Rift Valley fever (RVF) is a zoonotic arboviral disease that is a threat to human health, animal health and production, mainly in Sub-Saharan Africa. RVF virus dynamics have been poorly studied due to data scarcity. On the island of Mayotte in the Indian Ocean, off the Southeastern African coast, RVF has been present since at least 2004. Several retrospective and prospective serological surveys in livestock have been conducted over eleven years (2004–15). These data are collated and presented here. Temporal patterns of seroprevalence were plotted against time, as well as age-stratified seroprevalence. Results suggest that RVF was already present in 2004–07. An epidemic occurred between 2008 and 2010, with IgG and IgM peak annual prevalences of 36% in 2008–09  and 41% , respectively. The virus seems to be circulating at a low level since 2011, causing few new infections. In 2015, about 95% of the livestock population was susceptible ). Monthly rainfall varied a lot (2–540mm), whilst average temperature remained high with little variation (about 25–30°C). This large dataset collected on an insular territory for more than 10 years, suggesting a past epidemic and a current inter-epidemic period, represents a unique opportunity to study RVF dynamics. Further data collection and modelling work may be used to test different scenarios of animal imports and rainfall pattern that could explain the observed epidemiological pattern and estimate the likelihood of a potential re-emergence. Rift Valley fever (RVF) is a viral zoonotic disease mainly present in Sub-Saharan Africa, transmitted by mosquitoes and primarily affecting livestock . The epidemiology of the disease is not fully known, mainly because of data scarcity. In Mayotte, an island close to Madagascar, RVF has been present since at least 2004, and retrospective and prospective surveys have been conducted to collect data from livestock over 11 years. Our work uses this 2004–15 dataset to describe the past and current epidemiology of RVF on the island. Results suggest that the disease was endemic between 2004–07. An epidemic occurred between 2008 and 2010. Since 2011, the virus appears to be circulating at a low-level causing few new infections. Modelling work may be used to explain the observed epidemiological pattern, and estimate the likelihood of a potential re-emergence. Phlebovirus, Family Bunyaviridae) primarily affecting domestic livestock . Epidemics of RVF in livestock mainly cause abortions and neonatal deaths. In humans, symptoms are usually non-specific causing an influenza-like syndrome, but sometimes infection can also result in meningo-encephalitis, haemorrhagic fever and death [Rift Valley fever (RVF) is a zoonotic arboviral disease . The hypotheses underlying RVF virus emergence is the concomitance of (i) the presence of susceptible livestock, (ii) an increase in vector abundance  thereby facilitating virus amplification and transmission, and (iii) the presence of the virus itself. The virus may be imported through the movements of infectious animals or arthropod vectors, or be present locally. RVF virus may persist between epidemics at a low level in livestock causing sporadic cases, or be maintained in potential local reservoirs. The latter includes vertical transmission in mosquito eggs and a large range of wild mammalian hosts, such as buffaloes, rodents and bats, although no good evidence exists on the latter hypothesis [RVF is transmitted to mammals mainly by mosquito bites  of RVF serological data, merging the 2004–08 dataset presented in Cetre-Sossah et al. , the 2012 (about 35km North-South and 10km East-West), with a high population density  serological data were collated from different sources. The 2004–08 data were retrospective serological surveys ; whilst All sera were tested for IgG ) or IgM ) antibodies by ELISA. For RVF, IgG antibodies are detectable in the blood of infected animals approximately 10 days post-infection (PI), and are believed to remain detectable for several years PI. IgM are detected earlier (approximately 5 days PI), but remain detectable for only approximately 3 months PI [Cetre-Sossah et al. 2012 [These surveys  aimed at further exploring the geographical extent of past RVF infections, and analysed sera collected between June 2007 and May 2008, from herds sampled across the island. Finally, surveys 1–4 explored whether RVF had been present in Mayotte before the introduction of the 2006–2007 eastern African Kenya-1 lineage. These tested sera were randomly selected among sera collected for brucellosis prophylaxis (between 2004 and 2007), which were stored at the Veterinary Services offices. For surveys 1–5, sera were tested for IgG only.The first longitudinal study was conducted in small ruminants between June 2008 and February 2009. A total of 272 animals were sampled on the first occasion in June 2008, from 13 herds. These herds were selected across the island. Three months later, in September 2008, 76 animals were resampled, drawn only from herds negative in June 2008; and 79 animals from these herds were sampled again in February 2009  of Mayotte. Before 2015, animal sampling in this study was not subject to the approval of ethics committee nor to specific national of international regulations at the time of sample collection. Consent for blood sampling on a herd was obtained from its owner verbally after information in French  or Shimaore  was given. The animals were bled without suffering. No endangered or protected species were involved in the survey. For the 2015 data, all procedures on living animals were approved by the London School of Hygiene Animal Welfare and Ethical Review Board.Rainfall and temperature are known to drive vector abundance and competence. The climate in Mayotte is marine tropical, with little temperature variation (average year round 24–34°C), but important rainfall (1500mm on average per year). Two main seasons are observed: hot rainy (December to March), and dry cool (June to September) . MonthlyThe total dataset included the results of 5720 samples from 3529 animals , sampled from and collected over 11 years (October 2004 to June 2015). All 5720 blood samples were tested for IgG, and 26.5% (n = 1513) were tested for both IgG and IgM. The dataset used the data from surveys 1 to 5, 6c, 7a and 8 , definedfrom the aod R package [y to June of year y+1. This temporal aggregation  allowed estimating annual prevalences by capturing each rainy season as a whole, as opposed to aggregating by calendar year.To look for the potential variation of RVF seroprevalence over the 2004–15 period, monthly and annual IgG and IgM prevalence estimates were plotted against time. For both IgG and IgM, monthly and annual prevalence were estimated as the number of RVF positive animals per month (or year) divided by the number of animals tested in that month (or year), accounting for clustering using the one-stage cluster sampling method varbin,  package . When anThe livestock national identification database implemented in 2008  providedAfter examining the annual IgG and IgM prevalences , and IgGPhase 1 is the period with less information available. It includes the first four epidemiological years 2004–05, 2005–06, 2006–07 and 2007–08, during which IgG annual observed prevalence remained at 15% or less . Newly iPhase 2 (2008–09 and 2009–10) suggests recent transmission of the virus. Indeed, IgG annual prevalences were significantly higher compared to the rest of the study period, reaching their maximum average value of 36% in 2008–09 . In addition, Phase 3 (2010–11 to 2014–15) suggests a decrease in RVF virus transmission. IgG annual prevalence was significantly lower than during phase 2, at approximately 10–15% , and a sFinally, monthly rainfall pattern, and average monthly rainfall are shown in The analyses of serological data showed that Mayotte probably experienced an RVF epidemic in livestock around 2008–10. Peaks of above-average rainfall were observed during the epidemic phase, while variation of temperature was limited. The RVF virus seems to have remained endemic at a low level since 2011, causing few new infections. In 2015, about 95% of the livestock population was susceptible.Serological data were collected throughout the period 2004–15, although not in a standardised manner. Retrospective data were obtained from sera stored at the Veterinary Services office. These sera were collected prior to RVF detection on the island, and it was not possible to determine exactly how this sampling was conducted in livestock. Although these results are not of a comparable value to the SESAM dataset, it gives precious information on past RVF infections, and evidence of on-going RVF virus transmission in 2008 in animals (IgM positive), shortly after the newly imported RVF strain was sequenced from humans. From 2009 onwards, farms were sampled from the CAPAM official registry . Since MFew animals were sampled in surveys 1–4 , which rAe. aegypti to survive the dry season [There is seasonality in rainfall, with very dry months in June-September, and extremely wet months, especially from December to March . In our dataset, however, high IgM prevalences in 2009 with IgM positive animals across the whole island were detected during the dry season. This suggested a large epidemic but does not support a direct correlation between rainfall and new cases. In other ecosystems, such as in the Horn of Africa or Southern Africa, unusually heavy rainfall or an increase in vegetation density were observed from one to six months before the emergence of new RVF cases –21. TherCulex pipiens and Aedes taeniorhynchus, two RVF vector species, showed that temperature above 26°C favoured RVF virus amplification and transmission [Very little variation was observed in temperature over the period 2005–15. During the 2010 epidemic in South Africa, temperature above 25°C was the most important risk factor , and expsmission ,25. The There is no information on the virus lineage that circulated in 2004–07. The sequencing of the Mayotte 2008 lineage placed the virus into the East African clade that includes the Kenyan 2006–2007 and Madagascar 2008 lineages ,26. This(i) the presence of susceptible livestock, (ii) an increase in vector abundance , and (iii) the presence of the virus emerging from local reservoirs or newly introduced. The data presented here suggest that Mayotte currently meets two of the conditions for re-emergence, that are: (i) a high proportion of susceptible livestock that reached about 95% in 2015, and (iii) the presence of the virus, evidenced by the new infections observed in phase 3. Therefore, we hypothesize that with heavy rainfall, such as it was observed in 2008–10, RVF virus could re-emerge. Modelling work was done to assess whether climate pattern could favor RVF virus persistence in Mayotte, which appeared to be true even under very low transmission assumption [The working hypotheses underlying RVF virus re-emergence presented in the introduction are the concomitance of sumption . FurtherIn conclusion, this study has shown the value of repeated serological testing to explain RVF population dynamics in this island population despite limited resources. Linking these ongoing studies with additional data and modelling could also shed further light on the origin and re-emergence mechanisms of this virus.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Giant viruses have revealed a number of surprises that challenge conventions on what constitutes a virus. The Samba virus newly isolated in Brazil expands the known distribution of giant mimiviruses to a near-global scale. These viruses, together with the transposon-related virophages that infect them, pose a number of questions about their evolutionary origins that need to be considered in the light of the complex entanglement between host, virus and virophage genomes.http://www.virologyj.com/content/11/1/95.See research article:  Acanthamoeba polyphaga mimivirus was the first of the giant DNA viruses to be discovered, initially isolated in the search for the causative agent of pneumonia during a hospital outbreak in Bradford, UK ]. Wh]. WhEmil]. Wh]. WThe genome sizes of the NCLDVs vary greatly, from the 150 kb genomes of the poxviruses to the 2.5 Mb genomes of pandoraviruses . MoreovCafeteria roenbergensis virus (CroV) [[Giant viruses reproduce in ‘viral factories’, which are cytoplasmic compartments of the host cell that can be as large as the nucleus Figure . Accompa (CroV) .Virophages are related to a class of eukaryotic DNA transposons called Mavericks (or Polintons). These genomic parasites share a set of four core genes , as welGene flow has played a central role in the evolutionary history of virophages. Integrated virophages have been found in a mimivirus genome, and virophage genes also share similarity to genes in other DNA transposons, such as a class of linear plasmids called transpovirons that are also found in mimiviruses . Some vOne proposal to explain the endogenization of virophages is that it could have been positively selected for, since the association with a virophage is beneficial to the host cell (owing to its interference with the replication of the large DNA virus) . The suThe discovery of giant viruses has crossed some of the boundaries between viruses and cellular life, although ribosomes remain a distinguishing feature. The conflict between giant viruses and their hosts, with the former also infected by virophages, alongside genomic invasions with related transposons, is reminiscent of Darwin’s tangled bank, recapitulated at the microscopic scale in a droplet of water. Elucidating the role of gene flow between these microscopic entities will reveal their evolutionary dynamics and aspects of the origins of viruses and cellular life.


The 2014–15 Ebola virus (EBOV) outbreak, originally reported during March 2014 in the Western African nation of Guinea, has shown itself to be resistant to traditional containment methods, with over 28,000 infections and 11,000 deaths over 18 months. Recently, news that a Scottish nurse had relapsed to EBOV disease with neurological symptoms at 10 months after recovery have astonished experts. The prolonged nature of the outbreak has led to questions whether EBOV can become endemic in the human population, an undesirable outcome due to the large amount of resources required to keep this virus under control. In this commentary, we discuss aspects EBOV disease with those caused by pathogens considered endemic in humans, as well as factors which may contribute to sustained EBOV transmission in humans.Ebolavirus and family Filoviridae, was historically regarded as a re-emerging pathogen, in which natural infections of humans are sporadic and unpredictable, often with several years passing by before the emergence of a new case. EBOV infection is associated with high case fatality rates (CFR) in humans (up to 90%), but past disease outbreaks due to EBOV lasted only several months on average, and fatal cases numbered at most in the hundreds , of the genus CDC.gov, . As suchCDC.gov, .Continued efforts by the local and international communities are having an observable effect in mostly confining EBOV transmission within the countries mentioned above, and the numbers of new weekly cases have been declining from a peak of over 700 in mid-September 2014 WHO.int, . Since ACan EBOV become endemic in the human population? If we contemplate other pathogens that are considered endemic within humans, such as Hepatitis B virus (HBV), Varicella Zoster virus (VZV) and human immunodeficiency virus (HIV), patients exposed to these viruses very rarely display major, specific symptoms early in the course of infection. In many cases the patients themselves do not know that they have been infected, and thus unknowingly transmit the virus to other susceptible hosts. In contrast, EBOV infection is typified by general, flu-like symptoms that rapidly progresses to severe hemorrhagic fever and multiple-organ failure within between 7–14 days, accompanied by very visible signs of disease such as rash (Feldmann et al., On the other hand, live virus has been found in the semen of convalescent survivors at least 3 months after infection (Rowe et al., −3 substitutions per site per year (Tong et al., Ebolavirus. Published studies have also shown that EBOV is not significantly evolving, despite undergoing extensive passaging in humans during the 2014–15 outbreaks (Hoenen et al., From an evolutionary standpoint, EBOV does not have a significantly higher mutation rate (1.23 × 10


The many different species in close proximity make zoological collections a unique environment for disease transmission. Bovine Viral Diarrhea Virus (BVDV) is of special concern with zoos due to the numerous exotic ruminant species that this virus can infect. BVDV occurs as both a non-cytopathic and a cytopathic strain both of which are capable of infecting exotic ruminants. The cytopathic strain causes mucosal disease (MD) and death. Infection with the non-cytopathic strain may produce persistently infected (PI) animals. PI individuals may show vague clinical signs, including abortion. Management of BVDV in zoos should focus on identification of PI individuals and prevention of infection of other animals of the collection. Variability makes serological testing as the sole method of screening for BVDV infection undesirable in exotic ruminants. Combination testing provides a definitive answer, especially in sensitive wildlife. Use of a combination of antigen-capture ELISA (ACE) with haired skin, Real Time-PCR (RT-PCR) on whole blood, and antibody detection via serum neutralization has the greatest potential to identify PI animals. An animal that is positive on both ACE and RT-PCR, but is negative on serology should be considered highly suspicious of being a PI, and should be isolated and undergo repeat testing 4–6 weeks later to confirm positive status. This testing methodology also allows screening of pregnant and newborn animals. Isolation or culling may need to be considered in animals determined to be positive via combination testing. These decisions should only be made after careful consideration and evaluation, especially with endangered species. Pestiviruses, within the family Flaviviridae, are viruses that may persist undiagnosed in exotic animals and zoological collections. To understand how this happens, and the unique concerns when addressing zoo animals, one must first understand some of the nuances of zoos and zoo collection management. There are few places in the natural world that rival the species density and diversity as modern zoos and wild animal parks. Interactions may occur in species that have overlapping natural environments, but rarely encounter each other in the wild. Modern zoos also serve as breeding facilities for endangered wildlife, and as a result may unintentionally provide perpetuating sources of infection or reservoir hosts for pathogens due to attempts to breed from very limited founder populations Mohamed, .Most public zoos in America and Europe have experienced a positive shift over the last 30 years away from traditional bare, concrete floored, and chain-link fence enclosures. These traditional displays allowed easy viewing by zoo patrons but may have further perpetuated the spread of diseases by increasing animal stress, which may result in weakened immune function, along with exposure to feces and other contaminants that can serve as sources of infection. Unfortunately, there are still privately owned facilities where animals exist in these conditions. The mixing of species and space limitations in a zoo can create a unique environment for transmission of diseases. Traditional mixed species displays, such as an “African Savannah” or “Asian Rainforest,” combining different species can inadvertently serve as sources of infection to novel hosts. Newer management practices, such as rotational displays designed to allow movement between separate, but shared display areas through the day have maximized space and environmental enrichment for zoo animals, but may also result in direct and indirect mixing of species in a manner that results in exposure to novel diseases Nolen, .Zoo veterinarians must remain vigilant for disease causing organisms capable of infecting multiple hosts. Obviously, the viral and bacterial diseases usually receiving the most attention for routine surveillance and prevention are those capable of causing zoonotic infection in humans. Infection of any zoo animal with a reportable disease having public health importance strikes fear in all associated zoo veterinarians and zoo administration  protecting the visiting public from disease exposure while protecting the animals from exposure from humans (2) protecting the collection from disease exposure from each other and (3) preventing the introduction of disease into regions around the zoo.Pestiviruses have been historically classified into four phylogenetic groups: bovine viral diarrhea virus serovar 1 (BVDV-1), bovine viral diarrhea virus serovar 2 (BVDV-2), classical swine fever virus (CSFV), and border disease virus (BDV) of sheep , white-tailed deer (Odocoileus virginianus), elk (Cervus elaphus), Japanese serow (Capricornis crispus), Canadian bison (Bison bison bison), water buffalo , roe deer (Capreolus capreolus), mouse deer (Tragulus javanicus), red deer (Cervus elaphus), bongo (Tragelaphus eurycerus), eland (Taurotragus oryx), wildebeest (Connochaetes sp.), nilgai (Boselaphus tragocamelus), axis deer (Axis axis), and barasingha deer (Cervus duvaucelii)  have been identified, and cytopathic and non-cytopathic biotypes are described according to their effects in cell culture  Brock, . In a doIn a zoo, the clinical signs of diarrhea and weight loss associated with MD will result in animals being held off displays, quarantined, and diagnostic testing performed to determine the cause. Unfortunately, by the time clinical signs are observed, the virus has already contaminated the environment. To reduce possible transmission, all incoming animals should be quarantined appropriately for their species and tested for BVDV. The identification of asymptomatic PI individuals that are new additions, as well as those existing within the collection will help to control new sources of infection. Asymptomatic PI individuals present the greatest threat to zoo collections, especially to mixed species collections or those utilizing rotational exhibits. The focus for management of BVDV in zoos should be determining infected, especially PI, individuals and taking appropriate steps to prevent and control the spread of the virus. While vaccination is utilized to prevent fetal infections in domestic cattle, vaccination for BVDV has not been well studied and remains unproven in exotic species. In addition, the diagnostic tests used for domestic cattle likely have not been validated in exotic ruminants, and as said before, there is no pathognomonic histopathologic lesion described for BVDV BVD, . DefinitTragelaphus spekii). In that same study Axis deer, Barbary sheep, Water deer, Dorcas and Fallow deer showed no evidence of antibodies in blood sera , 11 Barbary sheep, (Ammotragus lervia), 57 pygmy goats (Capra hircus), nine Angora goats (Capra hircus), 21 mountain goats (Capra aegagrus-aegagrus), seven llamas (Lama glama), eight Persian goitred gazelle (Gazella subgutturosa subgutturosa), seven Caspian red deer , two fallow deer (Dama dama), and two camels (Camelus dromedarius) in two Turkish zoos showed negative serum antibodies to BVDV  on haired skin with Real Time-PCR (RT-PCR) on whole blood  and antibody detection via serum neutralization has the greatest likelihood of identifying PI specimens and those that may be transiently infected Brock, . Haired Pregnant females with a screening serum antibody titer to BVDV may have been exposed to the virus within the first trimester of pregnancy and as a result be carrying a PI fetus. These animals should be quarantined until the offspring is born then thoroughly screened for BVDV. The offspring should be tested for persistent infection via whole blood RT-PCR in combination with ACE or immunohistochemistry on a haired skin sample (Walz et al., Because of risk of transmission, it is not wise to introduce any viremic animals to others that may be in the first trimester of gestation BVD, . DependiBVDV is a virus capable of infecting exotic ruminants, many of which are commonly housed in zoos. Animals persistently infected with BVDV present the greatest danger to a zoological collection. Because there is no cure for BVDV, management practices in zoo collections must focus on detection of PI individuals and the prevention of the spread of the virus. Veterinarians must realize that while convenient and economical, serologic testing alone is not sufficient to rule out BVDV infection in zoo animals. Combination testing utilizing ACE from haired skin with RT-PCR has the greatest likelihood of identifying PI specimens and those that may be transiently infected. This should serve as the current “Gold Standard” for thorough survey of zoological collections.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human OA chondrocytes were encapsulated and cultured for 28 days either in chitosan-alginate beads or in alginate beads. The beads were formed by slowly passing dropwise either the chitosan 0.6%–alginate 1.2% or the alginate 1.2% solution through a syringe into a 102 mM CaCl2 solution. Beads were analyzed histologically after 28 days. Interleukin (IL)-6 and -8, prostaglandin (PG) E2, matrix metalloproteinases (MMPs), hyaluronan and aggrecan were quantified directly in the culture supernatant by specific ELISA and nitric oxide (NO) by using a colorimetric method based on the Griess reaction. Hematoxylin and eosin staining showed that chitosan was homogeneously distributed through the matrix and was in direct contact with chondrocytes. The production of IL-6, IL-8 and MMP-3 by chondrocytes significantly decreased in chitosan-alginate beads compared to alginate beads. PGE2 and NO decreased also significantly but only during the first three days of culture. Hyaluronan and aggrecan production tended to increase in chitosan-alginate beads after 28 days of culture. Chitosan-alginate beads reduced the production of inflammatory and catabolic mediators by OA chondrocytes and tended to stimulate the synthesis of cartilage matrix components. These particular effects indicate that chitosan-alginate beads are an interesting scaffold for chondrocytes encapsulation before transplantation to repair cartilage defects.This  It is the most prevalent form of arthritic disease in the world. It is mainly characterized by the slow progressive degeneration and the loss of articular cartilage accompanied by modification of the subchondral bone and synovial membrane inflammation . In cartide (NO) .Since no cure has been discovered so far for OA, the management of OA is focused on the control of symptoms, i.e. pain and loss of articular function. The guidelines recommend a combination of non-pharmacological  and pharmacological modalities  as the most effective strategy to manage pain and disability associated with OA –10. An aAside these treatments, debridement , chondraOne promising approach to repair cartilage is the transplantation of autologous chondrocytes or stem cells into cartilage defect using natural or synthetic scaffold . To perfin vitro studies have showed that chitosan or modified chitosan promoted the expression of cartilage matrix compounds by chondrocytes [Chitosan is a linear natural polymer of D-glucosamine with a variable frequency of N-acetyl-D-glucosamine units that is the product obtained after chemical N-deacetylation treatment of chitin  which isdrocytes –33. Thesin vitro the biological behavior of human OA articular chondrocytes embedded in this biomaterial.The aim of this work was to prepare a biocompatible three-dimensional biomaterial by mixing chitosan and alginate, and to investigate Human cartilage was obtained from knee joints of 16 different OA patients, after total knee replacement surgery with a mean ± SEM age of 72.53 ± 7.09 and ranged between 61–83 years. Upon dissection, the femoral, tibial and patellar articular surfaces were evaluated for the pathological cartilage modifications according to the Moskowitz scale . All carCartilage was cut into small fragments and submitted to sequential enzymatic digestion with hyaluronidase, pronase and collagenase, as previously described . The cel6 cells/mL as previously described by Sanchez et al. [The alginate beads were performed by suspending chondrocytes in 1.2% (w/v) low viscosity alginate  solution at a density of 6 x 10z et al. .6 cells/mL. This mixture was slowly passed dropwise through a syringe into a 102 mM CaCl2 solution (Sigma). After instantaneous gelation the beads were incubated for 10 min in the CaCl2 solution and washed with saline solution. The chitosan used to prepare the beads had a molecular weight of 30,700 Da and 15.7 mol % of degree of acetylation. To visualize the chitosan distribution, some beads were prepared using similar chitosan labeled with fluorescein isothiocyanate . The viscosity of chitosan-alginate solution was 294 cP ). The osmolarity of the chitosan-alginate solution was 256.33 ± 1.53 (mean ± SEM) mOsmol/kg using the Osmometer automatic type 15 . The pH, measured at 19°C with Microprocessor pH-mV Meter pH 526  of chitosan-alginate solutions was 7.35 ± 0.05 (mean ± SEM). The preparation of CA beads was performed by passing droplets thought a syringe with a 25 G needle. This method produced spherical beads  beads, 1.33% (w/v) chitosan solution and 2.4% (w/v) alginate solutions were prepared separately by dissolving 1 g of ultrapure medical-grade vegetable chitosan  in 75 mL 0.166 M acetic acid and 2 g low viscosity alginate in 83.3 mL 0.16 M NaOH. The solutions were then mixed to obtain a solution containing 0.6% (w/v) chitosan and 1.2% (w/v) alginate. Chondrocytes were suspended in the chitosan-alginate solution at the density of 6 x 10al beads  with a m2 in a humidified atmosphere. The culture medium was changed twice a week and the collected supernatants were kept at -20°C until analysis. Beads were maintained in culture for up to 28 days.Nine beads were placed in each well of a 24 wells plate and cultured in 1 ml of DMEM  supplemented with 10% foetal calf serum (Lonza), 10 mM HEPES (Lonza), 100 U/mL penicillin (Lonza), 100 μg/mL streptomycin (Lonza), 2 mM L-glutamine (Lonza), 50 μg/mL ascorbic acid (Sigma), 20 μg/mL proline (Sigma) at 37°C with 5% COBeads and supernatants were collected after key intervals throughout the culture period . The beads were then dissolved in 1 mL 0.1 M citrate solution. The suspension was centrifuged  at 300 g for 10 min. With this method, two fractions were collected: the supernatant containing macromolecules from the further-removed matrix (FRM) and a pellet containing cells with their associated cellular matrix (CM) . These t2. After mounting on an aluminum stub, they were platinum-coated (20 nm) in a Balzers SCD-030 sputtering unit. Some of them were teared to visualize their internal structure. The observations were realized in a SEM Jeol JSM-840A  under a 22kV accelerating voltage.Chitosan-alginate beads were observed both in an environmental scanning electron microscope (ESEM) in low vacuum "wet" mode and in a classical scanning electron microscope (SEM) under high vacuum conditions. For environmental SEM, the wet beads were directly placed on steel stub cooled at 2°C by a "Peltier" unit in the chamber of a FEI ESEM-FEG XL-30. The observation pressure started at 7 Torr and was progressively decreased to 2 Torr leading to the slow dehydration of the sample. The images were obtained using a gaseous secondary electron detector (GSED-Electroscan) with a 500 μm pressure limiting aperture (PLA) and under 12 to 20kV accelerating voltages. For classical SEM, the chitosan-alginate beads were first dehydrated in ethanol then critical point dried in COTEM observation was also realized on CA bead containing chondrocytes after 28 days of culture. Beads were fixed for 2 h at 4°C in 2.5% glutaraldelyde in 0.1 M sodium cacodylate (pH 7.4) and then for 1 h at 4°C in 1% osmium tetroxide in 0.1 M sodium cacodylate, pH 7.4. Samples were then dehydrated with increasing concentrations of ethanol and propylene oxide and embedded in LX112 resin  before polymerization at increasing temperatures . For orientation purpose, 2 μm sections were performed, stained with toluidine blue, and observed by light microscopy. Ultrathin sections (50–70 nm) were cut, stained with uranyl acetate, contrasted with lead citrate, mounted on uncoated grids, and observed by TEM .2 was added to prevent disintegration of the beads. After washing overnight at 4°C in 100 mM sodium-cacodylate buffer, the beads were dehydrated in graded series of methanol, placed in a xylene wash before being embedded in paraffin. Five μm sections were cut with a microtome  rehydrated and stained with hematoxylin-eosin, alcian blue or safranin-O/fast green. To visualize chitosan-FITC, hematoxylin-eosin stained sections of beads were observed using H3 filtercube (Leica). Alcian blue and safranin-O/fast green staining methods were used to analyze cartilage tissue formation. For safranin-O/fast green staining, sections were counterstained with hematoxylin  and fast green (Merck) to visualize cells and cell nuclei, respectively. Safranin-O (Sigma) was used for visualization of glycosaminoglycans (GAGs) in red. Sections were also stained with alcian blue solution (1% in acetic acid) to visualize extracellular GAGs in blue.At the end of 28 days of culture, beads were fixed with 4% paraformaldehyde in 100 mM sodium-cacodylate buffer, pH 7.4, for 4 h at 4°C; 20 mM CaCl+1, which is formed when Cu+2 is reduced by protein in alkaline environment . Results were expressed in μg/mL after 3 days, 21 days and 28 days of culture in text.The DNA content was measured in the CM fraction using the fluorimetric method with PicoGreen dsDNA reagent . Total protein was also quantified in the CM fraction utilizing bicinchoninic acid (BCA) as the detection reagent for Cu2  and MMP-1; -2; -9; -13  were measured in different compartments  after key intervals throughout the culture time  according to the manufacturer’s recommendations. No cross-reactivity existed between IL-6 and IL-8 assays. Less than 0.5% cross-reactivity and interference was observed between MMPs. The production of hyaluronan and aggrecan were expressed in ng/μg of DNA in text and, respectively, in figure and in table after 3 days, 21 days and 28 days of culture. The production of IL-6, IL-8, and PGE2 were expressed in pg/μg of DNA and MMP-3 in ng/μg of DNA after 0–3, 17–21, 24–28 days of culture in table. Results of IL-6, IL-8, PGE2 and MMP-3 were also expressed in percentage of production in comparison with alginate considered as control (100%) in table.Hyaluronan , IL-6, IL-8, aggrecan, MMP-3 , PGE3PO4) was mixed with 100 μL of conditioned culture medium or sodium nitrite . The absorption was measured at 540 nm. The NO production was expressed in nmol/μg of DNA in text.The spectrophotometric method based upon the Griess reaction was used to quantify nitrite in different compartments after key intervals throughout the culture time . Griess reagent . Experiments were performed in triplicate and the obtained values were average for the purpose of statistical analysis. Statistical significance was determined using GraphPad Prism software, version 6 for DNA, total protein, hyaluronan, aggrecan, IL-6, IL-8, PGETwo different techniques, SEM and ESEM, have been performed to observe the ultrastructure of the beads. Classical SEM pictures were the most relevant for illustrating the aspect of the CA beads . The surChondrocytes cultivated in CA beads during 28 days were observed by TEM . CA beadTo identify chitosan trabeculae, we have labeled chitosan with FITC . Fig 3B In CA beads, DNA content in CM fraction remained constant over time (3 days: 5.1 (2.4–7.9) μg/mL vs 21 days: 5.2 (3.2–7.2) μg/mL vs 28 days: 5.4 (3.1–7.6) μg/mL). In contrast, DNA content tended to increase after 21 and 28 days of culture in A beads  μg/mL vs 21 days: 6.7 (3.3–10.1) μg/mL vs 28 days: 7.1 (4.1–10.1) μg/mL) days in comparison to 3 days. Total protein content in A or CA beads did not change with time  μg/mL vs 21 days: 164.3 (136.2–192.3) μg/mL vs 28 days: 166.7 (134.4–199.0) μg/mL; CA beads: 3 days: 127.5 (103.2–151.8) μg/mL vs 21 days: 129.6 (106.7–152.6) μg/mL vs 28 days: 141.6 (106.2–177.0) μg/mL).Hyaluronan and aggrecan production was measured in culture supernatant, FRM and CM fractions. Hyaluronan was detected in these three fractions . No sign2 (p = 0.0056) after 3 days of culture . In comparison with A beads, CA beads significantly reduced the production of IL-6 (p = 0.0012), IL-8 (p<0.0001) and PGE culture . This siMMP-1, -2, -9, -13 productions were not significantly modified by matrix composition and by culture times (data not show). In comparison to A beads, the production of MMP-3 was significantly decreased in CA beads  after 3 This work describes an original method to incorporate cells in chitosan enriched matrix and the influence of this tridimensional matrix on chondrocytes metabolism. This patented method  consistsAgaricus bisporus mushroom.In this new biomaterial chondrocytes were like in cartilage matrix: scattered, isolated from each other, spherical and surrounded by typical organization zones of the matrix. Moreover, microscopic analysis has revealed that chondrocytes were in contact with chitosan trabeculae especially with territorial matrix. This fact possibly explained the particular behavior of chondrocytes when they are cultured in this biomaterial. Using FITC-labeled chitosan and electronic microscopy, we have observed that chitosan trabeculae formed a network filled by alginate fibers. Taken all together, these results showed that CA beads were an original and interesting biomaterial. Indeed, all methods used so far did not allowed alginate, chitosan and cell to mix. An alginate scaffold surrounded by a shell of chitosan , 40 has in vitro and in vivo studies have shown the major role played by IL-6, IL-8, NO and PGE2 in cartilage degradation [2 production [2, NO and IL-6 production by murine BV2 microglia cells [This study also demonstrated that the presence of chitosan in the extracellular matrix affected beneficially human chondrocytes metabolism. In summary, chondrocytes cultured in CA beads produced more GAGs but less pro-inflammatory mediators and catabolic factors. Clearly, these findings indicated that chitosan may exert a positive influence on chondrocyte and promote matrix deposition. Indeed, the presence of chitosan promoted the accumulation of GAGs around the cells revealed by blue alcian and safranin O/fast green staining. In addition, using an immunoassay specific for the aggrecan, we have shown that aggrecan contained in the surrounding matrix was higher in CA beads suggesting that CA matrix may accelerate aggrecan deposition in the extracellular matrix. These results confirmed a previous work demonstrating that aggrecan gene expression by chondrocytes cultivated on a pre-coated 4% chitosan coverchip for 7 days was higher than on uncoated coverslips . In contradation –47 and sradation , 49. In oduction . These dia cells . Anotheria cells  showed tAn important decrease of MMP-3 production was also observed in the CA beads compared to A beads suggesting an anti-catabolic effect of chitosan. Indeed, MMP-3 was shown to be responsible for the proteolysis of extracellular matrix proteins, mainly aggrecan  and of tOne limitation of this study was the absence of chitosan beads alone as control. The reason is that it was not possible to prepare chitosan beads with embedded chondrocytes that were suitable to culture. Indeed, chitosan solution must be prepared by chitosan powder dissolution in acetic acid. Chondrocytes were then suspended in this acid solution that was no appropriate with cells viability.These results suggest that the CA beads could be used as cell carrier to repair traumatic or degenerative cartilage lesion. Indeed, if implanted in cartilage lesion, chitosan-enriched biomaterial could promote cartilage matrix synthesis by chondrocytes but also decrease the local production of inflammatory and catabolic factors. These multiple biological activities confer to chitosan a benefit compared to other biomaterials. For example, a matrix composed with collagen or with hyaluronic acid failed to decrease pro-inflammatory and pro-catabolic mediators synthesis by chondrocytes , 60.in vitro test highlighted beneficial and promising effects of this new biomaterial on human OA chondrocytes. They produced less inflammatory and catabolic mediators and maintained the synthesis of cartilage specific matrix components. All these biological activities are beneficial for cartilage repair.CA beads could be used as carrier for cell transplantation, particularly to repair degenerative cartilage defect. This S1 File(PDF)Click here for additional data file.


Human oncogenic viruses include Epstein–Barr virus, hepatitis B virus, hepatitis C virus, human papilloma virus, human T-cell lymphotropic virus, Kaposi’s associated sarcoma virus, and Merkel cell polyomavirus. It would be expected that during virus–host interaction, the immune system would recognize these pathogens and eliminate them. However, through evolution, these viruses have developed a number of strategies to avoid such an outcome and successfully establish chronic infections. The persistent nature of the infection caused by these viruses is associated with their oncogenic potential. In this article, we will review the latest information on the interaction between oncogenic viruses and the innate immune system of the host. In particular, we will summarize the available knowledge on the recognition by host pattern-recognition receptors of pathogen-associated molecular patterns present in the incoming viral particle or generated during the virus’ life cycle. We will also review the data on the recognition of cell-derived danger associated molecular patterns generated during the virus infection that may impact the outcome of the host–pathogen interaction and the development cancer. Seven human viruses have been found so far to cause approximately 10–20% of human cancers worldwide . Today, Germline-encoded pattern-recognition receptors (PRRs) recognize chemically distinct moieties in microorganisms or “pathogen-associated molecular patterns” (PAMPs) . PRRs cacytoplasmic PRRs include the retinoic acid-inducible gene (RIG)-I-like receptors (RLRs)    and C-tys (RLRs)  and the s (RLRs) , 20, as s (RLRs) . The dous (RLRs) . Recentl “ALRs”) . Importa “ALRs”) . Some of “ALRs”) . Finally “ALRs”) . Each of “ALRs”) .There is abundant information on the mechanisms by which the seven oncogenic viruses block the molecular pathways of the innate immune system at the level of intracellular adaptors, and the reader is referred to the several extensive published reviews in the specific sections below. However, much less is known on the recognition of these viruses by the sensors that physically interact with viral PAMPs. Here, we will focus on the latest findings on the growing list of innate immune sensors that have been implicated in sensing each known human oncogenic virus Figure . We beliHepatitis C virus is a single-stranded RNA virus, with an enveloped nucleocapsid of about 50 nm. It is transmitted via parenteral route, and there are millions of people infected with HCV worldwide, for which there is no available vaccine . During trans. This was shown to be the case as well by Dreux et al., who reported the transfer of HCV–RNA containing exosomes from infected cells to pDCs  in HCV genome. In contrast, the virus’ 3′ poly-U/UC sequence, short dsRNA regions, and 5′ triphosphate of the uncapped HCV–RNA are recognized by RIG-I . AOver evolutionary times, the battle between the oncogenic viruses and their hosts has arrived at a balance that ensures the survival of both organisms. However, with the current advances in vaccinology and drug development, it is plausible to imagine that we are potentially getting closer to limiting the impact these seven viruses have on the population of the world. Although a complete understanding of all of the complexity of interactions with the native host for all of the oncogenic viruses discussed in this review is still lacking, it is clear that the innate immune system is able to recognize their presence through a network of sensors. Undoubtedly, the understanding of virus interactions with the innate immune system will aid in the development of effective treatments against these pathogens. More research is clearly warranted to devise effective approaches to harness the tools of the innate immune system for elimination of these viral pathogens without negatively affecting their hosts.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C. gattii were first identified in 2004. Cryptococcus gattii is considered to be an emerging non-zoonotic infection affecting animals and humans in Oregon. We report a longitudinal environmental isolation of C. gattii after an Oregon dog was diagnosed with the disease in 2009.In Oregon, human and animal infections by Cryptococcus gattii was isolated twice from the same location with a span of one year between isolation dates. Cryptococcus gattii molecular types VGIIa and VGI were isolated in 2010 from soil and tree bark near the home of a 9-month-old dog which three months previously had an infection caused by C. gattii genotype VGIIa. The environment featured heavy growth of Douglas Fir trees. In 2011, a second set of soil and tree bark samples was collected in the same area and C. gattii VGIIa was again identified from the environment, along with genotypes VGIIb and VGIIc.C. gattii should be considered to expand the understanding of this emerging pathogen. Understanding the ecology and how the environment and other factors might modify the existing niches is important for assessing risk and for designing measures to protect human and animal health.The use of animal surveillance data to identify environmental niches of  Cryptococcus causes pneumonia, skin infection and meningoencephalitis [C. neoformans is the most common cause of fatal meningoencephalitis, especially in HIV patients, C. gattii has caused disease mainly in non-HIV patients [C. gattii infections emerged on Vancouver Island, British Columbia (BC). Nearly all infections were caused by C. gattii genotype VGII, but the strains lacked the genetic diversity seen among other VGII isolates from other countries and appeared to represent distinct clonal populations, dubbed genotypes VGIIa and VGIIb [C. gattii spread to the BC mainland. In Oregon, human and animal infections by C. gattii were first identified in 2004, and they have subsequently been identified in Washington State as well [Cryptococcus gattii genotype VGIIc, which had not been identified in BC, has been diagnosed in animals and humans in the US Pacific Northwest (PNW) and seems to be centered in Oregon [Infection by the fungus phalitis -7. Whilepatients . In 1999nd VGIIb . During  as well . Interesn Oregon ,13.C. gattii infection is inhalation of spores from the environment. Estimation of the incubation period became possible with the identification of a focal reservoir of the fungus on Vancouver Island; an analysis of early cases among residents of the BC mainland with discreet histories of travel to Vancouver Island found incubation periods ranging from 2 to 13 months, with a median of 6–7 months [The only known means of acquisition of C. gattii infection were reported in Oregon, but early attempts to isolate the fungus from the environment around Oregon case residences were unsuccessful [C. gattii infection in domestic animals suggested that exposure to the fungus must have occurred near the animals’ residence and presented opportunity for identifying ecological foci of C. gattii in Oregon.From December 2004 through December 2012, 82 human cases of ccessful . In 2008Cryptococcus, and the isolate was sent to Oregon State University, Veterinary Diagnostics Laboratory  for further identification. It was identified as C. gattii, genotype VGIIa. The dog lived in a rural area at 177 feet elevation, heavily populated primarily with Douglas fir trees (Pseudotsuga menziesii). The property is located less than 30 feet from an unpaved logging road where several times a day logging trucks carry recently harvested Douglas Firs , woody debris (10–20 g), soil (50 g), and water (50–100 mL) were sampled as described by Kidd et al. [l-3,4-dihydroxyphenylalanine (DOPA) medium and incubated at 35°C for up to two weeks. Between one and six colonies were picked from each environmental positive sample and all picked colonies were genotyped. C gattii was confirmed using l-canavanine-glycine-bromothymol blue (CGB) differential medium [Environmental sampling was conducted in June 2010 at selected sites and areas surrounding the home environment of the dog with d et al. . Samplesl medium , and evel medium .A year later, May 2011, samples of soil and tree bark were again collected in the same area and at an additional location along the logging road.C. gattii; one soil sample had genotype VGIIa isolates, and another had isolates of both VGIIa and VGI (Table C. gattii isolates of genotypes VGIIa, VGIIb and VGIIc were identified from the bark and soil samples taken along the logging road bordering the same property. The C. gattii VGIIa isolates from soil and bark were indistinguishable from the VGIIa isolate obtained from the dog.Of the 12 samples obtained in 2010 around the home of the positive veterinary case, two yielded C. gattii from the environment in Oregon from the environs of a positive case. As was the experience in Vancouver [C. gattii in this biogeoclimatic zone southward from BC almost 500 miles to the Willamette Valley of Oregon. In BC, C. gattii was generally isolated from acidic soil, and geographic differences in soil pH may influence the extent of colonization reported. Cryptococcus gattii soil colonization also was associated with low moisture and low organic carbon contents although one of our positive soil samples came from a standing puddle. The role that trees play in the life cycle of C. gattii is not known, but the association of the fungus with decaying wood, especially Douglas Fir in the Pacific Northwest, but also many other species of tree is suggestive of an endophytic existence [This is the only longitudinal record of ancouver , isolatixistence ,16.C. gattii has proven difficult. After extensive sampling around human cases in Australia, Ellis and Pfeiffer reported the first environmental isolation of the fungus in 1990 from eucalyptus trees and noted that the distribution of cases was similar to that of eucalyptus trees [C. gattii has been isolated from a variety of trees and from soil samples in Coastal Douglas Fir and in the Coastal Western Hemlock xeric maritime biogeoclimatic zones. Similar climatic areas are found in Oregon’s Willamette Valley [C. gattii zone may be expanding, accentuated by the increase of logging in the coastal temperate rain forest zone which continues to be widespread, generally beginning in the easily accessible lowlands and working upslope.Perhaps because of the long potential incubation period, during which individual human cases may be exposed to a variety of biogeoclimatic areas, pinpointing environmental niches for us trees . In BC, e Valley . The C. Three features are common to all coastal temperate rain forests: proximity to oceans, the presence of mountains, and as a result of the two, high rainfall .C. gattii from the environment has been challenging in other areas of the PNW [C. gattii isolates will better ascertain the natural source and genomic adaptations of the infection in the Pacific Northwest. Based on a recent study VGII study was highly diverse, demonstrating large numbers of mutational and recombinant events. The three dominant subtypes in the Pacific Northwest were low diversity and completely clonal [Isolating  the PNW ,17 (R. W the PNW . With thy clonal .C. gattii environmental niches, as they are ultimately the source of animal and human infections. Although rare, C. gattii appears to be an emerging pathogen and understanding the ecological niches and how environmental or other factors might modify the existing niches is important for assessing risk and for designing measures to protect human and animal health.The use of animal surveillance to identify environmental sources of the disease should be considered to expand understanding of This publication was supported by Cooperative Agreement Number 3U01CI000306 from the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention.No human subjects, human materials, or human data was used.


Despite a relatively long search for the origin of ebolaviruses, their reservoirs remain elusive. Researchers might have to consider testing alternative hypotheses about how these viruses persist and emerge to advance ebolavirus research. This article aims to encourage researchers to bring forward such hypotheses, to discuss them scientifically and to open alternative research avenues regarding the origin and ecology of ebolaviruses. Epomops franqueti), hammer-headed fruit bats (Hypsignathus monstrosus), and little collared fruit bats (Myonycteris torquata)). Consequently, the majority of the tropical belt of Africa is considered to be at risk for EVD outbreaks [The largest Ebola virus disease (EVD) outbreak that has ever been recorded is currently waning. However, the ecology of the causative agents of EVD (Bundibugyo virus (BDBV), Ebola virus, Sudan virus (SUDV), and Taï Forest virus (TAFV)) is still a mystery and future outbreaks cannot be predicted . The curutbreaks . Criticautbreaks . AdditioExcellent and enormous research efforts on bats have brought insight into filoviruses’ ecology ,3. Such A potential connection between ebolaviruses and rivers is just one proposed hypothesis to call for alternative research avenues and substantial broadening of multi-disciplinary collaborations. Rivers have their own unique flora and fauna in the tropical ecosystem and many ecological aspects of rivers that might be important key events for viral emergence have not been considered. Further, it might be presumed by many that ebolaviruses are limited to terrestrial animal hosts. However, aquatic or semi-aquatic animals could potentially represent reservoir hosts and provide ebolaviruses with an ecological link from an original aquatic environment to the terrestrial environment. Of course, rivers are found anywhere in tropical Africa and, typically, human settlements occur along rivers. However, the possibility of viral persistence in hosts in or by rivers and river beds should not be disregarded as part of ebolavirus ecology without good reasons. Viral persistence in local riverine hosts might have been the cause of the so-far unexplained high genetic similarity between EBOV isolates from human outbreaks in Luebo, Democratic Republic of the Congo, that occurred in two consecutive years . A systeEphemoptera, more than 3000 recognized species) are evolutionary ancient aquatic insects. They spend most of their lives as eggs or larvae in sediment and other material under water where they feed on biological material for up to two and a half years before they emerge from the aquatic habitat as adults, sometimes in large masses [In line with the river hypothesis, riverine insects that are occasionally present in bats’ diets or are otherwise overlapping ecologically may be providing the virus with an ecological link from an original aquatic or semi-aquatic environment to terrestrial hosts. Virus may be concentrated or perhaps even activated through particular bat feeding and digestion processes. Early research indeed investigated the potential role of insects as hosts of EBOV, although few experimental infections were done and no ebolavirus was identified in the subset of insect diversity that was collected during post-outbreak screenings ,15,16. Ne masses ,18. For e masses . This exRousettus aegypticus) as natural reservoirs, although some important questions remain unanswered [i.e., close to the cave mouth and relatively far down on the walls, overlaps with preferred spots for specific insects and predisposes them to infection.The ecology of filoviruses other than ebolaviruses ,21 is alanswered ,4. It apanswered ,23. Howeanswered ,24,25. IThe many “perhaps” and “possibly” in this article reflect the need to investigate a number of aspects of a potential complex filovirus ecology; it presents a unique challenge to unite researchers from a number of fields to synergistically discuss and explore new research avenues. The hypothesis that rivers are important for ebolavirus ecology may indeed prove wrong. However, such hypotheses may invite researches to take an ecological view and critically reconsider and review if there are other factors that influence filovirus disease outbreak patterns more than the general distribution of the various bats that so far have been implicated. EBOV, and other filoviruses, provides us with an opportunity to work on the One Health concept in the broadest sense by expanding multidisciplinary brainstorming to include experts in fields such as entomology, hydrology, geology, and riparian forest biology to discover factors and patterns that might not have previously been considered important for infectious disease emergence . Importa


Adherence to antiretroviral treatment (ART) is critical to maintaining health and good clinical outcomes in people living with HIV/AIDS. To address poor treatment adherence, low-cost interventions using mobile communication technology are being studied. While there are some studies that show an effect of mobile phone reminders on adherence to ART, none has reported on the costs of such reminders for national AIDS programmes. This paper aims to study the costs of mobile phone reminder strategies  to support adherence in the context of India's National AIDS Control Program (NACP).The study was undertaken at two tertiary level teaching hospitals that implement the NACP in Karnataka state, South India. Costs for a mobile phone reminder application to support adherence, implemented at these sites  were studied. Costs were collected based on the concept of avoidable costs specific to the application. The costs that were assessed were one-time costs and recurrent costs that included fixed and variable costs. A sequential procedure for costing was used. Costs were calculated at national-programme level, individual ART-centre level and individual patient level from the NACP's perspective. The assessed costs were pooled to obtain an annual cost per patient. The type of application, number of ART centres and number of patients on ART were varied in a sensitivity analysis of costs.The Indian NACP would incur a cost of between 79 and 110 INR (USD 1.27–1.77) per patient per year, based on the type of reminder, the number of patients on ART and the number of functioning ART centres. The total programme costs for a scale-up of the mHealth intervention to reach the one million patients expected to be on treatment by 2017 is estimated to be 0.36% of the total five-year national-programme budget.The cost of the mHealth intervention for ART-adherence support in the context of the Indian NACP is low and is facilitated by the low cost of mobile communication in the country. Extending the use of mobile communication applications beyond adherence support under the national programme could be done relatively inexpensively. Adherence to antiretroviral treatment (ART) is critical for ensuring good clinical outcomes in people living with HIV/AIDS (PLHIV) , 2. ThisIndia is home to 2 million of the 33 million PLHIV globally with a national HIV-infection prevalence of 0.3% , 8. To pMobile phone applications that support treatment adherence address forgetfulness; an important barrier to treatment adherence, even in the Indian context , 8, 11. While most published studies focus on the effects of the intervention, few have obtained and analysed primary cost data and estimated mHealth intervention costs at scale , 20. In The study setting included two tertiary level hospitals that implement the NACP through ART centres on their premises. St. John's Medical College Hospital, a private non-profit teaching hospital, in Bangalore, Karnataka, implements the NACP through a public–private partnership model. The ART centre at this hospital is responsible for the treatment and follow-up of approximately 1,500 PLHIV enrolled under the NACP. The other was a public tertiary care teaching hospital in Mysore, also in Karnataka. This hospital manages the treatment and follow-up of 2,500 PLHIV under the NACP.Karnataka with a population of 61 million and an HIV-prevalence rate of 0.63% is one of four HIV high-prevalence States in India . As of DThe mHealth intervention that has been costed in this paper was developed for use in the HIVIND trial. The HIVIND trial  is a ranThe mHealth intervention deployed in the HIVIND trial had two components; the first was an automated IVR call and the second was a passive, neutral, picture message (SMS). Every patient in the intervention arm of the trial received each component of the mHealth intervention once a week. Details of the intervention are reported in the HIVIND trial protocol .The mHealth intervention that has been costed in this paper is from the HIVIND trial. Hence, costs that were not considered included the additional costs incurred to follow the trial protocol and maintain the research quality in the trial context. These costs would not be incurred in a routine programme setting. In view of the relatively limited time period used for estimating costs, discounting was not applied on costs or benefits.We considered the mobile-solutions agency that developed and managed the mHealth intervention for the trial to be the most economical as they offered the lowest price through a competitive bidding process for developing and deploying the intervention. In the HIVIND trial with 631 patients, 316 received the mHealth intervention. Further, each 30-second IVR call was priced at 1.50 Indian Rupees (INR), while the SMS was priced at 0.20 INR. If the same intervention were implemented at scale, the prices would decrease to 0.24 INR per 30-second IVR call and 0.19 INR per SMS if the number of calls and SMSs were to exceed 500,000, that is, when patient numbers exceed 10,000.Costs for the mHealth intervention were studied from the perspective of the Indian NACP. Costs were collected based on the concept of avoidable costs specific to the mHealth intervention. The concept of avoidable costs refers only to the inclusion of costs that are contingent on the mHealth intervention, and all other costs were considered as sunk costs; that is, costs incurred even if the intervention was not undertaken. Sunk costs (e.g. costs of buildings) were not included in the study. The costs that were assessed were one-time costs as well as recurrent costs (the latter included fixed and variable costs). In addition, A sequential costing procedure was used. It involved first identifying the resource used in natural units (minutes of staff time and/or other relevant units), measuring the resource used and subsequently pricing the resource using standardized prices.Costs that are incurred only once during the entire programme (i.e. one cycle of the programme =5 years), primarily mHealth intervention–development costs.These are costs incurred every year. They include fixed costs and variable costs. Fixed costs do not vary with a change in the amount or quantity of an output or service . Variable costs are costs that are dependent on the quantity of output (e.g. the number of beneficiaries of a service).The costs studied were stratified at three different levels in the deployment of the mHealth intervention: (1) national-programme level (2) ART-centre level and (3) patient level.These were costs incurred for the development of the mHealth intervention, costs for recording the mHealth intervention in different languages  and costs for the development of the web interface . The web interface facilitated the technical monitoring of the mHealth intervention. The above costs were those paid to the mobile-solution agency in the HIVIND trial. Costs for equipment consisting of a laptop and a mobile phone used for monitoring the mHealth intervention and communicating with other ART centres deploying them were also considered under one-time costs.These included the costs for the maintenance of the hardware and the development and maintenance of the web interface. A proportion of the salary for the programme officer responsible for the nationwide deployment of the mHealth intervention through the NACP was also included. Overheads at the programme level were calculated as a proportion of fixed costs.These included the costs of equipment . These one-time costs were calculated based on costs incurred at the ART centre, St. John's Medical College Hospital.These were the fixed costs incurred by the ART centre, St. John's Medical College Hospital and included the staff costs: (1) 10% annual time of a centre functionary, that is, an ART medical officer responsible for the overall monitoring of the mHealth interventions; (2) 10% annual time of the data manager responsible for updating patient information, the follow-up of patient responses captured by the web interface and identifying and reporting issues that arose with the functioning of the mHealth intervention; and (3) overheads that were a proportion of fixed costs incurred at the ART-centre level.It should be noted that fixed costs are dependent on the scale of the intervention. When the intervention is scaled up to the national level, the recurrent fixed costs at ART-centre level will increase step-wise and will gradually change into recurrent variable costs.These included costs per patient for the mHealth intervention . These variable costs change based on the number of patients when the application is scaled up. The time spent by the ART-centre counsellor (i.e. one hour per patient per year) on the following activities was considered: (1) training patients to receive and respond to the mHealth intervention, (2) follow-up of mHealth intervention receipt with the patient, and (3) routine patient counselling for adherence at monthly follow-up visits at the ART centre. To obtain these costs, the counsellors in the HIVIND trial maintained diaries for the time spent with each patient on these tasks.Sensitivity analysis was carried out by varying (1) the number of patients and the number of ART centres ; and (2) the type of mHealth intervention deployed, that is, (1) only IVR, (2) only SMS or (3) both SMS and IVR used in combination.For the scale-up of the intervention, the presence of 22 official regional languages recognised by the 7th Schedule of the Indian Constitution, and English were considered. Different estimates for the numbers of ART centres and PLHIV on treatment were considered. The numbers of ART centres were arrived at from NACP reports of the existing number of ART centres and projected estimates of the number of ART centres for 2017 . WhiThe proposed budget for the NACP IV is approximately 128,240 million INR for five years . Based oFirst-line ART costs the Indian NACP approximately 5,000 INR/patient/year, while second-line ART costs 30,000 INR/patient/year . ApproxiWhile mHealth interventions are attractive for adherence support, they hold the potential for disclosure of HIV status if the mHealth intervention is intercepted by others . In addiA previous study in our setting in the year 2009 reported a mobile phone ownership of 73%; ownership was associated with being male, literate and employed . This meA vast, rapidly growing body of literature, describes the use of mHealth technology to support various facets of prevention and management of HIV infection in LMIC settings. While the most popular application of mHealth is for adherence support, the technology has also been successfully used to schedule hospital appointments and obtain diagnostic reports –35. mHeaCosts for the application could change based on the changes in currency exchange rates. Costs may differ depending on the provider of the technology . Additionally, the costs of IVR calls and SMSs may change over time.Costs to other national programmes will vary based on the costs of mobile phone services in those settings, as well as programme costs, staff costs and so on; therefore, the findings from our study are relevant only in the context of the Indian national programme.The ubiquity and low cost of mobile phones in India make mobile phone technology a potential tool to support treatment adherence and other aspects of HIV-related care in the Indian context. Previous studies in our setting indicated a preference for IVR calls for ART-adherence support and showed an improvement in ART adherence in PLHIV on treatment. Estimating the costs of the mHealth intervention was considered essential prior to deploying it at scale, especially as no prior estimates of such costs exist in the Indian context. The costs estimated in our study indicated that the cost of mHealth interventions for ART-adherence support in the context of the Indian NACP is low, regardless of whether calls, messages or both modalities are used. The study also provides some indication of programme costs for other HIV healthcare-related functions that the mobile phone could potentially be used to support. Given that there have been a number of pilot projects and trials in the area of mHealth and HIV/AIDS for some years now, there are increasing calls to scale up and integrate these projects into national HIV programmes.


Clinical evidence suggests that antibodies from reconvalescent donors (persons who have recovered from infection) may be effective in the treatment of Ebola virus infection. Administration of this treatment to Ebola virus–infected patients while preventing the transmission of other pathogenic viruses may be best accomplished by use of virus-inactivated reconvalescent plasma. Nearly all infections have been reported in West Africa. The outbreak has reminded public health systems of the minimal medical options available for the treatment of persons affected by this disease. Beyond the management of symptoms, no vaccine or proven causal treatment is available, and interventions that are in development remain at early stages.The largest outbreak of Ebola virus infection to date included 15,935 reported cases and 5,689 deaths as of November 26, 2014 . The use of whole blood transfusions is probably the least desirable choice. For this option, a donor would only be able to donate approximately once per quarter; thus, the number of treatment courses that could be collected from any donor would be fairly limited. In addition, the required matching of blood type (ABO) and antigen (Rh negative/positive) in a whole blood unit for transfusion would add a layer of complexity. Whole blood also cannot be treated by any of the currently approved virus-inactivation methods  through blood transfusions in the United States even after implementation of sophisticated nucleic acid testing schemes for the blood supply (http://apps.who.int/iris/bitstream/10665/135591/1/WHO_HIS_SDS_2014.8_eng.pdf). In addition, the economic aspects of such a testing endeavor would appear challenging.Many challenges are associated with establishing and operating a virus-testing laboratory in an environment that lacks the equipment infrastructure or trained personnel. Within these circumstances, it is difficult to ensure that predonation test results for “HIV, HBV, HCV, syphilis, and other locally transmitted infections, as applicable” would be generated within 48 hours, or otherwise repeated at donation, as recommended by interim guidance from WHO  pandemic (For these reasons, the interim WHO guidance on use of blood and plasma for treatment of Ebola virus infection would benefit from inclusion of a chapter on virus inactivation for plasma. As an effective and readily deployable technical approach, SD-inactivated plasma transfusions might even be proposed as the standard of care.In conclusion, although the continued development of long-term scalable solutions such as a vaccine for Ebola remains critical, existing technology and protocols could help fill the gap. Establishment of a supply of virus-inactivated reconvalescent plasma for treatment of persons infected with this virus may be the most feasible treatment option.


Apple stem grooving virus (ASGV) infection.Plant small RNAs (sRNAs) associated with virulent virus infections have been reported by previous studies, while the involvement of sRNAs in latent virus infection remains largely uncharacterised. Apple trees show a high degree of resistance and tolerance to viral infections. We analysed two sRNA deep sequencing datasets, prepared from different RNA size fractions, to identify sRNAs involved in sRNA analysis revealed virus-derived siRNAs (vsiRNAs) originating from two ASGV genetic variants. A vsiRNA profile for one of the ASGV variants was also generated showing an increase in siRNA production towards the 3′ end of the virus genome. Virus-derived sRNAs longer than those previously analysed were also observed in the sequencing data. Additionally, tRNA-derived sRNAs were identified and characterised. These sRNAs covered a broad size-range and originated from both ends of the mature tRNAs as well as from their central regions. Several tRNA-derived sRNAs showed differential regulation due to ASGV infection. No changes in microRNA, natural-antisense transcript siRNA, phased-siRNA and repeat-associated siRNA levels were observed.This study is the first report on the apple sRNA-response to virus infection. The results revealed the vsiRNAs profile of an ASGV variant, as well as the alteration of the tRNA-derived sRNA profile in response to latent virus infection. It also highlights the importance of library preparation in the interpretation of high-throughput sequencing data.The online version of this article (doi:10.1186/1471-2164-15-568) contains supplementary material, which is available to authorized users. Malus x domestica (M. x domestica), has a wide range of infectious agents, which include fungi, bacteria, phytoplasma, viruses and viroids. One such virus, Apple stem grooving virus (ASGV), is the type member of the genus Capillovirus (family Flexiviridae) [The domesticated apple, viridae) . It is aviridae) . ASGV inviridae) .During infection the replication of RNA viruses generate long dsRNA intermediate molecules that triggers the synthesis of small interfering RNAs (siRNAs) . FurtherWith the introduction of next-generation sequencing the knowledge of sRNA species has been extended beyond the well-characterised miRNA, trans-acting siRNA (tasiRNA) and natural-antisense transcript (NAT) siRNA (nat-siRNA) groups. Although sRNAs were shown to originate from tRNA before, Lee et al.  was the Transfer RNA cleaved in the D or T loop give rise to a second group of sRNAs, called tRNA-derived RNA fragments (tRFs). This group can be further divided into sRNAs stemming from (a) the 5′ end of mature tRNAs, (b) the 3′ end of mature tRNAs and (c) the 3′ end of immature tRNAs, called 5′-tRFs, 3′ CCA tRFs and 3′ U tRFs respectively . SeveralIn this study a next-generation sequencing approach was followed to identify sRNAs that are associated with a latent virus infection in apple plants. In addition to illustrating the vsiRNA profiles associated with an ASGV genetic variant the results from this study demonstrate the involvement of tRNA-derived sRNAs in plant-virus interaction. The lack of differential regulation of miRNAs, phasiRNAs, nat-siRNAs and rasiRNAs in leaf material is also shown.Two library preparation approaches were followed. The first approach made use of total RNA to produce a broad range library (BRL) for each sample, with individual sequencing datasets comprising of between 7,543,861 and 11,648,479 reads. Reads of 27 nt and longer contributed to 73% of all BRL reads. Since sRNAs involved in gene silencing are mostly considered to fall within the 17 to 26 nt size-range, a second narrow range library (NRL) was prepared for each sample using size-selected sRNAs to increase the sequence depth of these sRNAs. These libraries generated 7,235,867 to 14,896,610 high quality reads per sample. The size-range 17 to 26 nt in length represented 97% of all the reads and were used for downstream analysis. Figure The NRL data was first used to analyse the production of vsiRNAs, since these datasets were enriched for sRNAs in the size range known to be associated with vsiRNAs. Reads, which did not align to the apple nuclear, chloroplast or mitochondrial genomes, were mapped  onto the complete genomes of six ASGV isolates. sRNA read-mapping results for the pooled NRL sequencing data from the virus-infected samples are shown in Table The occurrence of mixed ASGV infection was analysed using the genomes of three isolates . These isolates each had an equal or higher sRNA read count Table  than theSince only one sample was confirmed to be infected with a single ASGV variant, only reads from this sample could be used to generate a complete vsiRNA profile of this variant. Figure Altogether, from the three BRL datasets of the infected samples, 0.24% of the non apple-derived reads (>16 nt in length) mapped onto the six ASGV genomes. Although less than the NRL viral reads, these reads still covered 97% and 82% of the ASGV-AC and P-209 genomes, respectively Table . SimilarGTC, and was represented by a total of 1,814,310 reads in the BRL datasets. In contrast, for the NRL dataset only 1.6% of all reads (17 tot 26 nt), originated from tRNAs.Previous studies have established that sRNAs are generated from tRNAs in a non-random manner and that they play a regulatory role similar to other sRNA species , 22, 23.CTG.sRNAs, originating from both 5′ and 3′ ends of mature tRNAs, were identified in datasets from both library types  seedlings, grafted onto MM.109 rootstocks. The viral status was confirmed by two multiplex RT-PCR reactions described in Menzel et al. [Apple mosaic virus detection were replaced with those described in Hassan et al. [Sample material was collected from three healthy and three asymptomatic ASGV-infected (as confirmed by RT-PCR), greenhouse-grown, l et al. . The prin et al. . See Addn et al.  was usedn et al. . For theReads from the three NRL virus-infected datasets were combined for vsiRNA analyses. Reads that could map with less than two mismatches onto the apple nuclear, chloroplast or mitochondrial genomes, obtained from the Genome Database for Rosaceae , 33, werArabidopsis thaliana, Brachypodium distachyon, Medicago truncatula, Oryza sativa and Populus trichocarpa) were retrieved from the PlantRNA database [Mature tRNA sequences of five angiosperms  as a result of ASGV infection are indicated. (XLSX 145 KB)Additional file 1: Table S1: tRNA-derived sRNAs identified in the BRL data. The 15 sRNA reads with the highest read counts associated with a tRNA are shown and those differentially regulated (|log2fold change| > =1 and padj < =0.05) as a result of ASGV infection are indicated. Table S4. Results for the differential expression analysis of the individual tRNA-derived sRNAs, based on BRL data. Table S5. Results for the differential expression analysis of clusters of sRNAs originating from tRNAs, based on NRL data. Table S6. Results for the differential expression analysis of the individual tRNA-derived sRNAs, based on NRL data. Table S7. Results for the differential expression analysis of apple miRNAs present in miRBase. Table S8. Results for the differential expression analysis of recently predicted apple miRNAs. Table S9. Results for the differential expression analysis of the cluster of nat-siRNAs originating from both strands of the overlapping region of NAT pairs. Table S10. Results for the differential expression analysis of the cluster of nat-siRNAs, from the first transcript, originating from the overlapping region of NAT pairs. Table S11. Results for the differential expression analysis of the cluster of nat-siRNAs, from the second transcript, originating from the overlapping region of NAT pairs. Table S12. Results for the differential expression analysis of all the sRNAs originating from a phased cluster. Table S13. Results for the differential expression analysis of phasiRNAs. Table S14. Results for the differential expression analysis of tasiRNAs. Table S15. Results for the differential expression analysis of the cluster of rasiRNAs originating from both strands of a repetitive sequence. Table S16. Results for the differential expression analysis of the cluster of rasiRNAs originating from the forward strand of a repetitive sequence. Table S17. Results for the differential expression analysis of the cluster of rasiRNAs originating from the reverse strand of a repetitive sequence. (XLSX 1 MB)Additional file 2: Table S3: Results for the differential expression analysis of clusters of sRNAs originating from tRNAs, based on BRL data. Additional file 3: Table S18: Diagnostic RT-PCR primers. Multiplex-primers used to determine the viral status of apple plants. (XLSX 14 KB)


Fevers of unknown origin constitute a substantial disease burden in Southeast Asia. In majority of the cases, the cause of acute febrile illness is not identified.We used MassTag PCR, a multiplex assay platform, to test for the presence of 15 viral respiratory agents from 85 patients with unexplained respiratory illness representing six disease clusters that occurred in Cambodia between 2009 and 2012.We detected a virus in 37 (44%) of the cases. Human rhinovirus, the virus detected most frequently, was found in both children and adults. The viruses most frequently detected in children and adults, respectively, were respiratory syncytial virus and enterovirus 68. Sequence analysis indicated that two distinct clades of enterovirus 68 were circulating during this time period.This is the first report of enterovirus 68 in Cambodia and contributes to the appreciation of this virus as an important respiratory pathogen. OvePlasmodium spp. Inclusion/Exclusion criteria, testing algorithm, specimen processing and testing were as described by Kasper et al. However the catchment area of the study changed as per Figure Between 2009 and 2012, 17,363 patients were enrolled and tested by PCR for influenza virus and dengue virus (upper respiratory swab (URS) and serum, respectively) and by Giemsa stain of whole blood for Patients enrolled at the same site within a two-week span with similar clinical symptoms were included in individual clusters. We selected five such case clusters for further analysis. Two clusters originated from Kandal, and one each from Kampong Speu, Steung Treng, and Kratie provinces Figure . AdditioA and B , human parainfluenza virus 1, 2, 3, 4A and 4B, human metapneumovirus, human coronavirus (hCoV) OC43 and 229E, human enterovirus (HEV)/rhinovirus (HRV)), and 1 DNA virus (adenovirus (ADV). All samples positive by MassTag PCR were confirmed by single agent PCR and dideoxy sequencing of the resulting amplification products.Total nucleic acid was extracted from 250 μl of URS using the EasyMag automated extraction platform (BioMérieux). Total nucleic acid (TNA) was eluted in 40 μl. cDNA was generated using 10 μl of TNA and SuperScript II Reverse Transcriptase kit (Life Technologies). All samples were screened by MassTag PCR using respiratory panels composed of 15 known respiratory viruses ,10. The Mass Tag PCR data were analyzed using non-parametric statistics. The confidence score was calculated using fold inter-quartile range distance of the positive control or the test sample from the 95th percentile cut-point of the negative control distribution for a tag. The score is similar to parametric z-score but is distribution free.The list of primers can be found in Table All EV-D68 sequences were deposited in GenBank under accession numbers KJ556320–KJ556335. All base pair coordinates are provided relative to the original 1962 EV-D68 isolate, the Fermon strain (GenBank accession no. AY426531). A Maximum-likelihood phylogenetic tree of EV-D68 based on a 339 nucleotide fragment of the VP1 gene was constructed using Mega 5.2 software.


Deciphering the information encoded in genomic sequences is a key step in modern biomedical research. Recent findings indicate that this endeavor can be far more complex than anticipated, even for relatively small viral genomes. Vaccinia virus, the prototypic member of the poxvirus family, was initially annotated to have approximately 200 open reading frames (ORFs) of 65 or more amino acids within its 200 kbp double-stranded DNA genome. This annotation has framed the molecular biological studies of vaccinia virus since then. To further decode information in the vaccinia virus genome, we carried out systematic genome-wide ribosome profiling recently published in the Journal of Virology . In riboThe most intriguing finding of this study was the existence of numerous unsuspected translation initiation sites, which were revealed by ribosome profiling of infected cells treated with the translation inhibitor Harringtonine or Lactimydomycin. Both inhibitors arrest the ribosomes at or near the translation initiation site on mRNA with single nucleotide resolution. In addition to the start sites of most annotated ORFs, approximately 600 additional putative translational initiation sites using AUG or alternative near-cognate codons were discovered. Most of these ORFs are embedded in previously annotated ones including truncated non-frameshifting ORFs and downstream frameshifting ORFs. Others are located in non-coding regions including a few upstream ORFs located in the 5′ untranslated region of mRNA, ORFs in intergenic regions and antisense-strand of the annotated ORFs. These findings suggest multifaceted translation of the vaccinia virus genome. In concert with our earlier findings of pervasive transcription ,4, theseMost of the newly identified ORFs are small in size, as they were below the previous arbitrary cut-off of 65-amino-acids. In fact, several recent studies have suggested that small ORFs encoded by vaccinia virus can perform essential functions in viral replication. The smallest protein characterized has only 35 amino acids and is required for virus entry into cells .de novo synthesis. Other poxviruses, such as monkeypox and molluscum contagiosum virus, also cause serious problems in humans. Host range and pathogenicity also contribute to the specificity and efficacy of poxviruses as oncolytic agents in combating cancers and as vaccine vectors for prevention of infectious diseases. There are numerous licensed veterinary vaccines based on recombinant poxviruses that express genes of other pathogens and clinical trials to prevent infection with HIV, influenza virus, and malaria are ongoing. Poxviruses are powerful tools in studying immune system as they encode many genes that engage numerous aspects of innate and adaptive immune systems. A better understanding of the roles of the newly identified ORFs may provide insights into poxvirus replication and help to develop strategies to prevent poxvirus infection and improve poxviruses as oncolytic agents and vaccine vectors.It is possible that these newly identified translation products contribute to the differences in host range and pathogenicity of poxviruses. The virus responsible for smallpox is human-specific and was responsible for more deaths than all other infectious diseases combined in history. Although eradicated three decades ago, the smallpox virus can still be potentially used as biological weapons from unsecured stocks, genetic engineering or


Bunyaviridae, the genus Orthobunyavirus was originally named Bunyavirus, for the type species Bunyamwera virus, first isolated in 1943 from the eponymous town in western Uganda. Originally, the vernacular term “bunyavirus” was used for viruses in this genus, but as more genera were added to Bunyaviridae (there are currently 5), confusion arose over whether “bunyavirus” referred to members of the genus Bunyavirus or family Bunyaviridae.The largest genus in the family Emerging Infectious Diseases are Inkoo virus and Chatanga virus .In 1995, the Bunyaviridae Study Group of the International Committee on Taxonomy of Viruses recommended adding the prefix “ortho-” (Greek for “correct”) to the genus name  to prevent confusion. Two orthobunyaviruses reported on in this issue of