confer geminivirus resistance in tomato, bean and tobacco plants [Chickpea chlorotic dwarf Pakistan virus (CpCDPKV), a member of Mastrevirus  a multi-target approach was adopted. The RNAi trigger sequences include those encoding the N-terminus of the Rep gene, the large intergenic  and the N-terminus of the movement protein gene [Phaseolus vulgaris) showed that the begomovirus Bean golden mosaic virus (BGMV) can also be suppressed by the expression of a hpRNA transgene derived from AC1 [During the last decade geminiviruses have emerged as one of the major causative pathogens of economically important crops such as cassava, cotton and tomato. As resistance genes are not always available in the relevant crop germplasm, biotechnological approaches have been used to generate transgenic plants to confer virus resistance. Several different strategies have been reported to confer virus resistance to transgenic model plants by expression of: (1) trunked viral proteins, (2) artificial zinc finger nuclease, (3) peptide aptamers capable of binding to viral proteins, (4) non-pathogen-derived antiviral agents and so on ,22-25. Ho plants -16,30-32from AC1 .J. curcus plants with high resistance to multiple virus infection. After two rounds of virus challenge, resistant plants were free of virus symptoms and no virus could be detected , thereby allowing us to ascertain resistance to related viruses. There is 95.3% nt identity between the ICMV-Dha and ICMV-SG strains if we include sequences that we used for the hpRNAi construction. Two other n Figure . In viewn Figure  and 7. LJ. curcus plants with resistance to ICMV via expression of a hairpin dsRNA with sequences homologous to five key ICMV DNA-A genes. Virus resistance was confirmed through two rounds of inoculation with J. curcus-isolated strains of ICMV, ICMV-Dha and ICMV-SG. The T1 progeny transgenic plants were virus resistant, confirming that the resistance phenotype was heritable. Transgenic plants generated from this study can be used in various Jatropha breeding programs.We generated transgenic F1-5: 5’-AGCGCCTCGAGCGTTTGAATCTAGACACGATGTGCTC-3’,F1-3: 5’-TGCAGTGATGAGTTCCCCTGTGCGTGAACATGTTAAACACCTCACCGAAATCCT-3’,F2-5: 5’-GATTTCGGTGAGGTGTTTAACATGTTCACGCACAGGGGAACTCATCACTGCAGC-3’,F2-3: 5’-GGAATGTTCCCCATTCCAAGGTATCGAATACCCTCAAGAAACGCCAGGTCTGAG-3’,F3-5: 5’-CAGACCTGGCGTTTCTTGAGGGTATTCGATACCTTGGAATGGGGAACATTCCAG-3’,F3-3: 5’-AGCGCAAGCTTTAGCTGGAATTGGGCCCTGGATTGCAGA-3’.We replaced the G10-90 promoter in pX7-GFP  with a sJ. curcus .The PCR fragment was inserted in the sense orientation into the t vector  to generJatropha curcas (JcMD) elite plants  [Seeds were obtained from ngapore) . Cotyledngapore) .Genomic DNAs were prepared with DNeasy plant mini kits  according to the manufacturer’s instructions. Approximately 50 ng of genomic DNA were used for PCR. The reactions were subjected to 94 C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes for 40 cycles. Primers P1 (5’-ATCTCCACTGACGTAAGGGATGAC-3’) and P2 (5’-GTTTAAGATCTACTTACGTAATCAAGC-3’) were used to check the occurrence of marker excision events. DNA fragments containing the hygromycin resistance gene were amplified by either P3-1 (5’-GAGGGCGAAGAATCTCGTGCTTTC-3’) and P4-1 (5’-TACTTCTACACAGCCATCGGTCCA-3’) or P3-2 (5’-GAAGAATCTCGTGCTTTCAG-3’) and P4-2 (5’-CAACCAAGCTCTGATAGAGT-3’). The product amplified by primers P3-1 and P4-1 was 885 bp, whereas the product amplified by primers P3-2 and P4-2 was 745 bp  and ran in ABI7900HT. Forward primer 5’-CTGCACAATGTGGGACCCTTTG-3’ and reverse primer 5’-CTTCGCCCTGATGACAGAGATC-3’ were used for the amplification of viral DNA-A of either ICMV-Dha (128–290 nt) or ICMV-SG (127–289 nt). All samples were run in triplicate and the data was analyzed with RQ manager at a preset threshold cycle value . The o et al. , the rel+ membrane  following standard procedures [Genomic DNA was digested with restriction enzymes and separated on 0.8% agarose gels. The gels were processed and resolved DNA bands transferred to a nylon Hybond-Nocedures . Membran


Oncorhynchus mykiss in three fresh water hatcheries  on the west coast of Norway. The fish (25-100 g) showed signs of circulatory failure with haemorrhages, ascites and anaemia. The histopathological findings comprised inflammation of the heart and red muscle and liver necrosis. The affected fish had a common origin. Disease and mortalities were also observed up to four months after sea water transfer. Microbiological examination did not reveal presence of any known pathogens. Based on histopathological similarities to heart and skeletal inflammation (HSMI) in Atlantic salmon, associated with piscine orthoreovirus (PRV), extended investigations to detect a virus within the family Reoviridae were conducted. By the use of primer sets targeting the PRV genome, a sequence with 85% identity to a part of segment S1 of PRV was obtained. Further analysis showed that the virus sequence could only be aligned with PRV and no other reoviruses both on amino acid and nucleotide level. Two PCR assays were developed for specific detection of the virus. High amounts of the virus were detected in diseased fish at all affected farms and low amounts were detected in low prevalence at the broodfish farms. Further investigations are needed to determine if the virus is associated with the new disease in rainbow trout and to further characterize the virus with respect to classification, relationship with PRV, virulence, pathology and epidemiology.In fall 2013, anorexia, lethargy and mortalities up to 10-12,000 dead fish per week were observed in rainbow trout  Oncorhynchus mykiss  is a North Pacific salmonid species which is farmed in many parts of the world. Rainbow trout is often farmed in fresh water dams and raceways. In Norway the main production is in sea locations. According to FAO-FishStat the global production of rainbow trout reached 850,000 tons in 2012, and is a growing industry. Norway produced approximately 71,000 tons rainbow trout in 2013 (Directory of Fisheries), which is a limited number compared to the production of Norwegian Atlantic salmon  of more than 1 million tons.Rainbow trout Flavobacterium psychrophilum is an important cause of mortality. In Norway, outbreaks of infectious pancreatic necrosis (IPN) in fresh water are reported. Some farms have experienced serious mortalities due to F. psychrophilum both in fresh water and in brackish sea locations , , , 17], , , 19], , .Reoviridae were initiated. Total RNA from tissue samples from hatchery A was extracted using QIAsymphony SP (QIAGEN) in accordance with manufacturer’s instructions using the QIAsymphony RNA kit. Amplification of PCR products was performed by one-step RT-PCR using primer sets targeting parts of the PRV genome as described by Garseth and co-workers . L. L25]. LA relatively high proportion of the diseased fish in the present study suffered from anaemia. Anaemia in salmonids, including rainbow trout, has previously been linked to erythrocytic inclusion body syndrome (EIBS) . ElectroAn association between the disease and the virus is supported by findings of high amounts of virus in clinically diseased fish at the acute and subacute-chronic stage. Low viral loads or in some cases no virus at all, were detected in fish in late stages of the disease, which is expected when the immune system manages to fight the virus. Moreover, at the affected farms, large amounts of the virus were detected in some apparently healthy fish within diseased fish groups. The presence of high amount of virus before onset of clinical disease is a typical observation for diseases involving viral agents. However, during our investigations, one of the samplings consisting of fish with typical pathological findings from farm A was negative for virus when examined by real time RT-PCR. Although one would expect those to be positive assuming that there is an association between the pathological changes and the virus, this observation could be explained by undetectable viral loads. On the other side, this may also indicate another cause of the disease than the PRV-related virus. Nevertheless, the fact that the disease display similarities with HSMI in salmon and the virus resembles PRV, can point to a link between disease and virus also in the rainbow trout.Controlled challenge trials are necessary to reveal the impact of the novel virus on fish health, establish the true cause of the new disease and describe the pathogenesis. Challenge trials with uncultivable viruses pose a challenge as filtered tissue from diseased fish must be used. This leaves a possibility that more than one infectious agent is present in the inoculum, and complicates interpretation of the results. New cell lines or other strategies of cultivating fish viruses are needed to enhance investigations of both the newly detected virus and other viruses for which cultivation methods are still lacking.Further studies to reveal the pathogenesis and tissue distribution of the PRV-like virus at different stages of the infection are needed. The tissue distribution at the acute and chronic stage of the infection may vary considerably. Blood was used for detection of the virus in the clinically healthy broodfish. The prevalence and amount of virus in these fish were low pointing to a possible carrier state. Longitudinal studies will be useful to reveal how long infected fish might harbor the virus and in which organs it accumulates.The fact that all diseased fish originated from the same broodfish source indicates a possible vertical component in the spread of the disease that needs to be further investigated.The spread of the disease described here points towards an infectious etiology. This disease is either triggered by the introduction of a new infectious agent, or by a change in the virulence of an already established agent. Diseases are often multifactorial, so a number of different factors including general health status of the fish and water quality may have contributed to the disease outbreaks. Whether the virus detected at the affected rainbow trout farms can be associated with the new disease or other health issues need further investigations. If so, it is of major importance to reveal the reservoir of the PRV-related virus and stop the spread into new areas. The PCR methods established in this study will be useful to map present distribution of the virus and examine historical material in order to reveal earlier distributions. Challenge trials are necessary to investigate the transmissibility, virulence and pathogenesis of the PRV-like virus both in rainbow trout and other possible susceptible species.


While intensity modulated radiotherapy (IMRT) in anal cancer is feasible and improves high-dose conformality, the current RTOG/AGITG contouring atlas and planning guidelines lack specific instructions on how to proceed with external genitalia. Meanwhile, the RTOG-Protocol 0529 explicitly recommends genital sparing on the basis of specific genital dose constraints. Recent pattern-of-relapse studies based on conventional techniques suggest that marginal miss might be a potential consequence of genital sparing. Our goal is to outline the potential scope and increase the awareness for this clinical issue.We present and discuss four patients with perigenital spread in anal cancer in both early and advanced stages (three at time of first diagnosis and one in form of relapse). Genital/perigenital spread was observed once as direct genital infiltration and thrice in form of perigenital lymphatic spread.We review the available data regarding the potential consequences of genital sparing in anal cancer. Pattern-of-relapse studies in anal cancer after conventional radiotherapy and the current use of IMRT in anal cancer are equivocal but suggest that genital sparing may occasionally result in marginal miss. An obvious hypothesis suggested by our report is that perigenital lymphovascular invasion might be associated with manifest inguinal N+ disease.Local failure has low salvage rates in recent anal cancer treatment series. Perigenital spread may pose a risk of marginal misses in IMRT in anal cancer. To prevent marginal misses, meticulous pattern-of-relapse analyses of controlled IMRT-series are warranted. Until their publication, genital sparing should be applied with caution, PET/CT should be used when possible and meeting genital dose constraints should not be prioritized over CTV coverage, especially (but not only) in stage T3/4 and N+ disease. Organ preserving combined modality treatment has proven to be as effective as radical surgery and has been introduced as standard therapy for anal cancer worldwide –4. Anal When compared to standard anterior-posterior/posterior-anterior (ap-pa) or 3D-conformal radiotherapy , intensity modulated radiotherapy (IMRT) provides similar coverage of the planning target volume (PTV) while better sparing organs at risk (OAR), reducing the dose to critical structures –19 and aThe Australasian Gastrointestinal Trials Group (AGITG) and the RTOG have recently established contouring-guidelines for IMRT in anal cancer , 27. Theafter IMRT and three cases of anal cancer with (peri)genital infiltration at the time of first diagnosis before IMRT treatment and discuss them in the context of recently published relevant data. All patients have consented with their regular informed consent to anonymous scientific analysis of their data.Anal cancer invading genitalia or spreading into lymphatic structures close to external genitalia has been observed several times at our department over the last 18 months. All patients had extensive clinical and imaging workup including FDG-PET/CT (positron emission tomography/computed tomography) and MRI (magnetic resonance imaging), for the first time providing the opportunity to obtain detailed objective visualization of genital/perigenital spread . As locaA 50-year-old male patient presented with T2N0M0 anal margin squamous cell carcinoma (SCC) in 04/2013, with the tumor reaching the posterior scrotal skin  and a boost up to 54 Gy to the primary tumor and PET-positive lymph nodes. Two concurrent and two sequential cycles of 5FU/MMC chemotherapy were administered. The second follow-up at four months with FDG-PET/CT showed an elevated SUV in most of the initially affected lymph nodes and a biopsy of the anal canal revealed persisting carcinoma. Salvage surgery was performed . Due to a postoperatively persisting lymphatic fistula in the left inguinal region, a course of obliterating radiotherapy was planned. The planning CT unexpectedly showed a small subcutaneous tumor in the right vulvar/perivulvar region. A control CT 4 weeks after radiotherapy showed a significant increase in size, suggesting locoregional relapse  guidelines recommend CT or MRI of the pelvis for evaluation of pelvic lymph nodes . A FDG-PAs for patient setup, radiation treatment in anal cancer can be performed in prone or supine position with both advantages and disadvantages in each position . RegardiIn the last decade, IMRT has become a widely used radiotherapy technique for various tumor entities such as head and neck, prostate or breast cancer . The steAlthough the primary endpoint of reducing grade 2 toxicity was not met, the initial data published for this RTOG-0529 multicenter trial suggest reduced grade 3 toxicity (likely even more important than the primary endpoint) with IMRT  when comexternal perianal failure had stages from T1N0 up to T3N0 and the two patients with vulvar and scrotal relapse had T2N2 and T2N3 stage, respectively. Both patients with genital relapses had also both inguinal relapse and finally metastatic disease. Though an ultimately precise analysis of in-field relapse vs. marginal miss was not possible in their series either due to methodical limits , they explicitly suggest that “three of five failures appear to be in-field and two marginal, primarily because of inadequate coverage anteriorly. Failures occurred up to 3 cm inferior to the anal verge and anteriorly into the scrotum or vulva. This highlights the need to respect a minimum of a 2-cm margin on the tumor and anal margin in the CTV, even if this makes meeting genital dose constraints difficult” [To better understand to what extent genitals can actually be spared from treatment, pattern-of-relapse analyses have to be performed. Two recent studies have reported such patterns-of-relapse in anal cancer after conventional, simulator based therapy. Relapses have mostly occurred locally in the area of the primary tumor or regionally in initially affected lymph node areas , 46 and Regarding IMRT, there is a body of retrospective data that reports local and regional control after IMRT for anal cancer, albeit still with relatively short follow-up, with a varying degree of genital sparing, a varying percentage of local and regional relapse and no explicit analysis to what extent genital sparing may have contributed to local and regional relapse , 47–49. relapse indicating marginal miss  showed a vulvar/perivulvar iss Fig. , IId, f.iss Fig. , IIb, c,before application of IMRT, therefore treatment volumes could be chosen appropriately. In case 1 (T2N0M0), scrotal infiltration could not be ruled out clinically and consequently the clinically infiltrated parts of the scrotum/scrotal skin were included into the CTV. In case 3 , clinically there was considerable infiltration of the left mons pubis, demonstrating a case of perigenital lymphovascular invasion. The right inguinal region had shown no sign of affected lymph nodes at the start of the treatment but the patient was considered to be at higher risk of having further (microscopic) lymph node infiltration or inguinal relapse. Therefore, radiation on the right side purposely was performed not only with 36 Gy but up to 45 Gy, in order to apply a sufficient radiation dose to both inguinal regions. Both patients have been in remission to date, likely as a consequence of the relatively large treatment volumes and target contouring being based both on clinical and PET/CT examination, the latter unfortunately not yet being a standard imaging modality in anal cancer. Case 4 was an advanced T4N3M0 tumor with initial involvement of inguinal lymph nodes on both sides with perivulvar lymphovascular invasion on the left side. The infiltrated genital area was included into the PTV. Due to persisting anal tumor and progressive lymph nodes salvage surgery was performed. She later received palliative chemotherapy due to multilocular progression and died a few months later in palliative care.The other three cases report genital/perigenital infiltration already at time of first diagnosis, Case 2 and case 4 therefore show cases with locoregional failure and subsequent systemic progressive disease. In the context of systemic progressive disease the issue of local failure is sometimes of minor prognostic importance, the consequences for the patient  may nevertheless be severe. However, it has also to be kept in mind that, as recently shown by Mai et al., as well as in other recent publications, local relapse is not salvaged any more at recently published rates and thus dramatically influences overall survival , which iIn conclusion, while IMRT in anal cancer is feasible and improves high-dose-conformality, our case reports show that in both early and advanced anal tumors there is a risk of genital/perigenital spread at time of first diagnosis, increasing the risk for marginal misses if target volumes are too small. Genital spread can occur in form of direct infiltration (which may not only occur in high T-stage) or perigenital lymphovascular invasion . Until detailed pattern-of-relapse analyses of controlled IMRT-series such as RTOG-0529 are available, PET/CT and possibly MRI should be considered as a staging tool, genital sparing should be applied with caution, and meeting genital dose constraints should not be prioritized above CTV coverage, especially (but not only) in stage T3/4 and inguinal N+ disease.All procedures followed were in accordance with the ethical standards; an approval by an ethics committee was not applicable. All patients have consented with their regular informed consent to anonymous scientific analysis of their data.


A large measles virus outbreak occurred across Vietnam in 2014. We identified and obtained complete measles virus genomes in stool samples collected from two diarrheal pediatric patients in Dong Thap Province. These are the first complete genome sequences of circulating measles viruses in Vietnam during the 2014 measles outbreak. Paramyxoviridae and causes measles, a highly contagious infectious disease. Measles mainly affects children age <5 years and was responsible for the deaths of 114,900 individuals in 2014  , a randoon 3.5.0 , followeon 3.5.0 . The woron 3.5.0 . The aveBoth patients were admitted to the Provincial Hospital of Dong Thap due to acute diarrhea, and the generation of whole measles virus genomes from these patients suggests that the episodes of diarrhea reported in these patients may be a consequence of measles virus infection, consistent with previous observations . In addiKU728742 and KU728743.The complete genome sequences of the MVs/DongThap.VNM/06_14 (D8) and the MVs/DongThap.VNM/08_14 (D8) isolates are available at GenBank under the accession numbers


The burden of cardiovascular disease (CVD) and CVD risk conditions is rapidly increasing in low- and middle-income countries, where health systems are generally ill-equipped to manage chronic disease. Policy makers need an understanding of the magnitude and drivers of the costs of cardiovascular disease related conditions to make decisions on how to allocate limited health resources.We undertook a systematic review of the published literature on provider-incurred costs of treatment for cardiovascular diseases and risk conditions in low- and middle-income countries. Total costs of treatment were inflated to 2012 US dollars for comparability across geographic settings and time periods.This systematic review identified 60 articles and 143 unit costs for the following conditions: ischemic heart disease, non-ischemic heart diseases, stroke, heart failure, hypertension, diabetes, and chronic kidney disease. Cost data were most readily available in middle-income countries, especially China, India, Brazil, and South Africa. The most common conditions with cost studies were acute ischemic heart disease, type 2 diabetes mellitus, stroke, and hypertension.Emerging economies are currently providing a base of cost evidence for NCD treatment that may prove useful to policy-makers in low-income countries. Initial steps to publicly finance disease interventions should take account of costs. The gaps and limitations in the current literature include a lack of standardized reporting as well as sparse evidence from low-income countries.The online version of this article (doi:10.1186/s12889-015-2538-z) contains supplementary material, which is available to authorized users. Cardiovascular disease (CVD) and CVD risk conditions, such as hypertension, type 2 diabetes mellitus (T2DM), and chronic kidney disease (CKD), are the leading cause of global morbidity and mortality . IncreasEfforts to improve prevention and management of NCDs in LMICs are increasing. Mortality reduction targets, such as the World Heart Federation’s 25 × 25 and the Lancet’s 40 × 30, are unprecedented in putting NCDs on the international agenda , 8. The Recent reviews of household burden or cost-effectiveness of CVD interventions in LMICs have been published , 10–13; Our primary objective was to present the level and variability in direct medical or programmatic costs for treating CVRD in LMICs. Our secondary objective was to identify gaps in knowledge and suggest future economic research needs to support policy development, resource allocation, and decision-making.st, 2000 until approximately July 1st, 2014. We screened eligible titles and abstracts to determine which articles would be included for final review. Two independent reviewers reviewed the full text articles. In addition, we examined the references of the included articles and prior reviews for potentially relevant studies. Broadly, the following criteria were used to determine inclusion: Included an economic evaluation Included evaluation of at least one low- or middle-income country (as defined by the World Bank) Referred to our previously standardized list of cardiovascular or related-related conditions Made available in English Published on or after January 2000.We conducted our review according to the PRISMA guidelines . We sear The study must have conducted at least one type of economic evaluation, including cost analysis, cost-effective analysis, or cost-utility analysis, with clearly presented unit cost data. We did not consider costs with health outcomes as a denominator, such as disability-adjusted life years (DALYs), quality adjusted life years (QALYs), or life-years saved (LYS). The study must have utilized either original unit cost data regarding a CVRD intervention delivered in a health systems setting, or unit costs from a credible and known source, such as WHO-CHOICE. We did not consider population-level interventions or policy costs. The study must have presented direct intervention costs  from the provider perspective, regardless of payer or indirect costs. Studies that considered multiple perspectives were only considered if they clearly delineated provider costs. Provider costs were defined as those delivering the services, such as those incurred by health systems, hospitals or clinics, governmental bodies, funders, or programs. The study had to meet certain quality standards, which includes having a description of the intervention and analysis, detailing the time and location of data collection, and clearly stating the year and currency of presented costs.We used both the Drummond Checklist and Mogyorosy and Smith’s 2012 literature review for guidance in creating the following guidelines regarding quality and inclusion , 16: TheTo inform the organization and synthesis of the literature across conditions and interventions, we created a conceptual framework of the clinical and public health considerations in CVD treatment Fig. . There aFor each eligible study we entered the following information into an electronic database: condition(s), country or region, target population, type of treatment or intervention, and level of care. We also noted study methods, such as sample size and strategy, exchange rate, discount rate, sensitivity analysis, and cost categories. The total treatment or intervention costs, defined as the total cost per patient, were extracted in their original currency and year. For acute events, we used the total cost per patient as presented. For recurring costs, such as hemodialysis treatments or ongoing hypertension management, we calculated the cost per treatment or per year. Differences in inpatient and outpatient costs were noted where reported and applicable. Of note, most of the included studies reported multiple costs, particularly if they discussed more than one CVRD intervention or more than one geographic setting.We converted all costs to US Dollars (2012), the currency and year in which we present all costs in this paper. When possible, we extracted the data in local currency units (LCUs), inflated it to 2012 rates using the World Bank consumer price indices, and converted it to 2012 US Dollars via World Bank rates. When costs were presented for regions, we used the largest LMIC country in the region as a proxy. For data presented in international dollars, we converted it back into LCUs using World Bank Purchasing Power Parity, then we followed our standard processes. Once the data were in a common currency, we grouped similar interventions and qualitatively compared the magnitudes and variability of total and input costs. We combined data into the two overarching groups to reflect our aforementioned framework: management of CVD risk conditions and treatment of acute and chronic CVDs. The former group included hypertension, hyperlipidemia, CKD, T2DM, and generic CVD prevention. The latter group focused specifically on ischemic heart disease, non-ischemic heart diseases, stroke, heart failure, T2DM sequelae , and end-stage renal disease (ESRD), which is due predominately to poorly controlled hypertension and T2DM.The results of the systematic search process can be found in Fig. The number of published articles for CVD costs in LMICs has increased dramatically since 2000, as seen in Fig. n = 37) were cost analyses, meaning they only collected and presented data on costs, not health outcomes. Thirty studies determined the total costs of interventions by reviewing hospital records while 29 studies used an ingredients approach, taking the sum cost of activities and resources used to find the total costs. Only one study used a questionnaire to collect data. Of the 60 studies, over half converted their costs in US Dollars or the hypothetical currency “International Dollars” for presentation (32 articles). Although it is difficult to measure the quality of a study objectively, economic evaluation guidelines recommend discounting future costs and conducting sensitivity analyses when assumptions are made. We noted that approximately one-third of the 60 studies conducted sensitivity analysis, and one-fourth used discounting in their methods; 11 articles used both.We obtained cost data from a variety of economic evaluations. Almost two-thirds of the articles . There were also brief mentions of pharmacological tobacco cessation measures, rheumatic fever and generic CVD prevention strategies such as polypill use. More than three-quarters of this data came from Asian countries, and more than half from China, India or Thailand.We found 14 unit costs for hypertension management in LMICs, with drug therapy ranging from $2.21–$76.29. Using general practitioners in community programs cost only $0.81–$8.67 per patient per year, compared to annual outpatient costs to manage hypertension, which ranged from $38.00 to $565.54 per patient. Only three studies mentioned the costs of managing dyslipidemia, which were similar to managing hypertension. One study looked at the costs of managing chronic kidney disease across China, Malaysia and Thailand, with China being more expensive than Malaysia by a scale of six to eight. We identified 30 unit costs for managing diabetes. Two studies produced four costs on diagnosing diabetes, the most expensive of which were based in tertiary hospitals. Seven studies presented ten annual per patient costs of managing diabetes in a clinical setting, which ranged from $56.25 in India to $2,479.82 in China; the costs were higher in cases with complications and sequelae. Nine studies also presented 16 costs on inpatient and outpatient treatment for diabetes. Inpatient stays per bed-day ranged from $59.92–$143.14 with an average length of stay of around 7 days. An outpatient visit ranged from $4.63–$34.28, and we also found a ten-fold variation in yearly outpatient costs.Our review also identified 83 cost estimates from 34 studies regarding the treatment of ischemic heart disease (IHD), stroke, heart failure, non-ischemic heart diseases, T2DM sequelae, and end-stage renal disease (ESRD).Treatment of IHD typically consists of diagnostic procedures, surgical or other advanced medical procedures, and post-operative and long-term outpatient care. We found 8 articles analyzing IHD treatment with a total of 18 unit costs. An inpatient visit averaged $8,800, with a range from $455 to $22,500. The highest inpatient costs were associated with longer hospitalization periods, more severe conditions, and surgical interventions. The costs of surgical procedures, including catheter-based procedures (stenting and angioplasty) or coronary artery bypass surgery (CABG), ranged from $4,000–$22,000 per patient per procedure depending on the diagnostics, the complexity of the procedure, and whether medical therapy and recovery costs were included in the total cost estimates.Our search returned 15 articles and 24 unit costs for both ischemic stroke (IS) and hemorrhagic stroke (HS), though many studies did not specify the type of stroke. In most settings, IS is the more common of the two, is generally easier to treat, and has a lower case-fatality rate. Four articles compared the costs HS and IS treatment –20. HemoFive studies reported costs of T2DM sequelae, namely, diabetic foot ulcers and retinopathy. Severity of these impairments and costs saved by screening drove the total costs of both, though in opposite directions –25. For Five studies reported costs of end-stage renal disease (ESRD), primarily long-term dialysis or renal transplantation. The cost of one session of hemodialysis for ESRD ranged $79 to $97 –28, whilOther types of CVD, such as heart failure, Chagas cardiomyopathy (CC), congenital heart disease, and rheumatic heart disease, are important contributors to the burden of NCDs in LMICs. However, we identified only a handful of studies on the cost of treating these conditions Table .Table 2UTreating CVD and its risk conditions is complex, due in part to the interrelationship between hypertension, diabetes, and ischemic heart disease, and the fact that multiple shared risk factors affect CVD health outcomes. The clinical heterogeneity of CVD can make treatment costs for a single condition much more variable than in the case of infectious diseases or some other chronic diseases; these factors also affect the mean and distribution of costs within and across similar conditions. For instance, CVD encompasses different types of heart and related diseases, such as hypertension, stroke, and heart failure, with different levels of severity, and associated care and management. There are numerous clinical protocols for treating complicated conditions, and treatment includes different combinations of drugs, diagnostics and imaging technologies, surgery and different requirements for inpatient care and follow-up visits, making comparisons between studies all but impossible. Additionally, there are large variations in clinical characteristics, capabilities, and practices among and within countries; it is possible to have a wide distribution of costs even within a hospital if they offer various levels of care, such as general, specialty, and intensive care wards .Another factor affecting the variation in costs was differences in methodology used across studies. There was a lack of standard reporting across the studies, most notably failure to describe cost ingredients. When studies disaggregated costs, there were not clear and common categories for input or activity cost categories. In addition, for disaggregated input costs, it was unclear when activities included personnel in the costs. Many studies provided costs for imaging, diagnostic or therapeutic services, without indicating whether personnel costs were included. Surgery was a common category, but descriptions of what resources comprised surgery or surgical procedures were absent from the majority of the studies. Cost data was most informative when disaggregated into categories and inputs, allowing the reader to better understand heterogeneity and make more useful comparisons. We also noticed a lack of clinical protocol reporting in publications. Reporting on clinical protocols underlying the CVD interventions may help readers evaluate the comparability and transferability of costs estimates across studies, depending on the study objective.Trends in the data emerged despite these limitations. It is clear that there is growing interest and concern about the prevalence and cost of treating cardiovascular and risk conditions at every national income level. BRICS countries and a few other populous Asian countries produced about three-quarters of the literature we identified, laying a foundation of evidence for the costs of treating CVRDs in LMICs. The current evidence confirms that is expensive to provide treatment for complicated or advanced cardiovascular conditions, with inpatient treatment for CVD easily costing upwards of $10,000 per patient Fig. . LookingOur review identified critical gaps in information and demonstrated the substantial heterogeneity in treatment costs for CVRD across the available data for LMIC countries. The most obvious gap was the need for more consistent methodology, with clear presentation of data on cost ingredients and drivers. Cost data are used to inform resource allocation and to improve efficiency; health policy makers thus need to understand the underlying causes of health expenditures in order to make informed decisions. We identified no longitudinal studies in LMICs, however several modeled studies looked at health costs over a longer period of time. We anticipate that longitudinal studies would be particularly revealing regarding costs of chronic conditions, such as hypertension and diabetes, which require lifelong management. In the absence of longitudinal studies, health decision-makers are currently allocating CVD resources with an incomplete picture of future costs.We found very few cost analyses of CVD conditions conducted in low-income countries. While these countries are likely the most heavily resource-constrained, they are also likely the least prepared to prevent and treat CVD conditions. We found no data on the cost of diabetes care in low-income countries. Middle-income countries, particularly upper-middle income countries such as Brazil, China, and South Africa, have produced research or publications regarding technologically complex interventions, revealing relatively higher health system capacity. As the double-burden of infectious and chronic conditions increases, and policy makers in low-income countries expand health resources for chronic conditions, published studies from other LMICs can provide economic evidence on CVD interventions.Because our analysis focused on the unit costs of clinical interventions, we did not include many of the WHO Best Buys for NCDs such as tobacco taxation and regulation, population salt regulation, or health and physical activity promotion. Population based prevention measures typically cost pennies per person, but the total cost to the government will depend on the population size. It was beyond the scope of this paper to compare the total costs of population based interventions with clinical prevention and treatment costs. The evidence in this review strongly suggests that costs quickly escalate with advanced CVRD treatment; future investment in population prevention of CVRDs could reduce the need for these expensive clinical interventions.We also excluded studies presenting the cost of implementing guidelines for initiating prevention strategies of CVRDs based on absolute risk , 33. WhiThe evidence for CVRD treatment and prevention costs in LMICs is limited, particularly in low-income countries. We recommend conducting economic studies alongside efforts to scale up treatment and preventions on CVRD in these settings. Initially standard methods of care or clinical protocol could be used to inform costs of CRVD treatment and prevention in order to estimate costs and their key drivers are across settings. There is also a need to estimate the actual resource use and costs in the provision of care, as such information can be used to improve efficiency and budgeting over time. In addition to following recommended costing analysis protocols , future The burden of CVRD in LMIC is already substantial and will continue to increase in the absence of concerted prevention efforts, which could contain costs by reducing future spending on CVD. At the same time, the prevalence of CVD is already very high in regions such as Latin America and South and East Asia. Hence, efforts to allocate NCD resources equitably will inevitably include a balance of both prevention and advanced treatments for existing cases of CVD. Ongoing efforts to understand the cost of delivering CVRD care in these regions will be critical to achieving universal health coverage and improving overall population health.


Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices.Total nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV.Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors.PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies.The online version of this article (doi:10.1186/s12985-016-0509-3) contains supplementary material, which is available to authorized users. Bunyaviridae family, is a mosquito-borne, biosafety level (BSL)-3 select agent of major public health and economic concern, affecting humans and livestock throughout Africa [Arenaviridae family, is a BSL-4 select agent responsible for approximately 500,000 infections yearly in West Africa [Both Rift Valley fever virus (RVFV) and Lassa virus (LASV) are highly pathogenic viruses endemic to Africa. RVFV, within the t Africa –10, 22 at Africa , 25. LASt Africa . Both ofResearch with either of these pathogens is hazardous, expensive, and limited to studies at select institutions by approved individuals. As such, BSL-2 infection models for both viruses have been developed: Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection. While these models have been used for multiple pathogenesis and therapeutics studies , 24, 26,PTV infection in mice  and hamsPICV causes a similar disease in hamsters , 14, 26 In this study, we designed two TaqMan-based real-time RT-qPCR assays for detection of PTV and PICV. These assays were characterized and evaluated using cell culture supernatant from PTV or PICV infected cells and mock clinical samples. Overall, these assays could benefit the scientific community using animal models as surrogates for RVFV and LASV infection as well as biosurveillance for PTV infections in humans.w/v) SeaKem ME agarose  and a secondary overlay containing 5 % neutral red . Vero E6 cells were maintained in complete Eagle’s Minimum Essential Medium  supplemented with 10 % (v/v) fetal bovine serum (ThermoFisher), 100 U/ml penicillin G (ThermoFisher), and 100 mg/ml streptomycin (ThermoFisher). Cells were incubated at 37 °C with 5 % CO2.The Adames strain of PTV and the CO AN 4763 strain of PICV were provided by Dr. Robert Tesh . Each virus was initially passaged in Vero E6 cells to generate stock virus from the cell culture supernatant. Virus stock titers were determined by standard plaque assay using 0.6 % (RNA from the cell culture supernatant was extracted using TRIzol LS (ThermoFisher) and the Qiagen EZ1 robot with the EZ1 Virus Mini Kit  according the manufacturer’s directions. Sensitivity testing was conducted for each assay using total nucleic acid isolated from cell culture supernatants from PICV and PTV infected Vero E6 cells. These supernatants were previously titered by plaque assay, so the limit of detection (LOD) was determined based on the number of PFU/ml before sample extraction with the EZ-1 kits. For analytical experiments, extracted RNA was serially diluted 10-fold into water, and the diluted RNA was run with each assay in triplicate. The preliminary LOD was determined based on 3/3 replicates being positive (<40 Cq), and 60 replicates at this preliminary LOD was conducted for LOD confirmation. For mock clinical samples, cell culture supernatant was 10-fold serially diluted in water, serum, and whole blood , and each dilution was extracted using TRIzol LS and the EZ1 (Qiagen) according to the manufacturer’s instructions.Primers and TaqMan-minor groove binder (MGB) probe pairs were designed for PICV and PTV using Primer Express version 2.0  and AlleleID 7.73 . Primer/probe pairs (see Table Taq DNA polymerase Kit (ThermoFisher) was utilized. 20 μl experiments were performed with 5 μl of extracted RNA described above, 10 μl of 2X SuperScript One-Step RT-PCR master mix, and 0.4 μl of SuperScript II Reverse Transcriptase and Platinum® Taq DNA Polymerase mix. The primers and probes used are listed in Table 4, and 0.25 mg/ml BSA were used. Reactions were run on the LightCycler 480 (Roche) with the following cycling conditions: 50 °C for 15 min (1 cycle); 95 °C for 5 min (1 cycle); 95 °C for 1 s and 60 °C for 20 s (45 cycles); and 40 °C for 30 s (1 cycle). A single fluorescence read was taken at the end of each 60 °C step, and a sample was considered positive if the Cq value was less than 40 cycles.For analytical and mock clinical analysis SuperScript One-Step RT-PCR System with Platinum For ddPCR analysis One-Step ddPCR Advanced Kit  was utilized. 22 μl experiments were performed with 5 μl of extracted RNA, 5.5 μl of One-Step ddPCR supermix, and 2.2 μl polymerase mix. The assay specific primers and probes were used  maintained at USAMRIID provided West Nile virus strain NY99 (UCC# Flavi022) and dengue virus serotypes 1–4 . Rift Valley fever virus, Lassa fever virus , Mozambique virus, Junín virus strain Espindola, Machupo virus strain Carvalo, Mobala virus strain Acar, and Heartland virus are all maintained at USAMRIID.Capture regions were confirmed with Sanger sequencing by amplifying the capture region with modified M13F (-20) and M13R (-27) sequencing primers. Forward and reverse primers used for amplifying sequenceable PICV are as follows 5’-GTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGAATTTAGCGGCCGCCATGTGTGGCCCCCATTT and 5’-CCTTTGTCGATACTGGTACATTACGCCAAGCTCAGAATTAACCCTCACTAAAGGGACTAGTCCTGCTCAGTTGTTAGGCAAAGTGGTCTT. Forward and reverse primers used for amplifying sequenceable PICV are as follows 5’-GTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGAATTTAGCGGCCGCCAGATAGCTGCTGCCATTTTACA and 5’-CCTTTGTCGATACTGGTACATTACGCCAAGCTCAGAATTAACCCTCACTAAAGGGACTAGTCCTGCGCTTTTAAGTTTCCCAGCCAAA. Cycle sequencing was run with the BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher), cleaned up with the Qiagen DyeEx 2.0 Spin kit (Qiagen), and run on the Applied Biosystems 3500xL Genetic Analyzer per manufacturers protocol.Cq values were calculated using the second derivative method with the Roche LightCycler 480 software version 1.5.1. GraphPad Prism v. 6.04 graphing software  was used to plot sample data and for statistical analysis.Initial evaluations identified optimal primer and probe concentrations for each assay combination, and preliminary downselection testing identified a final assay for each virus Table . Assays 6 to 10-1 PFU/ml with each assay identified the preliminary LOD in which all three replicates were positive  as the unit of measurement. Testing of eight, 10-fold serial dilutions from each virus ranging from 10ive Fig. . This teive Fig.  using 606 to 101 PFU/ml. For each matrix tested, water, serum, or whole blood, the LOD for PTV remained consistent at 1000 PFU/ml as determined by 3/3 replicates falling below the 40 Cq cutoff value  was used to quantitatively determine post-extraction the number of target RNA molecules (counts) per μl of sample. To directly correlate ddPCR results to previous experiments, the spiked 10-fold dilutions of water from Fig. Both RVFV and LASV are highly pathogenic viruses of significant public health and economic concern, and both are considered biothreat agents. Use of RVFV is limited to BSL-3 conditions, and LASV is restricted to the highest level of containment, BSL-4. Due to these restrictions and the limited access to these viruses, BSL-2 models for both viruses have been developed for pathogenesis and therapeutic studies. To date, the authors are unaware of published real-time RT-qPCR assays for either PTV or PICV, so we developed and characterized real-time RT-qPCR assays for both PICV and PTV.A series of primers and probes for each virus targeting the polymerase gene were optimized and tested for assay LOD and performance in multiple clinical matrices. Real time RT-qPCR analysis and ddPCR both demonstrate clinically relevant LODs, detecting as low as one RNA target molecule per reaction. These values are within the range of other real-time RT-qPCR assays developed by our group targeting filoviruses, arenaviruses, and New World hantaviruses . These a


Tooth morphology and development can provide valuable insights into the feeding behaviour and evolution of extinct organisms. The teeth of Theropoda, the only clade of predominantly predatory dinosaurs, are characterized by ziphodonty, the presence of serrations (denticles) on their cutting edges. Known today only in varanid lizards, ziphodonty is much more pervasive in the fossil record. Here we present the first model for the development of ziphodont teeth in theropods through histological, SEM, and SR-FTIR analyses, revealing that structures previously hypothesized to prevent tooth breakage instead first evolved to shape and maintain the characteristic denticles through the life of the tooth. We show that this novel complex of dental morphology and tissues characterizes Theropoda, with the exception of species with modified feeding behaviours, suggesting that these characters are important for facilitating the hypercarnivorous diet of most theropods. This adaptation may have played an important role in the initial radiation and subsequent success of theropods as terrestrial apex predators. Albertosaurus revealed unusual enamel cracks leading to a void in the dentine called an “ampulla” between adjacent denticles along the distal carinae, which were hypothesized to have dissipated stresses on the tooth during feeding45121415Carnivorous tetrapods exhibit varying degrees of dental specializations for grasping and tearing their prey items, including ziphodonty– blade-shaped teeth with well-developed serrated cutting edges34579Erupted homodont maxillary teeth  show a lA number of features were found only in theropod dinosaurs. The mantle dentine immediately surrounding the interdental fold is globular in theropod teeth, and dentine tubules that originate in the fold and curve towards the pulp cavity extend outward from the globular dentine . In tran45Surrounding the interdental fold is an area of clear dentine, originally thought to be a deep layer of enamel encircling the globular dentineThe interdental fold is continuous below each interdental sulcus, forming a 1 μm channel through the enamel  and 3. T18Allosaurus and Gorgosaurus  and 2345 cm−1 (asymmetric stretching ν3)) were recorded within the globular dentine and the type A carbonate of the enamel, but not within the primary dentine , Coelophysis bauri (CM 76863 and 87671), Carcharodontosaurus saharicus (ROM 52037), Troodon formosus (ROM 05089), Allosaurus fragilis (ROM 66106), and a tyrannosaurid (ROM 57981) were examined histologically. Unerupted teeth were taken from Allosaurus (UMNH.VP 23761), Gorgosaurus sp. (CMN 2225 and Daspletosaurus sp. RTMP 2012.012.0029). The teeth were prepared out of intact dentaries and maxillae, and had not erupted past the level of gumline. The Allosaurus tooth was completely unerupted, having not yet erupted past the level of the gumline, and the Gorgosaurus, Daspletosaurus, and other tyrannosaurid teeth were partially erupted and fully erupted. Other ziphodont animals examined include the shark Carcharodon megalodon (ROM 30530), from the Pleistocene of Florida, Varanus komodoensis (ROM R7565), an extant varanid lizard, Dimetrodon grandis (ROM 6039) from the Permian-aged Craddock Bonebed locality in Texas, USA, and a phytosaur (ROM 7981) from the Upper Triassic Howard County, Texas. Comparative material of Smilodon sp. (ROM 3288) from the Pleistocene of North America and a crocodilian (ROM 67512) from the Cretaceous Kem Kem beds of Morocco, Triceratops sp. (ROM 67669) from the Upper Cretaceous Hell Creek Formation, a hadrosaurid (ROM 58205) from the Cretaceous Oldman Formation, Alberta, and Spinosaurus aegypticus (ROM 65613) from the Cretaceous Kem Kem beds of Morocco were also cut.To examine the pervasiveness of ampullae in theropods, teeth from multiple taxa were examined. Thin sections were prepared following standard histological methodology, with some modificationGorgosaurus sp. tooth (ROM 30582) was performed using synchrotron-radiation-based infrared microspectroscopy (SR-IMS) at BL14A1 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan2 spot to be focused on samples; this is superior to the 40 × 40 μm2 spot achievable using a conventional global beam2-coated IR reflective low-e slides . The 1.5-GeV third-generation synchrotron was operating in top-up mode at 360 mA. The FTIR bench was configured with collimated infrared synchrotron radiation, providing external infrared synchrotron radiation to the FTIR spectrometer. The modulated synchrotron infrared light generated by the Michelson interferometer of FTIR spectrometer was directed into the infrared confocal microscope for microspectroscopy. The FTIR spectra were collected in the mid-infrared range of 4000–650 cm−1 at a spectral resolution of 4 cm−1 with the co-addition of 128 scans and an aperture of 20 × 20 μm2 and step size 10 μm. The optical systems of the FTIR microscope were purged by using dry nitrogen and an automatic atmospheric suppression function in OMNIC™ , which were employed to minimize infrared absorption by CO2 and water vapor in the ambient air. A single-beam background spectrum was collected from an area free of sample on low-e slide. Stage control and data collection were done using the spectrometer software coupled with a gating system that eliminated the noise during 60-s electron injection periods in top-up mode operation. Optical images were also obtained using a camera linked to the infrared confocal microscope.The Fourier Transform Infrared spectroscopy (FTIR) analysis of a cf. CM, Carnegie Museum, Pittsburgh, USA; CMN, Canadian Museum of Nature, Ottawa, Canada; ROM, Royal Ontario Museum, Toronto, Canada; RTMP, Royal Tyrrell Museum of Palaeontology, Drumheller, Canada; UMNH, Natural History Museum of Utah, Salt Lake City, USA.How to cite this article: Brink, K. S. et al. Developmental and evolutionary novelty in the serrated teeth of theropod dinosaurs. Sci. Rep.5, 12338; doi: 10.1038/srep12338 (2015).


Varicella-zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in nonimmunized populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and whether new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterized population cohort in Guinea-Bissau and produce a unique set of 23 full-length genome sequences, collected over 7 months from eight households. Comparative sequence analysis reveals that four distinct genotypes cocirculated among the population, three of which were present during the first week of the outbreak, although no patients were coinfected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible coevolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns, and (recent) natural history.IMPORTANCE VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common among young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or cocirculate during an outbreak. In this study, we have sequenced chickenpox viruses from an outbreak in Guinea-Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the 6-month outbreak period and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential coevolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations. The virus is transmitted in aerosols, resulting mainly from the rupture of fluid filled skin blisters which are characteristic of both chickenpox and shingles but also from virus shed from the respiratory tract. Virus inhaled by a susceptible contact replicates in the nasopharynx, spreading thereafter to cause the centripetal rash characteristic of chickenpox. Like other airborne virus infections, chickenpox is epidemic. Immunity is generally lifelong, with outbreaks mainly affecting susceptible birth cohorts. In temperate countries such as the United Kingdom and the United States, VZV is estimated to infect 60 to 90% of close and household contacts and, by age 10, more than 90% of the population are immune . In contte virus . Howeverte virus . While me virus 610, none www.bandim.org)  , 12. EpiThis study uses recently developed enrichment methods , 14 whicEthical clearance for the study was obtained from the Ministry of Public Health in Guinea-Bissau and the East London and City Health Authority. Participation was voluntary. We selected eight putative transmission chains comprising two to four members of the same household collected during the start, middle, or end of the outbreak in 2001 . PutativTotal DNA was extracted from each sample by using a QiaAMP DNA minikit (Qiagen) according to the manufacturer's instructions. DNA quantification was performed with a NanoDrop spectrophotometer, and samples with 260/280 ratios outside the range 1.7 to 2.1 and 260/230 ratios outside the range 1.8 to 2.2 were further purified using a Zymoclean Genomic DNA Clean & Concentrator (Zymo Research Corp.). Whole-genome amplification using GenomiPhi V2  was performed using 10 ng of starting material. Libraries were constructed in accordance with the standard SureSelect XT v1.5 protocols (Agilent). Enrichment for VZV sequences was performed as described previously , 14. SeqNC_001348) using BWA , which regresses the root-to-tip distance against the sampling date, was used to assess the “clock-likeness”  of the Guinea-Bissau VZV sequence data using neighbor-joined trees inferred by Mega v5.2 (Estimates of substitution rates were inferred using the program Beast v1.7.5 . The Beaega v5.2 . To asseega v5.2  whereby NC_001348) as a template. All PCRs were carried out using Herculase II fusion DNA polymerase (Agilent Technologies) according to the manufacturer's instructions. The cycling conditions were as follows: denaturation at 95°C for 2 min, followed by 35 cycles of amplification , and then a final extension step at 72°C for 3 min. PCR products were purified using a DNA Clean & Concentrator-5 kit (Zymo Research) according to the manufacturer's instructions. Products were Sanger sequenced in the forward and reverse directions.The VZV reiteration regions R1, R2, R4, and R5 were amplified and sequenced from Guinea-Bissau sample DNA using primers designed against the Dumas reference genome  or a single SNP (n = 14). Four viruses—Bandim 6, 13, 14, and 17— had synonymous mutations at positions in ORF62. Three viruses—Bandim 15, 16, and 17—also shared a synonymous mutation in ORF64. Using the sampling dates for all 23 samples, we calculated the substitution rate (excluding the repeat regions) to be 1.82 × 10−5 substitutions per site per year (−6 substitutions per site per year [−5 substitutions per site per year), one derived just from genogroup 5B samples (5.91 × 10−5 substitutions per site per year), and one obtained from a previous studies by Firth et al. . To valper year ). The daIn all but three cases, which differed by a single repeat unit (either TA or GA), the OriS sequences obtained by Illumina sequencing matched those previously obtained by PCR and Sanger sequencing  see Tab and is cTwo R5 alleles were observed that segregated completely by genotype and did not vary throughout the outbreak . AlthougTogether, these data are consistent with a pattern of lineage coevolution involving the R1 repeat region and the OriS  (−6 substitutions per site per year)  . Previouer year) –36. Althr year) – rather tr year) –. Based or year) –, we estir year) –. We origr year) –. Sequencr year) –. Howeverbind RNA –40. The The structure and sequences of the Guinea-Bissau clade 5 repeat regions R2 to R5 and OriS overlap the repeat sequences found in other clades . The exception is the R1 tandem repeat, the C-terminal end of which contains densely repeating aspartic acid and glutamic acid residues, making it more acidic and hydrophobic than the R1 repeats found in clades 1 to 4 . ORF11 is expressed as an immediate-early protein in keratinocytes  and has In summary, whole-genome sequencing of VZV has enabled us, for the first time, to study the dynamics of VZV transmission and evolution during a localized outbreak in Guinea-Bissau in 2001. These data have allowed us to accurately measure VZV short-term substitution rates and observe that all clade 5 viruses sequenced to date have a unique R1 repeat structure that codes for a more hydrophobic and acidic N terminus in the ORF11p. Guinea-Bissau clade 5 sequences cluster separately from clade 5 sequences obtained from European and U.S. subjects, suggesting a different evolutionary history, although sequencing of more African strains is needed to confirm this observation. Although most VZV remains highly conserved during epidemic transmission, our data imply that changes occurring in the R1 and OriS are associated with the evolution of new viral lineages.


Streptococcus pneumoniae isolates typically express one of over 90 immunologically distinguishable polysaccharide capsules (serotypes), which can be classified into “serogroups” based on cross-reactivity with certain antibodies. Pneumococci can alter their serotype through recombinations affecting the capsule polysaccharide synthesis (cps) locus. Twenty such “serotype switching” events were fully characterised using a collection of 616 whole genome sequences from systematic surveys of pneumococcal carriage. Eleven of these were within-serogroup switches, representing a highly significant (p < 0.0001) enrichment based on the observed serotype distribution. Whereas the recombinations resulting in between-serogroup switches all spanned the entire cps locus, some of those that caused within-serogroup switches did not. However, higher rates of within-serogroup switching could not be fully explained by either more frequent, shorter recombinations, nor by genetic linkage to genes involved in β–lactam resistance. This suggested the observed pattern was a consequence of selection for preserving serogroup. Phenotyping of strains constructed to express different serotypes in common genetic backgrounds was used to test whether genotypes were physiologically adapted to particular serogroups. These data were consistent with epistatic interactions between the cps locus and the rest of the genome that were specific to serotype, but not serogroup, meaning they were unlikely to account for the observed distribution of capsule types. Exclusion of these genetic and physiological hypotheses suggested future work should focus on alternative mechanisms, such as host immunity spanning multiple serotypes within the same serogroup, which might explain the observed pattern. Streptococcus pneumoniae is a major respiratory pathogen responsible for a high burden of morbidity and mortality worldwide. Current anti-pneumococcal vaccines target the bacterium’s polysaccharide capsule, of which at least 95 different variants (‘serotypes’) are known, which are classified into ‘serogroups’. Bacteria can change their serotype through genetic recombination, termed ‘switching’, which can allow strains to evade vaccine-induced immunity. By combining epidemiological data with whole genome sequencing, this work finds a robust and unexpected pattern of serotype switching in a sample of bacteria collected following the introduction of routine anti-pneumococcal vaccination: switching was much more likely to exchange one serotype for another within the same serogroup than expected by chance. Several hypotheses are presented and tested to explain this pattern, including limitations of genetic recombination, interactions between the genes that determine serotype and the rest of the genome, and the constraints imposed by bacterial metabolism. This provides novel information on the evolution of S. pneumoniae, particularly regarding how the bacterium might diversify as newer vaccines are introduced. Streptococcus pneumoniae is a human nasopharyngeal commensal bacterium and important respiratory pathogen. The ability of the pneumococcus to cause invasive disease appears critically dependent upon its polysaccharide capsule  or ATCC 700669 [EMBL accession code: FM211187]. Alternating orange and brown boxes indicate the individual COG sequences that comprise each transport system. Solid vertical black lines divide the sequences associated with different transport systems. Dashed vertical black lines divide the sequences associated with alternative alleles of the same locus. The likely substrates of each transporter are listed in (A) Maximum likelihood phylogeny, as displayed in (PDF)Click here for additional data file.S4 Figcps locus are grouped according to their inferred impact on serotype. (A) The number of base substitutions outside the cps locus introduced by recombinations overlapping with the cps locus. Values of zero indicate the recombination entirely lay within the cps locus; values greater than zero reflect the extent to which the recombination extended into the regions flanking the cps locus. (B) The number of base substitutions outside the cps locus introduced by recombinations occurring on the same branch of the phylogeny as a recombination overlapping with the cps locus. This shows the genetic diversity imported by recombinations that are likely to be contemporaneous, or near-contemporaneous, with a recombination affecting the cps locus.In each boxplot, the recombinations affecting the (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Tablecps locus are listed in the same order as in Recombinations affecting the (DOCX)Click here for additional data file.S3 TableS. pneumoniae TIGR4, or locus tag from S. pneumoniae ATCC 700669. The putative substrates are summarised from Bidossi et al.Each transport system is labelled with the relevant gene name, locus tag from (DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.