Dementia represents an economical burden to societies nowadays. Total dementia expenses are calculated by the sum of direct and indirect costs. Through the stages of the diseases, as the patients may require institutionalization or a formal caregiver, the direct costs tend to increase. This study aims to analyze the direct costs of dementia in Portuguese nursing homes in 2012, compare the spending between seniors with and without dementia, and propose a predictive costs model. The expenses analysis was based on (1) the use of emergency rooms and doctor's appointments, either in public or private institutions; (2) days of hospitalization; (3) medication; (4) social services use; (5) the need for technical support; and (6) the utilization of rehabilitation services. The sample was composed of 72 people, half with dementia and half without. The average annual expense of a patient with dementia was €15,287 thousand, while the cost of a patient without dementia was about €12,289 thousand. The variables “ability to make yourself understood,” “self-performance: getting dressed” and “thyroid disorders” were found to be statistically significant in predicting the expenses' increase. In nursing homes, in 2012, the costs per patient with dementia were 1, 2 times higher than per patient without dementia. Dementia encompasses a number of diseases and conditions with specific characteristics, such as cognitive impairment, functional, and behavioral  was around €160 billion . Out of this amount, 56% of the cases were attributed to informal care costs and the remaining 44% to direct costs   show changes that put them in the level of moderate or severe impairment.Cognitive changes may be associated with dementia or normal aging process. It was found that two-thirds of his sample had clinically definable cognitive impairment but they had no dementia  but they have the diagnostic category on clinical assessment of “cognitive impairment, no dementia” (CIDN).To assess the costs associated with dementia there are internationally defined protocols that include multiple dimensions.These protocols are standardized into six dimensions, namely, (1) the use of emergency rooms and doctor's appointments, either in public or private institutions; (2) days of hospitalization; (3) medication; (4) social services use; (5) the need for technical support; and (6) the utilization of rehabilitation services , through access to institutional documents, including daily occurrences to account for the number of trips to the emergency room, appointments in public or private institutions, hospital stay days, and consultations in rehabilitation services. The cost of six dimensions were analyzed.In this dimension, there was found the number of times each user attended emergency department, hospital visits and consultations at the health center. Consultations in private hospitals were also considered. The calculation of each episode was done by multiplying the number of emergency episodes/consultations with the actual price of each episode. Financial, values for each episode were obtained considering the actual prices of health care episodes provided under the National Health Service1.To analyze the hospitalization costs, the number of days that each user was hospitalized in 2012 was recorded. These data were multiplied by the daily price of admission, a value of €85The value that the medication represents for the patient was analyzed through access to monthly medication receipts. It was considered the charged value. These receipts are issued independently of costs to the inpatient service in nursing home.All subjects included in the study were part of the social response nursing home. For the assessment of this dimension, the reference value with families in nursing home for the year 2012 was considered. This amount is €930.06/user/month , however, we considered an amortization period of 8 years. To account for the cost of medical drugs and diapers , the monthly bills of the subjects were acceded.The number of sessions of physiotherapy, occupational therapy, podiatry or chiropractic treatments and the assistive devices used by the patients were checked in the records or with the institution's staff. Differentiation was made between the prices of physical therapy sessions in relation to the severity of dementia according to the stages defined by the BIMS in RAI-RUG. In this test, higher scores indicate cognitively intact and lower scores show severe impairment  intact cognition, (2) moderate, or (3) severe impairment  heating step, (2) training phase, and (3) relaxation phase. Using the assumptions presented above, a set of treatments that should be included in a physiotherapy session at each stage of dementia and for subjects without dementia were defined. The prices for each technique are the prices set by the National Health Service1. For sessions of podiatry and chiropractic the values that the user disbursed in private services were counted, as these treatments are not included in the National Health Service price list.Because this is a relatively cheap service when compared with a doctor's appointment and its price is not well established, by generalizing the values of occupational therapy, it is considered that a physiotherapy consultation costs €22. For occupational therapy sessions a value of €22 per session was considered, regardless of the level of severityStatistical analyses were performed using the program IBM SPSS version 19.0. The significance level α = 0.05 was considered. Frequencies (absolute and relative) were used to provide descriptive statistics of the socio-demographic variables. Means and Standard Deviation (SD) were used to analyze the data dimension of economic costs. Univariable and multivariable general linear models were performed in order to assess associations between dementia costs and the characteristics of the subjects (RAI-RUG variables).p < 0.05. Univariable logistic models were performed considering costs as dependent variables and each of the variables was significantly associated with the costs (26) found in the previous step. As a candidate for the multivariable model, we considered all variables significant at the 0.10 significance level in the univariable models. The final model includes all variables with p < 0.05.Were initially considered 400 variables of RAI-RUG in the model construction. Of all the variables included in the analysis, the total score was considered. Were included questions of these groups: (A) Identification Information ; (B) Hearing, Speech, and Vision ; (C) Cognitive Patterns ; (D) Mood ; (E) Behavior ; (G) Functional Status ; (H) Bladder and Bowel ; (I) Active Disease Diagnosis ; (J) Health Conditions ; (K) Swallowing/Nutritional Status ; (L) Oral/Dental Status ; (M) Skin Conditions ; (N) Medications ; (O) Special Treatments and Procedures ; (P) Restrains  , while a user without dementia presents an expenditure of €12.3 thousand (SD = 1288), a difference of €3000 between the two types of subjects.The mean ± SD was used to provide descriptive statistics for economic costs Table . AnnuallSocial services are the largest share of the costs. The values of this dimension are the same for both groups, enough so that it is difficult to distinguish between them. In Portugal the financial support provided by the state is fixed, regardless of the economic situation and the degree of dependence of the user , about 83% of the costs associated with technical aids. For the group of subjects without dementia, diapers accounted for about 69% of the cost of all technical aids.Except for the group without dementia, the size of the use of medication  takes up a larger volume than the use of technical aids . It should be noted that the size of technical aids includes the cost of diapers. The annual charge in diapers for the group with dementia was €19.5 thousand . There is a known relationship between dementia and lower control capabilities in the areas of verbal expression, auditory comprehension, repetition, reading, and writing , predictive costs were also tested. Since none of the interactions proved statistically significant, it was decided to not include any of this information in the final model.In models created the socio-demographic variables, as age and gender, were not found to be significant in the variable costs.This study is the first in Portugal to analyze the direct costs of dementia in nursing homes. A user in a nursing home with dementia presents a cost of €15,287, about €3 thousand more than the cost of a user without dementia in the same service. The prevalence of people with dementia in the four non-profit organizations analyzed is 17%. The latest data shows that in 2010 about 66,275 people lived in nursing homes  day centers specific to dementia, (2) home care services along with a location tracking device, and (3) respite care services.Specific day centers offer appropriate care, therapies, and activities catered to their patients that stimulate their participation and integration. Private sector in Madrid has specific day centers for Alzheimer's disease and other forms of dementia. On average, the monthly payment can vary between €175 and €935 (depending on schedules and contracted services)  is €239 (Ministry of Solidarity Employment and Social Security and Union of Portuguese Mutual Societies, The last alternative is respite care services. The total cost of implementing a group psychoeducational intervention of 11 sessions with an average of nine participants per session is €4123 (Caravau et al., The predictive model shows that only three of the variables led to increased costs of internment with subjects in nursing homes. Subjects who rarely or are never understood, those who need support in getting dressed, and those with clinical disorders of the thyroid are showing greater cost shares for inpatient care. It was appropriate that in future investigations were analyzed the relationship between the economic costs to the degree of dependence of the person. However, in this study was not possible to do so because it would be necessary to establish a minimum number of people for each group, about 20–30 people per group. There was no statistical capacity with this sample for this procedure.The costs of caring for a person with dementia are high and represent a major burden on the economy of the country, patients, and families. Knowing the real costs of an individual with dementia in nursing homes allows for a more effective management and efficient use of national resources.The present results may support the evaluation of the impact of policies, the prioritization of expenditures, and the adjustment of decisions based on the Portuguese reality.Overall, it is believed that this study has obtained a good approximation of the actual costs of a user with dementia. Studies such as this should be applied at a national level to fully understand a panoramic view of Portugal, and in future research it would be interesting to analyze the cost that represented the use of social resources to the Government, to the patient and the family.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.


Homo were surely bedeviled by blood-feeding arthropods, some of which doubtless carried zoonotic pathogens. However, the phenomenon of vectorborne human epidemic disease began only after humans began building settlements 15,000 years ago  was only possible because black rats (Rattus rattus), the host of the vector flea, had become extraordinarily successful at living off human garbage and nesting in our buildings.The earliest members of genus Anopheles gambiae mosquitoes in Africa and An. dirus mosquitoes in Southeast Asia thrive in the small puddles  created around villages. Best adapted of all are Aedes aegypti mosquitoes, the cosmopolitan vector of epidemic yellow fever, dengue, chikungunya, and Zika viruses. Their ecologic niche is nearly ours. These mosquitoes lay eggs in artifacts: water storage jars, roof gutters, flower pots, dog dishes, even upturned bottle caps. Their cognate species, Ae. albopictus, is only slightly less versatile, having an attraction to discarded tires. Evolution of blood-feeding arthropods to our changing environment and evolution of some zoonoses to exploit this advantage are major links in the emergence of obscure pathogens into epidemic threats and is a timely subject for this issue of Emerging Infectious Diseases.Two of the most important malaria vectors in the world exploit human activity to proliferate. Immature forms of Aedes species. Since its arrival in the Western Hemisphere ≈1 year ago, Zika virus, which had previously been associated with a clinically mild and inconsequential illness, is now increasingly suspected of being the cause of an alarming epidemic of neurologic birth defects and Guillain-Barré syndrome in tropical regions. Zika virus, the subject of several articles in this issue, reminds us of some of the impediments to responding to emerging vectorborne pathogens.Persistence of human yellow fever, the seeming inexorable expansion of dengue, and the surprising, explosive spread and severity of first chikungunya virus and now Zika virus bear testament to the threat posed by habituated 4 bases each replication. The chikungunya pandemic that began 10 years ago was fueled in part by a single, nonsynonymous base change that enabled that alphavirus to replicate more efficiently in Ae. albopictus mosquitoes (First, Zika virus belongs to the most prevalent class of emerging pathogens, the zoonotic single-stranded RNA viruses, which have mutation rates as high as 1 base/10Second, conditions enabling transition from vectorborne animal-to-animal transmission to arthropod-mediated human-to-human transmission are poorly understood. Like dengue virus, another flavivirus, Zika virus was likely originally a pathogen of subhuman primates. Between its discovery in a sentinel macaque in Uganda in 1947 and the first recorded epidemic 60 years later in Yap, Federated States of Micronesia, only 14 human cases had been reported, all from Africa and Asia (Third, the pathogenicity and transmission dynamics of vectorborne zoonotic pathogens are much more complex than those of directly communicable pathogens. It is not yet known if Zika virus will find sustaining, nonhuman hosts in the Western Hemisphere, as has yellow fever virus, or how wide the range of vector species will be. Pathogenicity and transmission dynamics will be factors in determining where Zika virus will become endemic and what will be the most suitable methods of control.Fourth, accurate diagnosis is key to surveillance and response. It might seem as if Zika virus sprang from nowhere, but almost certainly it must have been infecting many more humans in Africa and Asia than we had been aware. Our ability to serologically diagnose infections with emerging arboviruses is often compromised by close antigenic relationships within virus families. Zika, dengue, West Nile, and yellow fever viruses can co-circulate, not only among themselves, but possibly with unidentified or poorly characterized flaviviruses. The limitations of current diagnostics are a primary reason why the association between Zika virus and birth defects remained speculative so long.Fifth, vector control is a force multiplier that can reduce the risk from many viruses that would require the development of individual vaccines. However, insecticide resistance and application problems greatly impede effective implementation.The best defense is preventing a problem from growing into a threat. Fewer than 20 of the 86 known pathogenic arboviruses can be considered major causes of human disease, and 3 of these, West Nile, chikungunya, and Zika viruses, have emerged from relative obscurity within only the past 20 years (–The discovery of 3 highly pathogenic mosquitoborne viruses in China and the United States during the past 5 years (


Oliva spp. shell beads recovered from four archaeological cave sites Jerimalai, Lene Hara, Matja Kuru 1, and Matja Kuru 2, located in Timor-Leste, Island Southeast Asia. While Pleistocene-aged examples of modified marine shells used for personal ornamentation are common in African and Eurasian assemblages, they are exceedingly rare in Southeast Asia, leading some researchers to suggest that these Modern Human societies were less complex than those found further west. In Timor-Leste, the lowest Oliva bead to be recovered was directly dated to ca. 37,000 cal. BP, making it the oldest piece of personal ornamentation in Southeast Asia. Morphometric, taphonomic, use wear, and residue analyses of these beads alongside modern reference specimens, and experimentally made examples indicate that the Oliva shells were modified to be strung consecutively (as in a necklace), and while their mode of production changed remarkably little over the thousands of years they were utilised, an increase in their deposition around 6,000 cal. BP suggests that there was a change in their use coinciding with sea-level stabilisation. These tiny beads demonstrate that early Island Southeast Asian societies produced the same kinds of symbolic material culture we have come to expect from the more intensively studied African/Eurasian region, and that limited sampling and poor recovery methods have biased our perspectives of this region.In this paper, we describe 485  Representing a behaviour specific to humans whereby standardised items are displayed on the physical body to project symbolic meaning to members of the same or nearby groups, ornamentation in the form of beads or pendants made from marine shells and animal teeth, continues to be one of the best sources of information regarding social behaviour in early Modern Human communities e.g., –11). Unt. Unt11])This paucity of remnants of past symbolic behaviour has led some researchers to posit that the earliest Modern Human communities in Southeast Asia were materially less complex than those located in Africa and Europe. It has even been suggested that as biologically and behaviourally Modern Human populations moved away from their African homeland, they somehow ‘lost’ key technological features which separated them from more ‘archaic’ hominid groups . While sOliva spp. shell beads recovered from Jerimalai, Lene Hara, Matja Kuru 1 (MK1), and Matja Kuru 2 (MK2) in Timor-Leste. Found throughout the deposits of these four sites, the oldest example was directly dated to 38,246–36,136 cal. BP, while the most recent beads are only a couple of hundred years old. This collection demonstrates that early ISEA societies produced the same kinds of personal ornamentation as the more intensively studied African/Eurasian region, and that limited sampling and poor recovery methods have biased our perspectives of a region which in actuality houses a rich record of symbolic material culture.Significantly, recent excavations in Timor-Leste, Island Southeast Asia (ISEA), have unearthed numerous marine shell beads simply through utilising fine meshed sieves. The controlled excavation and recovery techniques, coupled with a methodical dating regime, including the direct dating of individual pieces of shell material culture, have resulted in the identification of early instances of shell fishhooks , and sheThe sites of Jerimalai  and Lene Hara  are located less than a kilometre apart and within a kilometre of the current coastline at the easternmost tip of Timor-Leste, southeast of the village of Tutuala . They arExcavation of Jerimalai was undertaken during July 2005, when two 1 x 1 m square test pits were dug in the eastern and central area of the shelter (Squares A and B). The Jerimalai deposit has an upper ceramic-rich and a lower aceramic horizon, and was excavated in approximate 1–5 cm (average depth of 2.2 cm) spits within stratigraphic units, where such could be identified. Wet screening of all cultural deposits through 1.5 mm mesh ensured recovery of even very small finds, including lithic micro-debitage and shell beads. Pottery occurred in the upper horizon, with stone artefacts, shell, and bone remains being found throughout the deposit. Owing to the fact that more than 50 kg of anthropogenic marine shell was excavated from these two squares, 3-D piece plotting of individual shell artefacts was not feasible, though finished beads and obviously worked pieces of shell were separated and individually bagged when identified during wet sieving and sorting in the field. Other pieces were only identified once back in the laboratory.Oliva shell beads which may represent heirloomed or reused artefacts deposited long after their creation (discussed below); and (2) the deposit becomes increasingly compact and carbonate cemented at depth, reducing the amount of bioturbation and other post-depositional disturbances that may affect the oldest layers.While the analysis of the Jerimalai cultural materials is ongoing, radiometric dating of the lower levels has already demonstrated that it is the oldest modern human occupation site in ISEA east of the Sunda Shelf , 25. TheAt Lene Hara, Test Pit F—the 1 x 1 m square from which all of the Lene Hara beads reported herein originated—was excavated in the lower northern chamber during 2002 –28. ThisThe cave sites of MK1  and MK2  are located adjacent to each other in an uplifted limestone ridge north east of the modern village of Poros, and only a few hundred meters north from Lake Ira Lalaro. They are approximately 370 m above sea level and around 8 km in straight-line distance from the north coast of Timor-Leste , with MKAt MK1, the oldest date returned was 16,355–15,566 cal. BP , though most of the deposit built up between 5600–4600 BP . ArchaeoHuman use of MK2 begins sometime prior to 35,000 years ago with an age estimate of 36,866–35,285 cal. BP 32,220±300 OZF-785) for a near basal unit (Spit 47) returned, though it should be noted that excavation was discontinued prior to reaching bedrock 0 OZF-785. A stoneBoth MK1 and MK2 appear to have been used largely simultaneously. MK2 constituted an attractive temporary campsite around c. 30,000 years ago, before abandonment during the Last Glacial Maximum (LGM) and then occasional reoccupation across the Pleistocene-Holocene transition. This site then appears to have become much less attractive to people, perhaps owing to an opening of a hole in the roof at the back of the shelter [Oliva shells as humanly modified items of adornment was based on the following: (1) evidence for human agency in their selection, transport, and accumulation at each of the four sites; and (2) the presence of clearly identifiable manufacturing marks and use wear demonstrably distinct from taphonomic traces. In order to differentiate between these natural and anthropogenic factors, two lines of investigation were followed: (1) the examination of low-tide collected live and dead specimens of Oliva spp., and (2) the experimental creation of Oliva shell beads. Review of published criteria for identifying anthropogenic alterations of shell beads, such as that provided in d’Errico et al. [The identification of the o et al. , Vanhaero et al. , Stiner o et al. , and Crio et al. , supplemOliva oliva  from Kawara Beach in north Queensland. This non-endangered species is abundant throughout the estuarine zone surrounding Kawara Beach, where collecting is permitted without need for permits from the Queensland government. These shells were gathered over several visits to the area over the course of two months. Additionally, a range of Oliva spp. specimens curated in the Archaeology and Natural History Osteology Reference collection held at the Australian National University were included in the study. These shells were examined using a Zeiss 2000-C stereo microscope fitted with an AxioCam MRc5 camera in order to identify the types, intensity, distribution, and frequency of natural damage present on beach collected specimens.Reference collection examples which were examined as part of this study included more than 80 newly gathered Oliva shell beads bearing anthropogenic alterations of known origin in terms of manufacture method and use type. Three alternative methods of apex perforation were explored using 15 of the Oliva oliva shells collected from Kawara Beach: (1) grinding on a sandstone slab; (2) percussion flaking, where the shell is held stationary and a stone is brought down against the apex; and (3) tapping, where the shell apex is forcibly tapped repeatedly on a hard surface . Each oOliva beads was individually examined using the same microscope set up, with anthropogenic traces mapped onto drawings of each shell, these drawings produced from high-resolution digital photographs of their dorsal and ventral surfaces and the use of an illustrating programme (Canvas XII). During this process, a red residue was observed on more than half of the shells . Oliva brettinghami  is a small species of Olive shell, typically reaching only 20 mm in length. Oliva spp. have a short spire with rounded shoulder, and range in colour from a very light cream to a deep cream/brown overlaid by a pattern of longitudinal zigzags in a darker tone [The Timor c region , though ker tone . These oker tone , themselOliva shells (such as storm surges) at the archaeological sites are precluded by the elevation of Jerimalai and Lene Hara above the shore, while MK1 and MK2 are far from the coast. Similarly, while each of the four sites contain substantial economic marine shell assemblages, it is unlikely that the Oliva shells were gathered for subsistence given that they average around 9 mm in absolute length. Thus, while easily collected live during low-tide, one would have to collect an enormous quantity to gain enough calories to make their collection and processing worthwhile. In this way, the Oliva spp. shells are similar to the Nassarius globosus and Nassarius pullus shells found in levels dated post 6,500 cal. BP at these same sites [Nassarius kraussianus shell beads recovered from the Middle Stone Age levels of Blombos Cave in South Africa [Natural accumulation of the me sites , as wellh Africa .Oliva appear to have been gathered as ornamental shell, that is, they were used exclusively for ornament-making (32). Most appear to have been collected live or shortly after death as very few exhibit the muted shell sculpture, pitting, and low intensity but widely distributed damage to their surfaces indicative of having rested on the shore for an extended period of time. This preference for undamaged shells which retained their natural shiny surface is reflected throughout the collection, with no changes in this preference identified either between the sites or through the stratigraphy.Instead, the Oliva spp. shell beads were identified and examined from Jerimalai, Lene Hara, MK1, and MK2  and continue throughout the stratigraphy to the most recent levels . An increase in their deposition occurred at 5,000–6,000 cal. BP, is evident in Jerimalai and Lene Hara, and perhaps also MK1.In total, 485 2 Tables . These aOliva shell into a bead required minimal modification involving only the removal of the apex so that a string could be passed through its internal structure. While a small number of the shells present evidence for having undergone beach wash, and consequently, likely exhibited a small perforation at the tip when collected, most show no evidence for having accrued natural damage prior to collection. As such, the vast majority of artefacts must have had their apex intentionally removed for use.Transformation of the raw As was mentioned above, three approaches to removing the shell apex were experimented with in order to identify which method/s were used by the ancient Timorese people: (1) grinding on a sandstone slab; (2) percussion flaking, where the shell is held stationary and a stone is brought down against the apex; and (3) tapping, where the shell apex is forcibly tapped repeatedly on a hard surface. Traces left from each of these approaches were found to be distinctive and distinguishable from one another, as well as from naturally (beach washed) perforated shells. Shells ground on the sandstone slab exhibited a clean horizontal surface covered with parallel striations indicating the direction of grinding , while rThe tapping-technique, was the only method of apex removal identified in the Timorese collection, including on the oldest bead recovered . Given the simplicity and ease of this method, something that could be done by young and old alike, it should not be surprising that it had such longevity.Oliva were manufactured—having had their apex deliberately removed using the tapping-technique—as, while superficially similar in appearance to beach worn shells, the apical holes in such shells only become large enough to pass a string through after having suffered significant damage from beach wash, resulting in additional holes appearing in the body whorl and an overall weakening of the entire shell. Thus, ‘naturally occurring’ Oliva beads are not robust enough to endure the intense use indicated by the use wear outlined below. Furthermore, not one of the archaeological examples exhibit evidence for having suffered the intensity of damage observed on the modern, beach worn specimens.Importantly, it was observed during experimentation that while traces of manufacture are initially clear to the observer, once the newly made bead is strung and used for even just a few hours, these traces become smoothed resulting in a perforation wall which is very similar at low magnification in appearance to a natural, beach worn opening . DespiteOliva shell outer lip, the Timorese beads exhibit faceting of either isolated sections of the lip extremities, or notches ranging in width from a couple of millimetres to covering half of the lip length  hang loosely at a 15 to 60 degree angle to the string owing to the height of the collumela. Beads located further up the string, on the other hand, will sit vertically with the sting passing through the canal. These differences in the placement of beads on a single, long strand will result in a number of the beads displaying heavier wear on the posterior sections of their columella, while on others wear is more pronounced around the canal. Such is the pattern observed in the Timorese collection. Consequently, this evidence suggests that the Timorese beads were strung as a beaded strand which was worn against the body or an item of material culture.Results of the stringing experiment found that those beads strung consecutively (as in a necklace) were accumulating wear in the same locations  and of the same type  observed on the archaeological examples. Additionally, those attached to a piece of cotton canvas  were begDuring experiments, several important observations were made which further elucidate how the archaeological specimens were strung—most interesting being that a sharp point was found to be necessary to string the beads in experimentation, as it was impossible to force the string through the tiny aperture without a point pushing it further into the shell structure. For the experimental beads, a metal needle point was used, and it is possible that the sharp point of a fish spine or bamboo splinter may have been similarly utilised for the archaeological examples. Fish bone has been found at each of these sites and is currently undergoing analysis.Oliva shell beads to LA-ICP-MS analysis which recorded a significant presence of iron in its composition. Red ochre was commonly observed caught within the sutures and internal structure of the shell beads from each of the four sites . Workedy Glover , and cony Glover : 119), aremoved”  were ideremoved” . A photoremoved” : 305) shOliva shell bead production in Timor-Leste begun in the first few thousand years after initial settlement, though apparently after the selection of Nautilus shell for ornamental purposes [Oliva shell beads largely mirrors those of the more coastal Jerimalai and Lene Hara shelters.The evidence recovered from Jerimalai, Lene Hara, MK1, and MK2 indicate that purposes . At thispurposes , 48, andpurposes , the shoOliva shell beads remains low and constant from their initial appearance in the archaeological record until levels dated to between 8,000 and 4,000 cal. BP where a sharp increase in bead numbers is found in Jerimalai and Lene Hara, with a similar pattern perhaps indicated at MK1. This increase in bead numbers ties in with a significant increase in net deposition rates for deposit sequence accumulation and quantity of shell deposited at all four sites from about 7,000 cal. BP. After around 4,000 cal. BP, frequency of Oliva shell beads diminishes back to a level similar to that observed for levels deposited prior to 8,000 years BP. Interestingly, the use wear observed on individual beads coming from these three analytical temporal periods  also reflects changes in use intensity. As shown in Figs Oliva shells shifted to a different type of use, but rather that they were simply worn far more intensively during this time than both before and after this period.Deposition of Oliva shell bead usage coincides with the initial appearance of Nassarius shell appliqués at these same sites [Oliva and Nassarius—while having been used in different ways, both indicate the increased use of marine shell for ornamentation at the time of sea-level stabilisation, and while it is tempting to simply correlate the two phenomena, the fact that it is not simply a case of more beads being deposited, but that each bead saw more intensive use individually argues against a simple case that such shells became more abundant or easier to collect from 8,000 cal. BP. Instead, it would appear that sea-level stabilisation and the associated increased productivity of the near shore coastal environment may have precipitated changes in settlement patterns and social interaction around Jerimalai and Lene Hara, resulting in increased use of these shelters by more people at any one time. With populations expanding in the area, it may have been that shell beads not only saw an increase in demand (to decorate more items/people) resulting in beads being used for longer, but also saw increased significance in mediating tensions brought about by a growing society—effecting increased use of the beads. After ~4000 cal. BP evidence for occupation at the sites declines dramatically and likely reflects the movement of everyday habitation out into open villages [Oliva shell beads had returned to a level similar to that previous to the major environmental change. Continuing analysis of both the artefactual and faunal assemblages recovered from these four sites will enable us to further elucidate the nature of these social and organisational changes in the future.This phase of increased me sites . These tvillages . Beads dOliva beads from Timor is invaluable. In particular, they demonstrate the potential abundance of such small pieces of symbolic material culture in ISEA when modern standards of archaeological recovery are pursued. Indeed, with new excavations being undertaken across the Southeast Asian region at this time, we expect to see a complete overturning of the notion that this region was somewhat of a cultural backwater in terms of symbolic production in coming years.As the tempo and mode of the emergence of personal ornamentation in the first instance, and its diversification throughout the Late Pleistocene/Holocene period in different locations is considered key for understanding changes in our ancestors’ social and cultural development, the information provided by the Oliva shell beads recovered from four archaeological sites located at the eastern end of Timor-Leste has allowed us to identify an important example of early marine shell bead use dating back to around 37,000 years ago. Microscopic analysis of the archaeological bead collection in combination with replicative experiments on modern Oliva shells indicates that the Timorese beads were strung consecutively creating a beaded strand which was worn against the body or an item of material culture coloured with red ochre.Analysis of more than 450 While commonly reported for both Africa and Eurasia, examples of symbolic material culture in Southeast Asia are exceedingly rare, though immensely important for exploring the development of Modern Human cultural diversity in this region. An increase in bead production between ca. 8,000 and 4,000 years ago indicates that Timorese communities appear to have been undergoing significant changes in settlement and social interaction, likely in response to the changing coastal environment. One of the most significant findings of this research, however, is the demonstration of the potential richness of symbolic material culture items in early Southeast Asian sites—in direct contrast to previous perceptions.


To the Editor: Middle East respiratory syndrome coronavirus (MERS-CoV) was first diagnosed in humans in 2012. Human-to-human transmission of MERS-CoV has been limited, and the transmission route is still unclear. On the basis of epidemiologic studies, involvement of an animal host has been suggested  at either 4°C or 22°C and stored them at –80°C at 0, 8, 24, 48, and 72 hours post dilution (hpd) in quintuplicate. Infectious virus titers were determined by endpoint titration on Vero E6 cells in triplicate (t test) and DMEM (p = 0.0311) but not in dromedary camel milk (p = 0.1414) or cow milk (p = 0.2895). Samples stored at 22°C showed a greater loss of infectivity than did samples stored at 4°C. Infectious virus titers decreased to <15% when samples were stored at 22°C for 48 hours . This decrease was significant by student 1-tailed paired t test analysis comparing t = 0 and t = 48 hpd (p<0.05). However, despite the reduction in virus titer, viable virus could still be recovered after 48 hours. Pasteurization of raw milk can prevent foodborne disease outbreaks caused by a variety of pathogens. We heat-treated dromedary camel, cow, goat milk, and DMEM samples for 30 min at 63°C, after which no infectious virus could be recovered  to a final median 50% tissue culture infectious dose of 10ecovered .CoV survival has been studied in phosphate-buffered saline and minimal essential media and, like MERS-CoV, human coronaviruses–229E and -OC43 and severe acute respiratory syndrome–CoV were able to survive in suspension at room temperature for several days (Human-to-human transmission of MERS-CoV is inefficient, and the transmission route has not yet been revealed. The predominant detection of MERS-CoV by quantitative PCR in nasal swab samples suggests the virus causes upper respiratory tract infection in dromedary camels (


We validated the feasibility and veracity of this pipeline with sequencing results of various types of clinical samples and public datasets. VIP has also contributed to timely virus diagnosis (~10 min) in acutely ill patients, demonstrating its potential in the performance of unbiased NGS-based clinical studies with demand of short turnaround time. VIP is released under GPLv3 and is available for free download at: https://github.com/keylabivdc/VIP.Identification and discovery of viruses using next-generation sequencing technology is a fast-developing area with potential wide application in clinical diagnostics, public health monitoring and novel virus discovery. However, tremendous sequence data from NGS study has posed great challenge both in accuracy and velocity for application of NGS study. Here we describe VIP (“Virus Identification Pipeline”), a one-touch computational pipeline for virus identification and discovery from metagenomic NGS data. VIP performs the following steps to achieve its goal: (i) map and filter out background-related reads, (ii) extensive classification of reads on the basis of nucleotide and remote amino acid homology, (iii) multiple k-mer based  The world contains a high diversity of human viral pathogens. There are approximately 300 recognized viral pathogen species, and additional species continue to be discovered. The identification of viral pathogens has a tremendous impact on infectious diseases, virology and public health. Nearly all of the outbreaks of public health issues over the last decade have been caused by viruses, including Severe Acute Respiratory Syndrome (SARS) coronavirus5Shotgun metagenomic sequencing of clinical samples for viral pathogen identification provides a promising alternative solution. Although metagenomics is typically applied to understanding genomic diversity from environment samples, this methodology has also revolutionized virology with comprehensive applications, including viral pathogen identification of infectious disease in clinical laboratories1011de novo sequence assembly or mapping to reference genomes, but will not process further to the specifics of virus identification and discovery. There are many bioinformatics tools developed specifically for virus detection from NGS data. In general, the strategies in these pipelines are computational subtraction to pathogen detection. Reads corresponding to the host  are first removed, followed by alignment to reference databases (DB) that contain sequences from candidate pathogens14151617de novo assembly. The majority of pipelines utilized to assemble metagenomics data were originally developed to assemble single genomes, However, single-genome assemblers were not aimed to assemble multiple genomes from metagenomics data which were with nonuniform sequence coveragesA key feature of latest NGS platforms is their speed. It takes minimum turnaround times about 8 hours for sequencinghttp://www.ncbi.nlm.nih.gov/refseq/) and their neighbor genomes in sense mode (Here we present VIP (Virus Identification Pipeline), a one-touch bioinformatic pipeline for virus identification with fairly self-explanatory results in Hypertext Markup Language (HTML). VIP performs extensive classification of reads against DB collected by Virus Pathogen Resource (ViPR)nse mode . Novel vhttps://github.com/lh3/wgsim). Receiver operating characteristic (ROC) curvesIn order to evaluate the accuracy of the two-steps classification strategy (sense mode), a query data set of 125 base pair (bp) reads was generated in-silicon. The viral reads were generated at different mutation rate ranging from 1% up to 40% from four known representative viruses which were Dengue Virus 2 , Epstein-Barr virus , Norovirus  and H7N9 (segmented negative-sense RNA) using wgsim  for our benchmark experiment. These samples were infected with viruses of diverse types and all of results were confirmed by independent experiments.To evaluate VIP, we first tested the ability of VIP to detect the presence of viruses in various samples generated from different sequencing platforms. As indicated in −8We selected 3 sets of data from SRA to evaluate the sensitivity and specificity of VIP to identify candidate virus in clinical metagenomics datasets. Note that the 3 datasets results have been confirmed by related experiments and the genbank accession number of each reference sequence was introduced. Sensitivity and specificity were used to evaluate the ability of classification algorithm. Sensitivity, or true positive rate (TPR), was calculated as TPR = TP/(TP + FN). Specificity, or true negative rate (TNR), was calculated as SPC = TN/(TN + FP), in which TP and TN represent true positives and true negatives, and FN and FP represent false negatives and false positives. The gold standard criterion for a correct viral classification was BLASTn alignment against the target viral genome at an E-value cutoff of 10NGS and VIP analyses were applied to analyze a clinical sample from 2014 Guangzhou Dengue outbreak to test the speed and feasibility of VIP. As is shown in The state-of-the-art NGS technology is becoming mature and is increasingly introduced in routine laboratories. The price of NGS is falling dramatically in some cases below the price of traditional identification methods. NGS has emerged as one of the most promising strategies for the detection of pathogens in clinical samples. Viral metagenomics provides comprehensive viral pathogen identification in a single assay. However, bioinformatics analysis for viral metagenomics has been the bottleneck when deploying NGS for virus identification due to the absence of a designated bioinformatician in most laboratories. Our goal is to establish a one-touch computational pipeline in routine laboratories for virus identification from metagenomics NGS data generated from complex clinical samples.de novo assemblies benefitted from the classification method in VIP generated longer and accurate contig to help construct the phylogenetic tree. Nevertheless, SURPI was proved to provide more comprehensive information for pathogen identification by searching against nt/nr while VIP focused on viruses.  for comprehensive identification of pathogen. Coverage map, a very attractive feature provided in SURPI, was also carried out in VIP with strategy as previously described. The major difference was the way to select the close reference. SURPI would map to all nucleotide reference sequences corresponding to that genus picked out during alignment. VIP will choose a close reference based on abundance of reference genome due to the proposed hypothesis that the genome coverage percentage was alongside with the sequencing depth. The approaches for assembly share little similarity. VIP implemented the “classification to assembly” while SURPI used the entire data set to perform assembly. Results suggested higher N50 were achieved by VIP. (Data not shown) Compare to other pathogens, like bacteria, fungi and parasite, most viral pathogens are RNA viruses which suggested the potentially high mutation rate or divergent genomes. It could be impossible to find a suitable reference genome during alignment procedure in some cases which undermined the coverage map and underscored the phylogenetic analysis. The construction of a phylogenetic tree allows us to visualize the underlying genealogy between the candidate divergent virus and existing reference sequences. The overall effective ses. see  and S4.de novo assembly strategy to generate longer contigs for identification of divergent viral sequences. Results of VIP can be easily accessible through web browsers providing intuitional information including the summary table, genome coverage map and phylogenetic tree figure. . Another powerful application by NGS for virus identification is a single step of high-throughput parallel sequencing of small RNAsVIP also makes significant contributions for virus discovery. We recently analyzed cell culture supernatants and found considerable amount of reads were classified under the orbivirus genus. The orbivirus reads, however, shared less than 50% identity with several specific segments. The phylogenetic report showed the one of the contigs was included in the branch of Tibet orbivirus. The Taken together, our results demonstrate that the proposed VIP combined with NGS has the advantages of simplicity, rapidity, universality and feasibility in the applications of virus detection and discovery. In addition to maintaining the software, we are currently upgrading VIP including development of a user-friendly graphical interface (GUI) based on stand-alone web interface. Therefore, VIP shows the great potential to be standardized and readily and freely accessible to a wider audience of scientists in routine laboratories. Bioinformatics analysis is no longer the weak link when applying NGS as a diagnostic tool for infectious diseases.The VIP is comprised of a series of Shell, Python, and Perl scripts in Linux and incorporates several open-source tools. VIP has a set of fixed external software and database dependencies and user-defined custom parameters. The pipeline accepts cross-platform results generated from 454, ion torrent and Illumina with a variety of formats such as FastQ, FastA, SAM and BAM alignment formats. Reads are handled by concatenating the files into a single file for streamlined analysis.http://picard.sourceforge.net). VIP will determine the encoding version of the input data. This is necessary to make sure those differences in the way the quality scores were generated from different sequencing platforms are properly taken into consideration during preprocessing. VIP can also accept FastA format raw NGS data. The quality control step, however, will only perform sequence-based strategies, such as the complexity and length as main factors. Generally, the quality control step is comprised of trimming low-quality and adapter reads, removing low-complexity sequences using the DUST algorithm and retaining reads of trimmed length >20 bp using PRINSEQRaw NGS short read data can be imported by trans-formatted into FastQ format using PICARD  complete genomes; (2) complete sequences; or (3) complete cds. Assigned reads are directly mapped to all nucleotide reference sequences selected using optimal BLASTn at reward/penalty score (1/−1). The optimal score strategy is most suitable for sequences with low conserved ratioThe construction of a phylogenetic tree allows us to visualize the underlying genealogy between the contiguous sequences and reference sequences. In order to perform a phylogenetic analysis of candidate viruses in a certain viral genus, a backbone with high quality and wide spectrum is indispensable. For a genus, sequences with Refseq standard sunder that genus and the reference sequence which is used to generate the coverage map are selected to carry out multiple sequence alignment to build a backbone using MAFFTde novo assembly step benefits from the classification method in VIP for significant reduction of complexity of reads. Still de novo assemblies from virus samples, especially RNA viruses, into a genome sequence is challenging due to extremely uneven read depth distribution caused by amplification bias in the inevitable reverse transcription and PCR amplification process during library preparations. We present the Multiple-k method in which various k-mer lengths are used for de novo assembly with Velvet-Oases3435de novo assembly into longer contiguous sequences, assigned reads and contigs are retained if they are the most appropriate empirical >1.5 × longer than the average length of the candidate reads. Finally the largest contig after de novo assembly is added into the backbone to generate phylogenetic tree by unweighted pair-group method with arithmetic means (UPGMA) and visualized by Environment for Tree ExplorationThe A 3.8 gigabase (Gb) human nucleotide database (human DB) was constructed from a combination of human genomic DNA, unlocalized DNA GRCh38/hg38), ribosomal RNA , RNA (RefSeq), and mitochondrial DNA (RefSeq) sequences in NCBI as of July of 2015. The viral nucleotide databases in fast mode were constructed from a combination of sequences in ViPR/IRD as of July of 2015. The viral nucleotide DB in sense mode consisted of 87071 entries was constructed by collection of all Refseq viral complete genomes and their neighbor genomes. The neighbor genomes were the complete viral nucleotide sequences which were non-RefSeq recorded from DDBJ, EMBL and GenBank. The viral protein databases were constructed from NCBI Refseq DB. The bacterial DB in sense mode was constructed from the collections of unique genome segments at species level within GOTTCHA/hg38, riVIP is tested on a common desktop PC with a 3.4 GHz Intel Core i7-4770 and 16 GB of RAM (running Ubuntu 14.04 LTS). Minimum hardware requirements for running VIP include a 4 GB of RAM, PC running Ubuntu 14.04 LTS (preferred). VIP and its external dependencies require about 500 MB of disk space. Reference data requires about 70 GB of disk space. During VIP runtime, up to 10 × the size of the input file may be needed as additional temporary storage.How to cite this article: Li, Y. et al. VIP: an integrated pipeline for metagenomics of virus identification and discovery. Sci. Rep.6, 23774; doi: 10.1038/srep23774 (2016).


A non-pathogenic Semliki Forest virus vector VA7-EGFP previously showed promise in targeting human tumor xenografts in mice, but the oncolytic capacity of the virus in canine cancer cells and the safety of the virus in higher mammals such as dogs, are not known. We therefore assessed the oncolytic potency of VA7-EGFP against canine cancer cells by infectivity and viability assays in two dog solid tumor cell lines. Furthermore we performed a 3-week safety study in two adult Beagles which received a single intravenous injection of ~2 × 10VA7-EGFP was able to replicate in and kill both canine cancer cell lines tested. No adverse events were observed in either of the two virus-injected adult Beagles and no infective virus could be recovered from any of the biological samples collected over the course of the study. Neutralizing antibodies to Semliki Forest virus became detectable in the dogs at 5 days post infection and remained elevated until study termination.Based on these results, testing of the oncolytic potential of attenuated Semliki Forest virus in canine cancer patients appears feasible.The online version of this article (doi:10.1186/s12917-015-0498-2) contains supplementary material, which is available to authorized users. Canine tumors resemble human malignancies at cellular, phenotypic, and genomic levels, and dogs may serve as a more realistic cancer model system since their larger body size is more amenable to different surgical techniques and enables better evaluation of biodistribution of drugs and biotherapeutics than laboratory rodent models –3. BecauTogaviridae, and some of them have shown promising oncolytic potential in preclinical model systems [Alphaviruses are enveloped, single-stranded, positive sense RNA viruses of the family  systems , 7. As a systems .In previous studies, oncolytic alphavirus VA7, which is based on an attenuated A7(74) strain of Semliki Forest virus (SFV), was capable of eradicating human brain tumors and melanoma xenografts in immunocompromised mice following just a single intravenous injection , 10, whiin vitro with SFV vector VA7-EGFP and assessed virus replication and cell killing. In addition, we performed a minimal safety study in non-tumor bearing adult Beagles using ~2 × 105 plaque forming units (PFU) of the unmodified parental virus SFV A7(74) given via intravenous infusion. Our results show, on one hand, that dog tumor cells are able to support replication of oncolytic SFV, and on the other hand, that a limited inoculum of virus does not elicit side-effects in healthy adult dogs. These results pave the way for clinical translation of attenuated SFV in canine cancer patients.We hypothesized that attenuated SFV can infect and kill canine cancer cells and that it can be safely given to dogs intravenously. To test our hypothesis, we infected, as a proof-of-concept, two canine osteosarcoma cell lines (Abrams and D17) P < 0.001 in all and D17: P = 0.0053, 0.008 and 0007, respectively)  to cell ratios (MOI = multiplicity of infection) with our reporter virus VA7-EGFP. Infection was productive in both cell lines, as seen by progressive increase in virus-expressed fluorescent reporter protein, concomitant with progressive cytopathic effects Fig.  and cellly) Fig. . The rat5 PFU of non-recombinant parental SFV A7(74), accounting for a degree of virus inactivation in saline during animal preparation. We kept the injection dose relatively low to assess potential self-amplifying viremia, which is a hallmark of alphavirus infection in susceptible mammalian hosts [Two adult female laboratory Beagles were administered by intravenous infusion approximately 2 × 10an hosts , 18. Nei6 PFU and diluted in DMEM. Virus recovery from these samples as shown by plaque assay was 6.25 × 104 PFU/0.1 g (6.25 % recovery) and 3.75 × 105/0.1 g PFU (37.5 % recovery) in dog 1 and dog 2, respectively. Thus, using the lower recovery rate, the smallest theoretical amount of feces giving at least one plaque in the assay was 400 PFU/g. Live virus could not be recovered from any of the analyzed serum, urine, or fecal samples. The theoretical detection limit for liquid samples was 25 PFU/ml.In order to determine the sensitivity of our virus detection assay in feces, a negative baseline sample (0.1 g) from each dog was spiked with 10Levels of neutralizing antibodies (NAbs) in serum samples collected before virus injection and 9 and 21 days post infusion were determined by plaque inhibition assay.NAbs were not present at baseline, while NAb titers of 1250 on day 9 and 250 on day 21, respectively, could be detected in both dogs. Our results thus indicate that both dogs had obtained virus and mounted a humoral immune response in the absence of detectable viremia.Necropsy and histopathology showed no specific findings associated with the virus administration. No virus antigen was detected in any of the organs analyzed.Oncolytic viruses carry great potential as broadly active cancer therapeutics, as they simultaneously interfere with multiple cell survival signaling pathways and trigger inflammatory responses, which, in turn, may expose the tumors to the immune system. In the present study, we have assessed the potential of a promising oncolytic candidate, attenuated SFV, to target canine cancer. Like most human and rodent cancer cell lines in culture , also thin vitro may not correlate to therapy efficacy in vivo, rendering extensive in vitro killing assays unwarranted [in vivo, as infection triggers local inflammatory responses principally mediated by type I IFN to which the tumor cells may respond [8 PFU/ml, lasting 1–4 days [post infusion, suggesting that healthy dog tissues are poorly permissive to SFV, resulting in absence of viremia and viral shedding. In theory, the used input dose of approximately 2 × 105 PFU could have been too low to give rise to detectable viremia. While we cannot completely exclude this possibility, we do not believe it to be the case as very few viral particles are required for productive infection in susceptible host. Depending on the route of inoculation , as little as 3–8 PFU constitute the 50 % protective dose for attenuated SFV strains against lethal SFV strain challenge, with productive replication and 100 % protection reached with 20 PFU administered peripherally [While using a broader panel of dog tumor cells may reveal relative degrees of permissiveness to SFV, we have noticed that tumor sensitivity to lysis arranted , 19, 20. respond . Paracri respond –24. Inde1–4 days , 25–27. pherally , 22, 28.pherally , 30. Thepost injection, but still all dogs developed antiviral antibodies from day 5 onward [The neutralizing antibody response observed in both dogs in the present study indicated productive infection by the attenuated SFV, although no infectious virus was recovered from any blood samples or from the secretions. This is in line with the results from a recent study in healthy dogs injected with vesicular stomatitis virus, where infectious virus could not be detected in blood at any time 5 onward . In a st5 onward . So far,5 onward . Also in5 onward , 35 or a5 onward –38. Howepost-mortem.Oncolytic adenovirus has so far showed excellent safety in tumor-bearing  and healAedes mosquitos endemic in the tropical areas [In light of the poor capacity of SFV to replicate in dogs upon intravenous injection, clinical application of the virus in canine cancer patients would most likely entail intratumoral virus injections ensuring successful delivery of the virus into the tumor mass. This route has occasionally proven to be therapeutically superior over systemic administration even in susceptible hosts , 13. Theal areas . Hence, in vitro, and parental A7(74) strain was safe causing no visible symptoms or distress in the laboratory Beagles. No virus could be detected in any of the organs, blood or secretions analyzed. Our findings support proceeding with SFV mediated oncolytic virotherapy to first stage clinical studies in canine cancer patients.We report here the first proof-of-principle data for Semliki Forest virus as a candidate oncolytic agent in dogs. As our results demonstrate, live attenuated SFV VA7-EGFP was able to replicate and kill canine osteosarcoma cells in vitro studies has been also described earlier [post infection (p.i)., cell debris spinned down, and the supernatant sterile-filtered using 0.2 μm filter before freezing the virus in -70 °C in work aliquots.Generation of the plaque-purified replication competent, attenuated A7(74) strain of SFV, kindly donated by Dr. R. E. Shope at the Yale Arbovirus Research Unit , has been described previously . Generat earlier . VirusesTwo canine osteosarcoma cell lines, Abrams  and D17  , were teAbrams and D17 cells were plated on 48–well plates at a density of 25,000 cells/well. The next day, the cells were infected with VA7-EGFP at MOI of 0.01, 0.1, and 1. Growth medium was used as sham treatment and all samples were prepared as triplicate with identical results. Virus infection was monitored by fluorescence microscopy and cytopathic effect was evaluated by phase contrast microscopy  24 and 48 h p.i. Finally, crystal violet staining was performed to reveal cytopathic effect at the final time point of 48 h p.i. and stained plates were imaged with digital camera. For quantification of cell viability, crystal violet from stained wells was dissolved in 250 μl 10 % acetic acid for 20 min in room temperature in a shaker. Samples from dissolved dye were transferred to 96-well plate and absorbance was measured at 595 nm wavelength with plate reader .ad libitum. The experiment was approved by the National Animal Experiment Board of the Regional State Administrative Agency of Southern Finland (ESAVI/3231/04.10.07/2013). Dog 1 weighed 6.7 kg and dog 2, 12.9 kg.Two healthy adult female HsdRcc:DOBE Beagle dogs  were infected with A7(74) strain of SFV. Both dogs were 1 year old retired breeding females. The dogs were housed together in standard conditions, fed twice daily, and given water Before virus administration, an intravenous catheter was placed into the cephalic vein and 10 ml of sterile saline was administered to ensure vein access. As SFV is pH-sensitive and could potentially suffer from inactivation in physiological saline which lacks buffering capacity, we chose to inject 1 million PFU of the virus in order to expose the dogs to a minimally infective dose (see Discussion). Virus was diluted in 50 ml of saline  and kept on ice until given to the dogs via intravenous infusion over 15 min (200 ml/h). The infusion line ran through tempered water bath (37 °C) before entering the vein. After virus administration, the virus-saline bag and infusion line were flushed with 25 ml of saline at the same infusion rate to ensure that the dogs received the entire dose of virus. Altogether the infusions lasted for approximately 23 min.The dogs were monitored for adverse events according to the Veterinary Cooperative Oncology Group - Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 . AttitudThe sample collection schedule is shown in Fig. One milliliter blood was collected for hematology before, and 1, 2, and 4 days after virus administration and then three times weekly. In addition, 6 ml of blood were collected into serum tube for clinical chemistry and the assessment of SFV antibody titers before virus infusion and then, 9–21 days after virus administration. Serum was separated as described earlier. Hematological parameters were determined by an automated analyzer  and serum was assayed by a clinical chemistry analyzer . Urinalysis was done once a week by dipstic  read with automated reading device  Specific gravity was determined by refractometry, and a standard volume (0.5 ml) of urine sediment was examined under a light microscope using 10× and 40× objectives after centrifugation (5 min 600 g). Urine protein-creatinine ratio was measured by a clinical chemistry analyzer (Konelab 30i). Samples for SFV antibody titer were frozen first at -18 °C and transferred the same day into -80 °C until analysis. Hematology, chemistry panel, and urinalysis were analyzed the day they were collected.et al. [et al. [6 PFU of SFV A7(74) per 0.1 g of baseline fecal samples of the two dogs diluted in 10× (w/v) amount of medium. The virus-feces-medium mixtures were then vortexed, sterile filtered, serial diluted (from 10−1 to 10−6) in growth medium, and titered as described above.Sera obtained from the dogs were titered for SFV A7(74) on Vero cells according to Ruotsalainen et al. . Serial  [et al. . Urine set al. [NAbs were measured from the serum samples collected on baseline, and 9 and 21 days after the virus infusion according to Ruotsalainen et al. . BrieflyNecropsy and histopathological examination were performed by a board-certified pathologist (MOA). Representative samples of all selected organs  / mean A595(uninfected sample)) × 100. The data was plotted and analyzed with GraphPad Prism . Statistical analysis was done using unpaired, two-tailed


There is an increasing need to develop novel bioassay methods for low-cost, rapid, and easy-to-use multiplex detection of pathogens in various fields ranging from human infectious disease diagnosis, drinking water quality control, to food safety applications. Due to their unique advantages, magnetic and optomagnetic bioassay principles are particularly promising for biodetection platforms that will be used in developing countries. In this paper, an optomagnetic method for rapid and cost-efficient qualitative biplex detection of bacterial DNA sequences is demonstrated. Within less than two hours, the assay gives an answer to whether none, both, or only one of the bacterial DNA sequences is present in the sample. The assay relies on hybridization of oligonucleotide-functionalized magnetic nanobeads of two different sizes to rolling circle amplification (RCA) products originating from two different bacterial targets. The different bead sizes are equipped with different oligonucleotide probes, complementary to only one of the RCA products, and the read-out is carried out in the same sample volume. In an optomagnetic setup, the frequency modulation of transmitted laser light in response to an applied AC magnetic field is measured. The presented methodology is potentially interesting for low-cost screening of pathogens relating to both human and veterinary medicine in resource-poor regions of the world. Novel bioassay methods for low-cost, rapid, and easy-to-use multiplex detection of pathogens are increasingly demanded in diverse fields ranging from human infectious disease diagnostics , 2, drinV2 = V2' + V2'', of the output voltage from the photodetector, normalized with respect to the total intensity of transmitted light, V0. We consider two data analysis approaches; firstly V2'/V0 and secondly the phase angle response, ζ = arctan (V2'/V2''), vs. frequency of the AC magnetic field. Beads bound to DNA coils will respond differently than free beads, which will be reflected in the shape of the curves. We are able to show that each combination of presence/absence of the two targets has a distinct phase angle signature, thereby enabling a straightforward qualitative biplex read-out. It should be noted that in previous work [In this paper we present an optomagnetic method for rapid and cost-efficient qualitative biplex detection of bacterial DNA sequences. Within less than two hours, the assay gives a qualitative answer to whether none, both, or only one of the bacterial DNA sequences is present in a sample. The assay begins with a padlock probe ligation and rolling circle amplification (RCA) protocol (about 70 min) for highly specific target recognition and isothermal enzymatic amplification. The resulting two types of RCA products in the form of macromolecular coils of ssDNA (here denoted DNA coils) with a repeating sequence motif are mixed with magnetic beads (exhibiting Brownian relaxation behavior) of two different sizes, each functionalized with a detection oligonucleotide complementary to only one type of DNA coil. It should be noted that the padlock probe ligation reaction constitutes highly specific target recognition since both the 5' and 3' ends of the linear padlock probe are designed to bp next to each other on the target strand. After an incubation step of 20 min and transfer to a disposable cuvette, the coil-bead sample is measured using an optomagnetic system (measurement time about a few minutes) in which the sample, while being subjected to an alternating current (AC) magnetic field , is illuminated by a laser beam and the transmitted light is collected by a photodetector. The field-induced sample response is measured as the second harmonic component, ous work  we demonSequences of targets, padlock probes, and detection oligonucleotides can be found in Supporting information, Table S1.Escherichia coli (EC) was conjugated to 250-nm beads , and detection oligonucleotide for Vibrio cholerae (VC) was conjugated to 100-nm beads (Brownian nanofluid (BNF)-Starch avidin, Micromod, Germany). The two bead batches were further diluted with PBS to a final concentration of 400 μg/mL, after which they were mixed at a volume to volume ratio of 1:2 to get final approximate concentrations of 130 and 270 μg/mL of 250- and 100-nm beads, respectively.Two 200-μL batches of oligonucleotide-functionalized magnetic beads were prepared according to protocols described in detail in Supporting information, Section S1. Detection oligonucleotide for Target recognition and RCA were performed essentially as previously described in ref. , for detThe optomagnetic setup comprises a Blu-ray laser source, a photodetector, a pair of electromagnets, a sample holder, a current source, a data acquisition (DAQ) unit, and a computer; for details, see Supporting information, Section S3. A schematic illustration of the setup is shown in Supporting information, Fig. S1. An AC magnetic field is applied using the electromagnets, and the frequency modulation of the photodetector voltage signal is measured using a lock-in amplifier with the magnetic field frequency as the reference. The magnetic beads respond to the applied AC field by the formation and disruption of chain-like superstructures. The modulation of transmitted light by this chain formation/disruption dynamics is measured as the second harmonic component of the photodetector voltage signal, divided into its in- and out-of-phase components. Theory behind the optomagnetic measurement principle can be found in Supporting information Section S4 as well as in ref. . In partFifteen μL of bead solution was gently mixed with 15 μL of DNA coil solution of different compositions and concentrations. The solution was incubated for 20 min at 55 °C, after which it was diluted with 30 μL of a 50–50 buffer mixture (50 v/v % of 1 × PBS pH 7.4 and 50 v/v % of hybridization buffer). The final solution was transferred to a cuvette and measured using the optomagnetic setup in the frequency range 1–200 Hz with an AC magnetic field amplitude of 2.58 mT.V2'/V0) and out-of-phase (V2''/V0) vs. frequency spectra for four selected DNA coil concentration combinations, respectively. It can be observed that all four curves have clearly distinct signatures, different in shape and/or peak frequency. For no DNA coils present  a comparably narrow V2' peak is seen at a frequency corresponding to one in-between the two individual free-bead peaks, which could be considered as the sum of the peaks for 250- and 100-nm beads. When measuring on samples with only 250-nm beads and EC-coils , the free-bead peak is located at 15 Hz, whereas the free-bead peak for 100-nm beads  is located at 40 Hz. It is reasonable to assume that the result from measuring on a sample containing both bead sizes would be represented by a peak at a frequency somewhere in between those two values. When DNA coils in high enough concentration are present, the free-bead peak is suppressed in favor of the formation of a new bound-bead peak at a lower frequency. This V2' peak is tentatively explained by the formation of chains by beads bound in DNA coils. As can be seen in Supporting information, Fig. S3A and B, the bound-bead peak for 250-nm beads in EC coils appears at 5 Hz whereas the bound-bead peak for 100-nm beads in VC coils appears at a slightly higher frequency. Using this information, the interpretation of the curve shapes and peak frequencies of V2' spectrum represents the sum of the contributions from EC coils with bound 250-nm beads and free 100-nm beads. Accordingly, the curve shows one peak at 37 Hz representing the free 100-nm beads and a signature of one low-frequency peak representing the response from EC coils with bound 250-nm beads. For the sample EC 0, VC 1, the V2' curve is a sum of the response from free 250-nm beads and VC coils with bound 100-nm beads. Since these two peaks overlap much more than for the case of the EC 1, VC 0 sample, the resulting curve is much narrower. In the EC 0, VC 1 curve, a small residual peak at 50 Hz can be seen, corresponding to the response from a small amount of free 100-nm beads. The explanation for the residual free 100-nm bead peak being located at a somewhat higher frequency compared to the measured free-bead peak at 40 Hz could be explained in terms of the existence of a small part of the bead size distribution  having few or no oligonucleotides, therefore unable to bind to DNA coils.Panels A and B in V2' peak amplitude upon increasing DNA coil concentration) with regards to SDs of the measured samples. Thus, we proceeded by considering the phase angle ζ = arctan (V2'/V2'') vs. frequency spectrum. In ζ vs. frequency spectra belonging to the four samples in In recent studies  we have E. coli and V. cholerae DNA sequences in an optomagnetic measurement setup. Within less than two hours an answer can be obtained. The presented methodology is potentially interesting for low-cost screening of pathogens relating to both human and veterinary medicine in resource-poor regions of the world.We have demonstrated qualitative biplex detection of


Plants represent a safe, efficacious and inexpensive production platform by which to provide vaccines and other therapeutic proteins to the world’s poor. Plant virus expression vector technology has rapidly become one of the most popular methods to express pharmaceutical proteins in plants. This review discusses several of the state-of-the-art plant expression systems based upon geminiviruses that have been engineered for vaccine production. An overview of the advantages of these small, single-stranded DNA viruses is provided and comparisons are made with other virus expression systems. Advances in the design of several different geminivirus vectors are presented in this review, and examples of vaccines and other biologics generated from each are described. Over the past twenty years, great advances have been made in the construction and generation of plant-derived vaccines. Vaccines and other biopharmaceutical proteins produced from plants are safe, efficacious, and can be easily scaled up for mass production. Plant-made vaccines may provide a select advantage for specific demands that may not be readily addressed through conventional vaccine production. These range from providing inexpensive vaccines for the world’s poor who reside in developing countries to stockpiling vaccines against pandemic infectious diseases and even to generating plant made biologics for the field of personalized medicine ,2.Initially, vaccines and other pharmaceutical proteins were generated from transgenic plants, however, recent advances in plant virus molecular biology have yielded an alternative means of transiently expressing proteins through the use of virus expression vectors which are engineered to be delivery vehicles. Plant virus expression vectors offer many advantages for foreign protein expression over stably transformed plants; these include greater expression levels over a short period of time, the ability to generate proteins which may impede plant growth, as well as reduced biocontainment issues and related public perception concerns related to genetically modified crops. The deletion of virus movement and coat proteins disables plant to plant movement of virus expression vectors, and as a result, the possibility of pharmaceutical proteins being transmitted to weedy relatives is greatly reduced. Conversely, stable transgenic lines have the advantage of providing a permanent genetic resource which lacks significant variation in foreign protein expression and can be stored as seed .Agrobacterium is also frequently used as a means to introduce virus vectors to plants ..cis-actiIt has been suggested that geminiviruses originate from a common ancestor along with other small, circular, single-stranded DNA viruses of related genomes, including nanoviruses, circoviruses and cycloviruses. This group of viruses infect a diverse array of organisms, ranging from plants and mammals to birds, fish and insects ,8. The cGeminiviruses replicate via a rolling circle mechanism and use a replication initiator protein, Rep, that initiates replication at a highly conserved stem-loop structure located between two major open reading frames of the genome. The replication strategy of geminiviruses has been reviewed extensively and will be only briefly described here ,18. FollBegomoviruses and Curtoviruses, and betaC1, which is encoded by betasatellites that are associated with a number of begomoviruses ..N. benthet al., (2013), have developed a technology based on the mastrevirus Tobacco yellow dwarf virus (TYDV) that offers an interesting new twist on previous geminivirus expression vector constructs  requires the presence of the helper viruses tomato leaf curl virus (ToLCV) or beet severe curly top virus (BSCTV). An expression vector was engineered from this betasatellite by replacing the βC1 ORF with segments of either the CaMV 35S or the petunia ChsA promoter. The authors showed that they could silence reporter gene activities in transgenic plants expressing by 35SGUS and in nontransgenic petunia plants expressing ChsA by supplying this betasatellite in the presence of helper virus .Recently, the Bean yellow dwarf geminivirus vector has been used to deliver sequence-specific nucleases and DNA repair templates to facilitate homologous repair of double-stranded breaks within plant sequences ,51. RepaPlant virus expression vectors have been engineered to function as rapid, inexpensive and robust platforms for vaccine production. The implications of this and other technologies related to molecular pharming in plants are substantial. Geminivirus vectors offer select advantages over plant RNA virus expression vectors, for example, they are able to function in a much broader range of plants, and thus provide more choices of the production system to be used. Geminiviruses lack the stability issues that their RNA virus vector counterparts experience . Geminiv


Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. Most models of influenza virus assembly postulate a direct interaction between the viral matrix protein M1 and the cytoplasmic domains of the envelope glycoproteins hemagglutinin (HA) and/or neuraminidase (NA) as a driving force for virus assembly ,3. HowevThe most pronounced feature of the cytoplasmic tail of HA is the attachment of fatty acids to (mostly) three conserved cysteine residues ,11. Althi.e., HA from the A/Udorn/72 (H3N2) (Udorn) strain in the background of the A/WSN/33 (H1N1) (WSN) strain. It was subsequently shown that deletion of cytoplasmic acylation sites from HA of the Udorn virus is lethal for virus replication, but recombinant virus could be rescued and grew to only moderately depressed titers by expression of M1 from the WSN strain -methionine/cysteine . Virus preparations were subjected to SDS-PAGE under non-reducing conditions and fluorography using 1 M salicylate. The dried gel was exposed to X-ray film and bands intensities were analyzed with Bio-1 D software .Virus particles were produced by infecting MDCK cells in a 15 cm dish at an m.o.i. of >2. At 24 h after infection, the supernatants were harvested, cell debris was removed (2000× 6. RNA was then extracted with the Invisorb Spin Virus RNA Mini Kit (Stratec) and cDNA synthesized with the Maxima H Minus First Strand cDNA Synthesis Kit . 4 µL 5× RT-Buffer, 1µL dNTPs (10 mM), 1 µL Oligo(dT)18 Primer (100 µM), 1 µL Maxima H Minus Enzyme mix and 13 µL RNA were used per sample, which were incubated at 50 °C for 30 min. The reaction was terminated by incubation for 5 min at 85 °C.In order to analyze an equivalent number of virions the supernatants from infected MDCK cells were adjusted to an HA titer of 2in vitro transcribed, M segment-derived RNA transcript. The reaction product was directly used in a quantitative RT-PCR with the following primers specific for the influenza A virus M gene segment: Forward primer (5′→3′): AGATGAGTCTTCTAACCGAGGTCG, reverse primer (5′→3′): TGCAAAAACATCTTCAAGTCTCTG (Invitrogen). Real-time RT-PCR was performed utilizing a TaqMan probe, OneTaq DNA polymerase (NEB) and the ICYCLER-IQ5 Multicolor Real Time PCR Detection System . One assay with a total volume of 25 µL contained 5 µL 5× OneTaq-buffer, 16 µl RNAase free water, 0.5 µL dNTPs (100 µM), 0.25µL forward and reverse primer (100 µM), 0.75 µL probe , 0.25 µL OneTaq DNA polymerase and 2 µL cDNA as template. Temperature profile: 3 min, 95 °C (1×); ) (40×). Fluorescence values (FAM) were collected during the annealing step. A standard curve was generated by using serial dilutions of an Whereas the crystal structure of the ectodomains of various HA subtypes has been determined, very little is known about the remaining part of the molecule, the cytoplasmic tail, the transmembrane domain (TMD) and the linker region that connects the TMD to the ectodomain. Based on the amino acid sequence as well as on the 3D structures of their ectodomains the 18 antigenic subtypes of HA are divided into two evolutionary groupings: group-1  and group-2  ,31. To iAs obvious from The transmembrane region contains 26–27 amino acid residues and is (by definition) demarcated on both sides by either charged  or large and hydrophilic residues (N or Q). Its N-terminal part that contacts the outer half of the lipid bilayer is highly conserved, both between all subtypes and more pronounced within group-1 and group-2 HAs. The mean conservation value (the relative height of logo stacks) of the N-terminal conserved cluster is ~93% for group-1 (amino acid positions 529–542) and group-2 HAs (531–543). The high conservation of amino acids suggests that this region is not only a membrane anchor  but might play a further, more specific role, for example binding to hydrophobic ligands. In accordance, we have recently identified a cholesterol-consensus motif in group-2 HAs that comprises the completely conserved residues Tyr 527 and Lys 528 in the linker region and Leu 532 and Trp 533 at the beginning of the TMD . In contThe cytoplasmic tail (10–11 amino acids) is again highly conserved. Noteworthy, five residues Gly 557, Cys 561, Ile 563, Cys 564 and Ile 565 (WSN numbering) are almost invariant, not only within one group but also through all HA subtypes and are therefore likely to be of functional importance. Ile 563, the palmitoylated C564 and I565 form a hydrophobic patch at the C-terminal end of the tail that is also conserved in HA of Influenza B virus . C561 loThe location of the third acylation site is also not completely conserved. However, as is obvious from their consensus sequences, each HA subtype has at least three cysteine residues . In mostBased on the described sequence conservation, we created a set of mutants to investigate which amino acids in the cytoplasmic tail of HA are required for or affect virus replication . The who3H-palmitate when they are expressed from a plasmid in CV1 cells [Having verified the mutation by sequencing, the mutant HA plasmid together with seven plasmids encoding the other viral proteins were transfected into HEK 293T cells and three days later the supernatant was tested for virus particle release by a plaque assay in MDCK cells. For four mutants, HA with deleted cytoplasmic tail (G557stop), HA with deleted C-terminal palmitoylation sites  and the mutant with a non-conservative substitution in the C-terminal hydrophobic patch (I563Q), we never (five transfections) rescued infectious virus particles, although control experiments using plasmids with wild-type HA done in parallel showed that transfection was successful. Note that the failure to generate infectious virus particles with HA G557 stop and HA I563Q is not due to a complete blockade of acylation since these HA mutants are efficiently labeled with V1 cells . In summary, both palmitoylation sites in the cytoplasmic tail, but not the stearoylated cysteine at the end of the TMD of H1 subtype HA are indispensable for virus replication.i.e. to arginine, asparagine, threonine, glycine and surprisingly also to cysteine (specified by another codon (TGC) than the one in wild type HA (TGT)), were never observed. We can only speculate why a reversion to cysteine did not occur but this region in the viral genome is part of a larger signal for packaging of Ribo-Nucleoprotein Particles (RNPs) into budding virus particles [The other mutations in HA did not prevent rescue of infectious virus, which was amplified for further characterization. After each amplification step, RNA was extracted from virus particles, cDNA was prepared and ~300 nucleotides at the 5′ end of the HA gene were sequenced to ensure that the mutation is still present. During subsequent passage of recombinant virus in MDCK cells, we observed nucleotide reversions at three of the mutated codons. For the mutant having a substitution in the TMD (G547I), we always (five attempts) observed a single nucleotide exchange from ATT (isoleucine) to AGT (serine). A partial exchange of T to G, reflected by superimposed peaks for the respective bases, was already visible in the viral genomes prepared from the transfection supernatant and the resulting virus G547S completely outgrew G547I after two amplifications , it apparently confers a fitness advantage since the revertant virus completely outgrew the mutant one in just two amplification steps  to HA titer ratio revealed that the relative infectivity of the mutants was reduced to 0.8 (G547S), 0.7 (G557A), 0.2 (AC1) and 0.01 (G557E) relative to wild type virus (1.0). The other mutants showed  identical titers to wild type virus, no reduction in specific infectivity (I563L and Q560E = 1.8); and no difference in plaque size was obvious between these mutants and wild type WSN.To analyze the replication kinetics of the viruses, MDCK cells were infected with wild type WSN or one of the mutants at a low multiplicity of infection (m.o.i.), and the supernatants were collected at various times and assessed for virus titer by plaque assay. Overall, differences in growth properties between wild type and mutant virus were rather subtle as seen in the growth curves calculated from three independent infections A–C. For 6 and subjected aliquots to SDS-PAGE and Western blotting with polyclonal antiserum against fowl plague virus, which cross-reacts with WSN M1  equal proportions in the virus preparation from wild type virus and from the mutants Q560E, G557E and Ac1 + L559C. The mutants G557A and I563L contain more M1 (~60%) at the expense of both HA and NP whereas M1 is reduced (~20%) and both HA and NP are increased in G547S. In contrast, Ac1 contains more NP (43%) and less M1 and HA. Although the effects seem to be large for some mutants we want to express our skepticism about the reliability of the data since even for wild type virus the relative ratios of HA, NP and M1 varied between different preparations .In order to estimate the relative amount of proteins incorporated into virus particles we metabolically labelled virus infected cells with [6 (t) value (28.2–28.6) compared to wild type WSN (27.5). However, a statistically significant difference was obtained only for the mutant I563L that has a Ct value of 29.1. One cycle difference in the Ct value indicates a two-fold difference in the amount of DNA suggesting that the content of RNPs in mutant I563L is reduced to ~30% compared to wild type. Finally, to quantify the amount of RNPs  present in virus particles, we performed quantitative real time PCR with M1 specific primers on virus samples adjusted to a HA titer of 26 C. The reIn summary, we have shown here that the cytoplasmic tail of H1 subtype HA is essential for virus replication since WSN virus with tailless HA could not be generated. This is in contrast to a previous study, where viruses containing tailless HA from the Udorn strain (H3 subtype HA) could be rescued in the background of a WSN helper virus and even revealed only little growth defects . Except Individual amino acids in the tail of HA from WSN had very different effects on virus growth. Deletion of the two cytoplasmic palmitoylation sites and non-conservative substitution of the adjacent isoleucine by glutamine completely prevented rescue of infectious virus . This isIn contrast, recombinant viruses with mutations in other conserved amino acids showed no altered morphology of virus particles , some onVVSLGAISFWMC554) is plotted as a helical wheel, G547 is located together with other small amino acids  on the same side of the helix as the stearoylated cysteine 554. One might speculate that these small residues are located on the outer surface of the trimeric TMD where they might form a groove which binds stearate to align it with the TMD helix as previously speculated [3) by the large residue isoleucine (volume of 170 Å3) might disrupt the assumed interaction and, therefore, a spontaneous reversion to serine (volume of 100 Å3) confers a growth advantage.However, although all effects are rather small , the repeated occurrence of amino acid reversion suggests that conserved residues in the tail and in the TMD confer a growth advantage . The moseculated . Alternaeculated . In bothHowever, our results are difficult to reconcile with the assumption that the cytoplasmic tail of HA alone recruits M1 to the viral assembly site, at least if this is assumed to occur by hydrophilic binding forces, such as salt bridges or hydrogen bonds. The hydrophobic patch ICI (563–565) at the C-terminal end of the tail plus the second palmitoylation site in close proximity (561) likely anchors the tail to the inner side of the plasma membrane . This asNevertheless, there is clear evidence that HA of both Influenza A and B virus at least functionally interacts with M1 during budding ,37. M1,