Using target capture of viral nucleic acid and next-generation sequencing, we generated the complete genomes of two novel human parainfluenza virus 1 isolates. Isolates AR001 (accession no. KX570602) and NM001 (accession no. KX639498) were collected 3 months apart from pediatric patients with acute respiratory infection from Arkansas and New Mexico, respectively. Paramyxovirinae of the Paramyxoviridae family (Acute respiratory infections (ARI) are one of the leading causes of morbidity and mortality in young children, accounting for ~25% of hospital admissions  and almoe family . There ae family .2) of 98%. A chest X ray revealed cardiopulmonary disease. He required only supportive care and was discharged home after evaluation. Patient 2 (NM001) was a 6-year-old otherwise-healthy female who presented on 16 December 2015 to the University of New Mexico Hospital in Albuquerque, NM, with 10 days of productive cough, intermittent fevers up to 104°F, and a SaO2 of 89%. Her chest X ray documented bronchial wall thickening and hyperinflation consistent with a viral infection. She was hospitalized for one day on the general pediatric floor and required 5 h of supplemental oxygen.Here, we present the complete genome sequences of two novel hPIV1 isolates collected 3 months apart from two pediatric patients with differing clinical courses. Patient 1 (AR001) was a 5-year-old male with no significant past medical history seen at Arkansas Children’s Hospital in Little Rock, AR, on 9 September 2015 after having a nonproductive cough for 6 days with rhinorrhea. The patient was afebrile and had an oxygen saturation in arterial blood  ResVir respiratory viral panel probe set. The UNM ResVir panel contains 5,683 hybridization probes designed to be complimentary to coding sequence (CDS) regions of 24 human respiratory viruses. Next-generation sequencing was performed on an Illumina MiSeq using v3 chemistry and paired 75-bp reads. Sequencing of AR001 and NM001 resulted in 83,471 and 919,050 sequencing reads aligning to the hPIV 1 RefSeq reference NC_003461, respectively. No other viruses were detected. Consensus genome sequences were generated by reference-guided alignment to NC_003461 and annotated using the VIPR Genome Annotator  revealed that both AR001 and NM001 grouped with two strains isolated in France in 2010 to 2011 (accession numbers KF530218 and KF530224) and a 2011 strain from the United States (accession no. KF530213). Taken together, these data indicate that although related, these two hPIV1 isolates, obtained from Arkansas and New Mexico 3 months apart, exhibit significant genomic differences, which may underlie the patients’ different clinical phenotypes.KX570602 and KX639498, respectively.The whole-genome sequence of isolates AR001 and NM001 have been deposited in GenBank under the accession numbers


Ticks, as a group, are second only to mosquitoes as vectors of pathogens to humans and are the primary vector for pathogens of livestock, companion animals, and wildlife. The role of ticks in the transmission of viruses has been known for over 100 years and yet new pathogenic viruses are still being detected and known viruses are continually spreading to new geographic locations. Partly as a result of their novelty, tick-virus interactions are at an early stage in understanding. For some viruses, even the principal tick-vector is not known. It is likely that tick-borne viruses will continue to emerge and challenge public and veterinary health long into the twenty-first century. However, studies focusing on tick saliva, a critical component of tick feeding, virus transmission, and a target for control of ticks and tick-borne diseases, point toward solutions to emerging viruses. The aim of this review is to describe some currently emerging tick-borne diseases, their causative viruses, and to discuss research on virus-tick interactions. Through focus on this area, future protein targets for intervention and vaccine development may be identified. The virus is particularly virulent for humans but has little or no pathogenic effect on livestock. Infection following a tick bite is followed by a short incubation period of between 3 and 7 days leading to a brief febrile illness characterized by fever, headache and myalgia. Haemorrhagic manifestations of disease develop 4–5 days after the development of fever and include bleeding from the nose, gastrointestinal system and urinary tract. The fatality rate ranges from 3 to 30%. Treatment is supportive, including replacement of fluids and blood constituents. There is no licensed antiviral treatment or licensed vaccine for CCHF.The virus now known as Crimean Congo Haemorrhagic fever virus (CCHFV) was first described during an outbreak of haemorrhagic fever in Red Army soldiers in the Crimea during the final years of the Second World War Ergönül, . This viHy. marginatum, a species found throughout the Iberian Peninsula, southern France, Italy, the Balkans, and Turkey. In 2010, the virus was detected in Hy. lusitanicum ticks in Spain   was isolated following an outbreak of haemorrhagic fever amongst villagers living in the Kyasanur Forest area in 1957  and the black-faced langur (Semnopithecus entellus) are considered sentinels for the presence of KFDV at a site  is a recently described tick-borne virus within the genus Argasid tick Ornithodoros savignyi and the Ixodid tick Hyalomma dromedarii  causes fatal encephalitis in a proportion of humans that become infected with it. Between 2013 and 2015, 8 cases of POWV encephalitis were reported from hospitals in Massachusetts and New Hampshire  contribute to the maintenance of virus  with infected ticks during the expansion of the Russian fur trade.Preliminary virological analysis suggested an association with tick-borne viruses such as (RSSEV) pointed researchers in the direction of ticks as the vector. Subsequent field surveillance detected POWV in pools of B. burgdorferi, across many States in the USA . Therefore, factors that influence rodent abundance such as predator decline, mediated by the red fox (Vulpes vulpes) for example, may have greater impact on disease transmission by ticks  , a flavivirus in the tick-borne encephalitis group, is a genetically distinct lineage (subtype) of POWV that can cause neuroinvasive infection in humans in parts of North America. DTV was originally isolated from the Rocky Mountain wood tick, Asfivirus and family Asfarviridae, and consists of a double-stranded DNA genome coding for ~150 proteins. The genome is surrounded by a protein capsid and a host-derived envelope. Virus particles are very robust and can survive for days in the environment or months within pork meat. ASF was first described in Kenya and is found across sub-Saharan Africa  and warthogs (Phacochoerus africanus) being infected following infestation with Argasid ticks of the genus Ornithodorus, particularly O. moubata. Infections in wild species do not result in clinical signs of disease. The second cycle involves infection of domestic pigs (Sus scrofa) that are highly susceptible to infection and shed virus in excreta. Initial infection is by tick bite but is then amplified by pig-to-pig transmission, either through contact or consumption of contaminated food. ASF is a notifiable disease in Europe  and significant effort is directed at preventing its introduction through import control of livestock and food stuffs. Outbreaks resulting from importation have occurred in domestic pigs on a number of occasions across Europe. Significantly in the case of Sardinia, following introduction in the late 1970s. Repeated attempt to eliminate the disease have failed and the disease is endemic  is the causative agent of African swine fever (ASF), an acute haemorrhagic fever that causes severe morbidity and high mortality in domestic pigs. ASFV is classified within the genus Thomson, . The epiO. porcinus SGE of ASFV infection in pigs  from North America in the nineteenth century (Ružek et al., D. reticulatus tick population but was rarely, if ever, encountered by humans. By introducing an exotic mammalian host that was highly susceptible to infection and regularly handled by humans, clusters of haemorrhagic disease occurred. A similar scenario appears to have resulted in the introduction of POWV in Eastern Russia and the activities of the farming industry in Eastern Europe has propelled the spread of ASFV. A further human factor that can increase detection rates of emerging tick-borne diseases is awareness by clinicians. Once a cluster of cases of disease have been reported, further cases come to light. However, a lack of disease awareness among clinicians and veterinarians can hamper the control of a disease epidemic, which could potentially be the case for ASFV where clinical signs can be similar to those observed with other diseases of swine (Guinat et al., Human factors are a major influence on disease emergence. The paradigmatic example of this is the emergence of Omsk haemorrhagic fever virus in central Russia due to the importation of muskrats (Tick-borne diseases continue to cause a burden to both animal and human health. As the twenty-first century progresses there will certainly be more examples of tick-borne virus spread and novel virus emergence. New viruses associated with ticks are being discovered as a result of human infection, such as Bourbon virus (Kosoy et al., NJ conceived the review. KM, LJ, LP, and NJ wrote the paper. All authors reviewed the final version of the manuscript.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.


We report the results of a rapid assessment of Zika virus awareness among key clinical specialties in Singapore.Between June 6 and June 19, 2016 we conducted an online survey of doctors working in obstetrics and gynaecology, neonatology and paediatrics in Singapore. The survey included 15 multiple choice questions to measure respondents' knowledge of Zika virus in four domains covering clinical and public health.A total of 110 survey responses (15% response rate) were obtained, 82% of respondents worked in the public sector. Overall, the median respondent score was 9.4 (Max score=15), with substantial variation (range: 3.5 - 14.7). Microcephaly and Guillain-Barré syndrome were recognised as causal complications of Zika virus infection by 99% and 50% of respondents respectively.  Clinical features which could help differentiate Zika from Dengue were less well understood with 50% and 68% correctly identifying conjunctivitis and low grade fever respectively. Worryingly, 14% favoured non-steroidal anti-inflammatory drugs as part of treatment, without first excluding dengue as a diagnosis. Also, only 36% of respondents were aware of the current recommendation for preventing sexual transmission of Zika virus. Fewer than 50% were aware of the need for ophthalmological evaluation as part of congenital Zika virus infection.Our assessment demonstrates that there is good awareness of the clinical manifestation of Zika virus disease among key specialty doctors, but confusion with Dengue disease remains. It also highlights knowledge gaps in the prevention of sexually-transmitted Zika virus infection and the clinical management of congenital Zika virus infection in newborns. Our study identified strategic areas to improve communication to front-line doctors during public health response to the Zika epidemic.  To date, 70 countries report ongoing mosquito-borne transmission of Zika virus, while microcephaly and other central nervous system malformations putatively associated with Zika virus infection have been reported from 20 countries.On February 1, 2016, the World Health Organization (WHO) declared a Public Health Emergency of International Concern (PHEIC) in relation to the then suspected association between Zika virus infection and microcephaly.2Aedes mosquito populations and year-round, endemic transmission of dengue and chikungunya viruses, is at potentially high-risk for introduction of Zika virus. The first imported case of Zika virus was reported in May 13, 2016 in a 48 year-old man who had recently returned from Sao Paulo, Brazil. Subsequently, a local autochthonous Zika virus outbreak was detected in July/August 2016.10Singapore, a tropical country with extensive international travel links, established Since the PHEIC declaration, guidelines for the testing and clinical management of Zika virus disease and associated complications have been issued and repeatedly updated by WHO and the US Centers for Disease Control (CDC). In Singapore, the Ministry of Health (MOH) has sent out three information updates via email as well as letters to all registered doctors as of June 1, 2016, which included public health information such as mandating the notification of suspected Zika virus disease and interim clinical management guidelines for Zika virus disease in pregnant women.Mass media and information access in Singapore are easily and conveniently accessible. As a dengue-endemic country, the risk of a Zika epidemic occurring has been reported by local media and the government. Medical practitioners are familiar with accessing information via the internet, including US CDC and WHO websites. Therefore, levels of Zika virus awareness among medical practitioners in Singapore should be representative of a standard that is realistically achievable in high-income countries as well as lower-resource settings with similar infrastructure for disseminating and receiving health information.We report the results of a rapid assessment of Zika virus awareness among key clinical specialties in Singapore, with the aim of identifying gaps in knowledge and opportunities for improving communication on Zika virus disease management and control policies.Study Participants and SitesBetween June 6 and June 19, 2016 prior to the Zika virus outbreak, we conducted an online survey of doctors working in obstetrics and gynaecology, neonatology and paediatrics in Singapore. A link to the online survey was disseminated by email to the relevant doctors at KK Women’s and Children’s Hospital and Singapore General Hospital, two large public hospitals in Singapore. Together they cater for approximately 50% of women’s and children’s hospital care in Singapore. To capture responses from doctors working in the private sector, the survey was also sent to members of the Obstetrics and Gynaecology Society of Singapore and the College of Paediatrics and Child Health, Singapore. The survey was anonymous and could be completed in about 10 minutes on a computer or mobile device. In order to make the survey educational, respondents were given a summary of relevant recommendations after they responded to each question. Respondents did not receive any incentive to complete the survey.Approval for this study, including waiver of informed consent, was obtained from the SingHealth Centralised Institutional Review Board (application number 2016/2251). The survey was anonymous so only anonymised data were used for analysis.Data CollectionThe survey included 5 questions regarding respondents' age group, sex, primary specialty, professional designation  and practice setting (public or private sector). In addition, 15 multiple choice questions were included to measure respondents' knowledge of Zika virus in four domains: (1) Clinical Presentation, Diagnosis and Management; (2) Transmission and Prevention; (3) Clinical Management of Pregnant Mothers Potentially Infected with Zika and Infants with Microcephaly; and (4) Ministry of Health Requirements for Notification of Suspected Zika Virus Infection. Of necessity, the survey was not an exhaustive set of questions, but was designed to capture general awareness of key knowledge and guidelines under these four domains. Respondents had to provide a response to every question in order to complete the survey.Data AnalysisWe summarised correct responses to each question using frequencies and percentages. In addition, survey responses were given a score of 0 to 15 based on the number of questions that were answered correctly. For some questions, respondents could choose more than one option (e.g. "Which types of mosquitoes are known to transmit Zika virus?"), so each question contributed a maximum of 1 mark to the total score, weighted by the number of possible correct options for that question. Scores were computed overall and for each of the four questionnaire domains. Statistical analysis was performed using Stata, version 11 (Stata Corporation).To compare levels of knowledge across domains, we normalised scores for each domain by dividing each respondent's score by the maximum possible score for that domain, such that scores for each domain were scaled betwen 0 and 1. We summarised score distributions using the median, range and interquartile range, and compared scores between categories of respondent age group, sex, medical specialty, professional designation and public/private practice using boxplots and the Kruskal-Wallis test. Response rates were estimated using the number of email addresses the survey link was sent to as the denominator.A total of 110 survey responses were obtained from emailing the survey to 747 doctors working in the key specialties of obstetrics and gynaecology, neonatology and paediatrics in Singapore. This represented an overall response rate of about 15%. Respondent characteristics are presented in Clinical Presentation, Diagnosis and Management Low-grade fever, rash and muscle or joint pain were each identified as typical symptoms of Zika virus disease by over 68% of respondents; fewer than 50% recognised conjunctivitis as part of the typical clinical presentation. Although signs of haemorrhage/bleeding and high-grade fever are not classical presentations of Zika virus disease, 6% and 37% of respondents respectively selected these. Microcephaly and GBS were recognised as a causal complications of Zika virus infection by 99% and 50% of respondents respectively.The majority of respondents recognised blood (83%) and urine (48%) as primary and secondary samples for Zika virus testing. However, while 67% were aware that polymerase chain reaction (PCR) should be the first-line test for Zika virus identification within the first week of fever onset, 20% stated that both PCR and serology would be expected to be positive. In addition, 29% believed PCR and 40% believed both PCR and serology would also be expected to be positive beyond one week.Nearly all respondents mentioned fever relief medication and fluid rehydration for management of Zika virus disease, but 14% favoured non-steroidal anti-inflammatory drugs, which are generally not recommended without first excluding dengue as a diagnosis.Clinical Management of Pregnant Mothers Potentially Infected with Zika and Infants with Microcephaly82% correctly identified CDC's recommendation to test infants (or cord serum) for Zika virus, but only 16% recognised the need to additionally test for dengue virus. 42% believed specific testing of infant cerebrospinal fluid (CSF) for Zika virus was warranted; CDC guidelines indicate that CSF should be tested only if collected for other purposes.11In addition, while 94% of respondents knew that cranial ultrasound was indicated for infants with suspected congenital Zika virus infection, only 57% recognised the need to test for other infections such as rubella, syphilis, toxoplasmosis and cytomegalovirus. Fewer than 50% were aware of the recommendation for ophthalmological evaluation within 1 month after birth.Transmission and Prevention Aedes aegypti as a mosquito vector for Zika virus, but only 32% knew that Aedes albopictus can also transmit the virus. Sexual contact and blood transfusion were recognised as alternative routes of transmission by 75% and 48% of respondents respectively, but 19% and 16% incorrectly mentioned droplet spread and saliva as transmission modes, and 34% mentioned breastfeeding.Most respondents (85%) identifiedAedes mosquito bites among pregnant women travellers, such as use of insect repellent and wearing long-sleeved clothing. However, there were misconceptions regarding the location and timing of Aedes biting activity; 58% would incorrectly recommend staying away from beaches or forested areas and 47% would incorrectly recommend travellers to avoid going outdoors at night.Nearly all respondents were aware of personal measures to avoid Only 36% of respondents were aware of the current recommendation for pregnant women and their partners returning from Zika-affected areas to abstain from sexual intercourse or use condoms appropriately throughout the duration of the pregnancy.Ministry of Health Requirements for Notification of Suspected Zika Virus Infection Among respondents, 81% were aware of the need to notify suspected cases of Zika virus disease even in the absence of microbiological confirmation, but there was lack of clarity regarding the definition of a suspected case of Zika virus disease; only 22% of respondents were able to correctly identify local MOH's definition of possible exposure to Zika virus.Domain-level KnowledgeOverall, the median respondent score was 9.4, with substantial variation (range: 3.5 - 14.7). Compared with the Clinical Presentation, Diagnosis and Management domain, median normalised scores were lower for domains on Transmission and Prevention (p<0.001) and Clinical Management of Pregnant Mothers Potentially Infected with Zika and Infants with Microcephaly (p<0.001) . There wTo our knowledge, this was the first such rapid assessment of key clinical specialty awareness of Zika virus disease guidelines at a national level globally. Our assessment demonstrated that there was good awareness of Zika virus disease signs and symptoms among doctors, but highlighted gaps in awareness of certain recommendations, particularly ones referring to the prevention of sexually-transmitted Zika virus infection in pregnant women, and the management of infants with possible congenital Zika virus disease. Our study identified opportunities for targeted improvement of communication around Zika virus guidelines among front-line doctors.There was a high level of awareness among our sample of doctors with regards to three of the four common signs and symptoms of Zika virus disease. These three signs and symptoms are similar to those for dengue, which is endemic in Singapore. It was likely that the numerous public health and educational campaigns on dengue may have aided the translation of clinical dengue knowledge to Zika virus disease. This could also explain the lack of awareness that conjunctivitis, which is less common in dengue cases, is an important distinguishing clinical feature of Zika virus disease. Similarly, 35% of respondents incorrectly chose ‘high-grade fever’ as a feature of Zika virus disease. These findings suggest that while knowledge of signs and symptoms of Zika virus disease that are similar to dengue may be good, there remains a gap in understanding of clinical features that could help differentiate Zika virus disease from dengue. This was not surprising since most information outlets have repeatedly reported the similarities between the Zika and dengue viruses, which belong to the same flaviviridae virus family and share common mosquito vectors. Furthermore, messages issued by most public health bodies to clinicians provide standard case definitions that do not highlight the distinguishing features of Zika virus disease. There may be a need to change the way such messages are conveyed, with more emphasis given to differentiation rather than a standard listing of signs and symptoms.We detected similar confusion in the clinical management of Zika virus disease that could be attributed to its perceived similarity to dengue. While >85% of respondents correctly stated fever relief medication and fluid rehydration as the main treatment options, a small minority (7%) wrongly chose platelet transfusion as a possible treatment for Zika virus disease. Worryingly, 13% stated that they would consider NSAIDS for managing Zika virus disease. This is not routinely recommended, as consumption of NSAIDS could have grave consequences if a patient with dengue was misdiagnosed as having Zika virus disease. When stratified by medical specialty, the highest percentage selecting this option worked in Obstetrics and Gynaecology (23%). We were not able to exclude the possibility that the respondents who selected this option were aware of the need to exclude dengue before prescribing NSAIDs.Two-thirds of respondents knew that PCR would likely be positive during the first few days of disease onset, but one-fifth wrongly stated that both PCR and serology testing would be positive. In a follow-up question with a more complicated scenario involving disease onset more than 8 days ago, 40% stated that both PCR and serology would be expected to be positive, while 25% wrongly selected PCR as the test of choice. Taken together, these findings suggest there is a need to improve the level of understanding for Zika virus disease diagnostics. Understanding the utility of PCR and serology in relation to the onset of symptoms is critical to minimise the potential for false positive and false negative diagnoses.,6,14Nearly all respondents correctly identified microcephaly as a condition linked to Zika virus disease. However, only 50% were aware of the confirmed link with GBS.5Aedes vectors, such as preference for night biting or beach/forest settings. This was surprising considering the numerous dengue education campaigns implemented by the authorities in Singapore, although the focus of these campaigns has been primarily on source eradication to prevent mosquito breeding.15Our analysis also points to gaps in knowledge regarding recommendations to prevent Zika virus infection in pregnant women. In particular, only one third of respondents were aware of the current guidelines recommending that pregnant women and their partners returning from Zika-affected areas practise safe sex or abstain from sexual intercourse for the duration of the pregnancy. It was reassuring to find that a high proportion of doctors (>90%) gave correct advice in terms of recommending the use of repellents and long-sleeved clothing to minimise the risk of mosquito bites. However, about half of respondents had misconceptions regarding biting behaviour of LimitationsWe intentionally targeted the survey only at the clinical specialties of obstetrics and gynaecology, neonatology and paediatrics since the nature of the Zika epidemic was most relevant to their patient cohort. Furthermore, in Singapore, pregnant mothers are routinely managed and followed up by obstetricians rather than primary care physicians. Therefore, our results should be considered ‘best case’ scenarios in terms of knowledge levels amongst doctors.We chose to conduct an online survey as this is a rapid and convenient mode of administration. KK Women’s and Children’s Hospital is the only public hospital catering specifically only for neonatal, paediatric, obstetrics and gynaecology care in Singapore. We believe that our email list reached a significant proportion of doctors practising in the relevant specialties by also including the Singapore General Hospital  as well as those in private practice via the medical societies and colleges. However, we cannot be sure that all the relevant doctors in Singapore were aware of the survey. Of note, participation from the private sector was low, with doctors in private practice comprising only 18% of respondents. We did not detect any obvious difference between public and private sector doctors but our study was not powered for such detailed analysis. However, we do not think awareness and knowledge levels would be lower amongst private practitioners as they also receive the same updates via email and SMS from the Ministry of Health.We restricted the time period of the survey to two weeks, to minimise the possibility of official guidelines being altered during the survey period, as public health authorities are continuously reviewing and updating recommendations. During the survey period, the WHO updated its recommendations on sexual transmission of Zika virus, but these did not influence any of the questions in our survey.Our sample size also precluded robust comparison of knowledge between clinical specialties and medical designations, although this was not a primary objective. Despite this, our survey has enabled rapid identification of gaps in knowledge that can be acted upon to improve local as well as global preparedness for management and control of Zika virus disease.RecommendationsOur rapid assessment identified good levels of Zika virus disease knowledge among doctors in key clinical specialties of obstetrics and gynaecology, neonatology and paediatrics in Singapore. The findings provide a reference threshold in terms of knowledge level among doctors in settings with robust infrastructures to access information for a rapidly-evolving epidemic. Knowledge levels in lower-income countries with limited public health systems and access to information via the internet may be much lower. We have identified specific key areas for targeted improvements when communicating Zika virus disease knowledge to frontline doctors. We recommend that future guidelines emphasise clinical signs and symptoms which differentiate Zika virus disease from similar conditions such as dengue, particularly in countries with endemic dengue virus transmission. Similarly, greater emphasis in information sources on not using NSAIDS as a treatment option unless dengue has been excluded is also needed. There is an urgent requirement to improve doctors' knowledge on the need to advice at risk patients to practise safe sex or abstain from sexual intercourse for the duration of the pregnancy. Similarly, it is important to highlight the importance of ophthalmic evaluations in congenital Zika virus infection. In addition, there is still a need to educate doctors in key specialties regarding the appropriate use of Zika virus diagnostic tests and their current limitations, both to minimise the risk of misdiagnosis and to help patients make informed decisions.The authors have declared that no competing interests exist.Clarence Tam: clarence.tam@nus.edu.sgData are available through Figshare at https://figshare.com/s/2ff4859475c8914d8770


Diabrotica virgifera virgifera virus 2 (DvvV2), was identified in the western corn rootworm, Diabrotica virgifera virgifera LeConte, through transcriptome sequencing and confirmed by reverse transcription-PCR. Here, we report the near-complete nucleotide sequence and the genome organization of DvvV2.The genome of a novel small RNA virus, tentatively named  Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a significant pest on maize in the United States, causing severe economic loss, and now threatens Europe (de novo assembled using Trinity (Diabrotica virgifera virgifera virus 2 (DvvV2), the second small RNA virus identified from this analysis (Western corn rootworm (WCR), s Europe . Given ts Europe , alternas Europe . All lib Trinity . Contigsanalysis . The preThe positive-sense, single-stranded RNA genome identified comprises 10,465 nt, excluding the poly(A) tail, and encodes a 3,427-amino acid polyprotein (nt 76 to 10386). The genome is AT-rich (57.7%). A 75-nt 5′ untranslated region (UTR) was confirmed by RT-PCR, while the 3′ UTR comprises 79 nt, followed by the poly(A) tail, as confirmed by 3′ RACE.Solenopsis invicta virus 3  , an unclassified virus, Nylanderia fulva virus 1 , an unclassified ssRNA virus isolated from the tawny crazy ant    with 32%KY070327.The genome sequence was deposited in GenBank under accession number


As the Zika virus epidemic continues to spread internationally, countries such as the United States must determine how much to invest in prevention, control, and response. Fundamental to these decisions is quantifying the potential economic burden of Zika under different scenarios.To inform such decision making, our team developed a computational model to forecast the potential economic burden of Zika across six states in the US  which are at greatest risk of Zika emergence, under a wide range of attack rates, scenarios and circumstances. In order to accommodate a wide range of possibilities, different scenarios explored the effects of varying the attack rate from 0.01% to 10%. Across the six states, an attack rate of 0.01% is estimated to cost $183.4 million to society , 0.025% would result in $198.6 million ($119.4 million and $79.2 million), 0.10% would result in $274.6 million ($130.8 million and $143.8 million) and 1% would result in $1.2 billion ($268.0 million and $919.2 million).Our model and study show how direct medical costs, Medicaid costs, productivity losses, and total costs to society may vary with different attack rates across the six states and the circumstances at which they may exceed certain thresholds . A Zika attack rate of 0.3% across the six states at greatest risk of Zika infection, would result in total costs that exceed $0.5 billion, an attack rate of 1% would exceed $1 billion, and an attack rate of 2% would exceed $2 billion. Our work forecasts the cost and economic burden of the Zika virus across six states in the US which are at the greatest risk of Zika . We evaluated the burden by varying attack rates, scenarios, and circumstances. All costs are reported in 2016 dollars. This is the first study to determine the potential economic impact of the Zika epidemic in the US across a wide range of possible scenarios . There has previously been debate regarding resource allocations to address the Zika epidemic. Our results are important for decision makers because they identify thresholds at which the economic impact of Zika on these six US states would exceed $0.5, $1.0, $1.5, and $2.0 billion. For example, a Zika attack rate of 1% across Alabama, Florida, Georgia, Louisiana, Mississippi, and Texas, the six states at greatest risk of Zika infection, would result in total costs that exceed $1 billion. Our study also reveals the primary determinants of Zika-associated costs, which can help guide future data collection and control efforts. As the Zika virus epidemic continues to spread internationally, countries such as the United States must determine how much to invest in prevention, control, and response. Fundamental to these decisions is an understanding of the potential economic burden of Zika. For example, former US President Barack Obama proposed allocating $1.8 billion towards Zika response programs, which included $828 million distributed towards the Centers for Disease Control and Prevention (CDC) and $210 million towards the US Department of Health and Human Services Response Activities, the majority of which was proposed to be allocated to address Zika on the continental US.[Evaluating the potential impact of the Zika epidemic can be difficult without the aid of computational approaches. Zika infection can lead to a constellation of symptoms that include fever, rash, joint pain, conjunctivitis, muscle pain, and headache that havThrough substantial adaptation and extension of our previously published model, we develPregnant women can be infected or uninfected. Based on CDC guidelines, if uninfWe estimated costs from the third-party payer perspective  and societal perspective . In addition, we calculated the proportion of direct medical costs that would be covered by Medicaid. Direct medical costs include the compliance-adjusted costs for all hospital visits and medical procedures for Zika-infected pregnant and non-pregnant persons, non-infected pregnant women, microcephaly and other CNS disorders in neonates, and GBS cases.Productivity losses resulted from both healthcare visits and time missed due to illness, with median national wage per hour serving as a proxy for the rate of productivity losses. For the Since productivity losses are a substantial piece of the total economic burden associated with a Zika outbreak, sensitivity around these costs are important. As a conservative analysis, we confined productivity losses to those of employable age (16+). We used state-level employment statistics to determine the percentage of the working-age population employed either full-time or part-time in the civilian labor force. We conveAedes aegypti and Aedes albopictus mosquito vectors [This study consisted of running various hypothetical scenarios to explore the impact of varying attack rates (from 0% upwards) in each of the six states . We focused on the Gulf Coast states and Georgia given their suitability of environmental conditions to the  vectors , 29 and  vectors  Addition vectors , 19 OtheThe model covers a Zika epidemic that spans 230 days—a duration determined by fitting the Zika-related microcephaly outbreak in Northeast Brazil using caThe key clinical outcomes vary by attack rate for the six states . For theOf relevance to local, state, and federal health care system planners is the Zika-related increase in medical resource utilization. We estimated that an attack rate of 0.01% would produce 770 outpatient visits for Zika-infected non-pregnant persons, 350,464 ultrasounds for pregnant women, and 1 amniocentesis for pregnant women, whereas for attack rates of 0.025%, 0.5%, 2% and 10% would result in 1925, 350,411, and 3; 38,501, 348,7464, and 60; 154,002, 343,489, and 240; and 770,011, 315,449, and 1202, respectively. Under the base case assumption where 60% of pregnant women seek additional screening for infection (screening compliance), each additional 1% increase in attack rate would translate into 3,505 additional screening visits.Direct medical costs remained relatively insensitive to attack rates for all states,  as the mDirect medical costs vary by the population at risk and the attack rate . SymptomBecause of different population sizes and birth rates, each state would be expected to incur very different total costs as well as costs per case, at the same attack rate. For example, at attack rates of 0.05% and 0.75%, Mississippi would incur direct medical costs of $5.1 million ($1.8-$10.6 million) and $9.6 million ($3.0-$31.9 million), respectively, while Texas would incur direct medical costs of $54.0 million ($18.7-$100.2 million) and $99.8 million ($30.9-$333.1 million), respectively .Because Medicaid costs are a subset of direct medical costs, they are also relatively insensitive to variations in the attack rate . At low As can be seen in Total costs are comprised of direct medical costs plus productivity losses Figs  and 6. WGiven the tremendous uncertainty surrounding potential attack rates for Zika in the US, our model and study demonstrated how various outcomes and costs would vary based on attack rate and other circumstances. Our study identified the thresholds at which the economic impact of Zika on the US Gulf Coast would exceed $0.5, $1, $1.5, and $2 billion. For example, in the base case scenario, an attack rate of approximately 0.3% would exceed $0.5 billion, an attack rate of 1% would exceed $1 billion, an attack rate of approximately 1.3% would exceed $1.5 billion, and an attack rate of 2% would exceed $2 billion in total costs.Without details regarding the Zika-prevention measures that would be implemented and how effective these may be, it is unclear what percentage of these costs may be averted. Possible targets of Zika control investment could be improved vector control, more extensive Zika screening, and accelerated vaccine development. Assuming a third of the costs could be averted, an attack rate of 1% would justify investments in the $1 billion range. Such an attack rate would be substantially lower than those observed in French Polynesia  62–70%), Yap IslaSince the US lacks a comprehensive surveillance system for Zika, no one knows the exact geographic spread of this novel endemic to date, requiring us to rely on assumptions and hypothetical scenarios. As both Zika and dengue are transmitted by the same mosquito, one possThe economic impact of infectious diseases is not always readily apparent and may be significantly underestimated, especially when manifested in life-long clinical outcomes. Since Zika may not pose an appreciable risk of mortality to infected adults, it is possible to overlook for the full extent of its impact. The most publicized effect of Zika has been the risk of microcephaly to children born to Zika-infected pregnant women. The costs of microcephaly are borne out over decades and thus may be difficult to evaluate without quantitative analyses. Our understanding of Zika is rapidly evolving as it becomes apparent that its clinical outcomes include not only microcephaly, but an array of other congenital impairments, such as vision-threatening ocular lesions. In additAs we aimed to be conservative in our estimations, our model in many ways may underestimate the economic burden of Zika. Since there is a dearth of previous estimates of the economic impact of microcephaly, we used the costs associated with autism as a proxy. However, the costs of autism are likely to be lower than those of microcephaly. Microcephaly can result in a number of clinical sequelae such as significant sensory and motor impairment that would require greater medical and personal care than most cases of autism. Furthermore, many cases of microcephaly and other CNS disorders may be difficult to diagnose until infants fail to reach milestones; weeks or months after birth. These initially undiagnosed cases will have substantial health burdens, for which the true cost is not yet incorporated into our calculations. In addition, we did not account for the potential lost productivity and medical costs associated with stress due to fear of Zika and concern over family and friends suffering sequelae of Zika infection, as demonstrated by other studies on previous epidemics such as H1N1 and EbolThe goal of this study was not to predict the course of the Zika epidemic, but to delineate how the economic burden may vary under different attack rates and circumstances. By definition models are simplifications that aim to distil systems down to the most pertinent relationships and key factors without including extraneous detail. While our data-driven analyses drew from a variety of sources and locations, the current literature on Zika is limited and new data continues to emerge. For instance, there are wide discrepancies on the reported risk of microcephaly in infants born to Zika-infected mothers. Therefore, we performed sensitivity analyses to determine how such uncertainty may affect results and to identify needs for future data collection. Additionally, we used laboratory testing costs from CMS reimbursements, which may differ state to state. Third-party payers typically have financial agreements with medical providers for a different fee for each medical charge and can be different from Medicaid reimbursement. Therefore, our Medicaid costs are an approximation due to data restrictions. Awareness of the risk for Zika virus may affect the behavior of pregnant women and couples trying to conceive. However, we did not include any costs associated with the termination of pregnancy (either spontaneous or planned) as the likelihood of this due to Zika is unknown or not well established. This analysis focused on six states in the continental United States at high-risk of Zika infection. While Zika infections may be expected outside of this area, our model chose to conservatively focus on these regions. In addition, our model focuses on attack rates that are distributed across the state and are varied across a wide range to account for the potential impact of vector control activities. With this in mind, the entire population of a state, including pregnant women, falls under the at risk definition provided by the CDC. However,To inform Zika funding options currently under review, our team developed a computational model to forecast the potential economic burden of Zika under different scenarios and circumstances and the circumstances under which these costs would exceed different thresholds. Our analyses indicate that the health and economic burden of even low attack rates of Zika in the Continental US would be both substantial and enduring.


Since early reports of the recent Zika virus outbreak in May 2015, much has been learned and discussed regarding Zika virus infection and transmission. However, many opportunities still remain for translating these findings into field-ready sensors and diagnostics. In this brief review, we discuss current diagnostic methods, consider the prospects of translating other flavivirus biosensors directly to Zika virus sensing, and look toward the future developments needed for high-sensitivity and high-specificity biosensors to come. Amidst the recent Zika epidemic, rising public health concerns have led to extensive research aimed at uncovering the underlying mechanisms of Zika virus (ZIKV) infection and transmission pathways –3. AccorBetween its first isolation as a zoonotic pathogen in Uganda 1947) and the first major human outbreak of Zika on Yap Island in Micronesia (2007), ZIKV has been primarily observed in Africa and the Pacific –7. Gener and the Flavivirus genus, ZIKV shares many common genetic sequences and protein structures with other high-interest flaviviruses, including Dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and Spondweni virus, its most similar relative .Fig.Fig19].FFor asymptomatic pregnant women who have traveled to high-risk areas for ZIKV and for symptomatic individuals within the first 2 weeks of symptom onset, the preferred detection method authorized by the FDA EUA is the Trioplex RT-qPCR assay, which is specific to DENV, Chikungunya virus (CHIKV), and ZIKV. In RT-qPCR, a patient sample is added to a buffered reagent solution containing target primers, reverse transcriptase , DNA polymerase (to amplify this cDNA), deoxynucleotides (dNTPs), and an intercalating fluorescent dye or fluorescent reporter Fig. . The ampFor later stages of ZIKV infection, antibody-based methods can be used. Typically, neutralizing antibodies to ZIKV develop in the human body within the first week of symptoms and continue to remain at detectable levels for up to 12 weeks. During this timeframe, serological assays can be performed to detect the patient’s anti-ZIKV IgM antibodies. However, due to the epidemiological and molecular similarities of ZIKV to other flaviviruses, IgM ELISA assays should be conducted for the antibodies formed against ZIKV, DENV, and CHIKV. In MAC-ELISA, a patient’s sample is added to a well plate pre-coated with antibodies to capture human IgM Fig. . A virusPRNT typically serves as a secondary test to IgM ELISA and measures the ability of a patient’s antibodies to neutralize a specific virus. In PRNT, serial dilutions of a patient’s serum are added to samples of a viral suspension, and each mixture is applied to a confluent host cell culture (e.g. Vero cells) Fig. . FollowiOutside of tests that the CDC offers, there are several private companies selling RT-PCR, ELISA, and lateral flow assay kits Table . Many reReverse transcription PCR (RT-PCR) has become the gold standard for molecular amplification and detection of viruses because of its high selectivity and relatively high sensitivity. Prompted by the 2007 ZIKV outbreak in Yap State, Micronesia, several RT-PCR methods have been developed to specifically identify a multitude of ZIKV strains independent of other flaviviruses. Published ZIKV-specific primer sets target highly conserved regions of the structural membrane (M) and/or envelope (E)  proteinsMany commercial nucleic acid amplification tests (NAATs) have been developed for ZIKV detection within the past year Table . BetweenTM assay (Luminex Corp.), uses a proprietary device to complete a series of steps, which include RT-PCR, followed by amplicon-particle hybridization, and final detection via an indicator molecule . Ex. Ex68]. Considerable research is still required to reach our goals of ZIKV sensing across an array of sample matrices with field-ready assay platforms. Fortunately, much has been learned over the past year about ZIKV on a molecular level, and thus many new opportunities have emerged for applying this knowledge toward treatment and diagnostics. Molecular identification of not only ZIKV, but also of other flaviviruses, hinges on the implementation of alternative techniques for amplicon production and detection. Strides have been made in the design of suitable primer sets specific to flaviviruses and ZIKV in particular; however, more development is needed for rapid, in-field detection. For antibody-based assay development, researchers may build their methods from existing cross-reactive assays, but adoption of forthcoming ZIKV-specific antibodies will be necessary for improved specificity. Above all, sensors that can be quickly and inexpensively assembled, screened for quality, and deployed will make the greatest impact in helping to understand and prevent the spread of ZIKV.


Zika virus (ZIKV) is an emerging pathogenic flavivirus currently circulating in numerous countries in South America, the Caribbean, and the Western Pacific Region. Using an unbiased metagenomic sequencing approach, we report here the first complete genome sequence of ZIKV isolated from a clinical semen sample. Since October 2013, an outbreak of Zika virus (ZIKV) has affected 68 countries across South America, the Caribbean, and the Western Pacific Region 35. HoweveA diagnostic semen sample provided 13 days post onset of symptoms from a patient returning from Guadeloupe was identified as positive for ZIKV RNA with a high copy number. ZIKV was isolated from the clinical semen sample in the C6/36 cell line. Isolation was not successful directly in the Vero cell line; however, infected C6/36 supernatant did propagate in Vero cells and produced cytopathic effect. Supernatant was removed for metagenomic analysis on days 5 and 7 in C6/36 cells and on day 3 in Vero cells. Eighty microliters of supernatant was inactivated in 320 µl of AVL buffer (Qiagen) and purified using the EZ1 virus minikit version 2.0 (Qiagen).RNA extracts were DNase digested and purified as described previously . cDNA waReads were quality trimmed to a minimum score of Q30 across the read. BWA version 0.7.5  was used9–Sequence data for the virus isolated from a clinical semen sample align well with other sequences of ZIKV from the outbreak, including those from the Caribbean region, where the patient acquired the infection. The characterization of ZIKV isolated from semen samples will help improve our understanding of possible viral polymorphisms resulting from infection in different cellular environments. The isolated virus will be available imminently from the National Collection of Pathogenic Viruses  and the KX673530.The complete genomic sequence has been deposited in GenBank under accession no.


Caliciviridae, a family of small icosahedral viruses encoding a positive-sense RNA genome. We have used cryo-electron microscopy and three-dimensional image reconstruction to calculate a structure of vesivirus 2117 virus-like particles as well as feline calicivirus and a chimeric sapovirus. We present a structural comparison of several members of the Caliciviridae, showing that the distal P domain of vesivirus 2117 is morphologically distinct from that seen in other known vesivirus structures. Furthermore, at intermediate resolutions, we found a high level of structural similarity between vesivirus 2117 and Caliciviridae from other genera: sapovirus and rabbit hemorrhagic disease virus. Phylogenetic analysis confirms vesivirus 2117 as a vesivirus closely related to canine vesiviruses. We postulate that morphological differences in virion structure seen between vesivirus clades may reflect differences in receptor usage.Vesivirus 2117 is an adventitious agent that, in 2009, was identified as a contaminant of Chinese hamster ovary cells propagated in bioreactors at a pharmaceutical manufacturing plant belonging to Genzyme. The consequent interruption in supply of Fabrazyme and Cerezyme  caused significant economic losses. Vesivirus 2117 is a member of the  The representative viruses of these genera are Norwalk virus (NoV), sapporovirus (SV), feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV) and Newbury-1 virus, respectively. The noroviruses and sapoviruses cause gastroenteritis in humans, while neboviruses have been shown to cause gastroenteritis in cattle. Vesiviruses and lagoviruses cause a range of symptoms in different animal species including stomatitis, conjunctivitis, respiratory illness and hemorrhagic disease [Caliciviruses are non-enveloped icosahedral viruses that have single-stranded, positive-sense RNA genomes. The  disease .Norovirus and Vesivirus, the VP1 protein is encoded by a second ORF and the minor structural protein, VP2, is encoded by ORF3, which is translated following ribosomal termination–reinitiation [Caliciviridae that encodes a fourth ORF, the product of which, referred to as VF1, is involved in the regulation of the innate response to infection [Calicivirus genomes are around 7.5 kb in length, encode up to four ORFs and are polyadenylated. The first ORF encodes the non-structural proteins and in the lagoviruses and sapoviruses the major capsid protein, VP1. In the genera itiation . The ORFitiation . Murine Caliciviridae exhibit a characteristic virion morphology of 32 cup-shaped depressions on their outer surface. The T=3 capsid is composed of three quasi-equivalent forms of the VP1 protein, termed A, B and C. These give rise to two classes of dimeric capsomere: A/B and C/C. Ninety dimers/capsomeres assemble to form a 35–40 nm spherical capsid that encloses the viral genome. A/B and C/C capsomeres differ only slightly in their conformations. An atomic model of RHDV shows the A/B capsomeres (described as bent) arranged around the fivefold symmetry axis, while the C/C capsomere, which adopts a flatter conformation, is located at the twofold symmetry axis [de novo RNA synthesis [Members of the try axis . The VP1ynthesis . The P dynthesis .9 virus particles were present in each millilitre of bioreactor fluid. Subsequently, the bioreactors were closed for decontamination, causing a significant delay in the production of Cerezyme and Fabrazyme, which affected around 8000 patients. Stock analysts estimate that the contamination and interruption in manufacturing may have cost Genzyme in the region of $200–300 million [Recently, vesivirus 2117 was identified as a contaminant of bioreactors containing Chinese hamster ovary (CHO) cells at Genzyme in both their Allston Landing and Geel sites. This led to an interruption in production of Cerezyme and Fabrazyme biopharmaceuticals for the treatment of patients suffering from Gaucher disease and Fabry disease, respectively. Vesivirus 2117 was first described as an adventitious agent from an unknown source following observations of cytopathic changes in CHO cells grown in culture . ElectroAdventitious agent testing is mandated for the prevention of contamination by viruses able to replicate in CHO cells and has led to the detection of five incidents in the past 20 years caused by murine minute virus, reovirus, Cache Valley virus, epizootic hemorrhagic disease virus and vesivirus 2117 . CurrentSapovirus), was first identified by EM in 1976 from stool samples of infants presenting with gastroenteritis. Sapovirus strains are classified into five genogroups (GI–GV) based on their capsid protein sequences. Currently, only GIII viruses can be propagated in cell culture; however, members of this genogroup cause porcine infections, while the other genogroups cause gastroenteritis in humans. As is the case for many members of the Caliciviridae, expression of a recombinant form of the major capsid protein is sufficient for the assembly of virus-like particles (VLPs), which exhibit the same morphology as virions [Another calicivirus, SV  and San Miguel sea lion virus (SMSV) groups. It has recently been proposed that structural similarities may be used to infer common heritage of diverse virus groups in the absence of genetic similarity [Vesivirus and, albeit at comparatively low resolution, similarities between vesivirus 2117 and structures for both sapoviruses and lagoviruses. The striking differences in P domain morphology within vesiviruses may indicate functional differences, perhaps related to receptor usage and entry mechanisms.Here we describe the structures of VLPs formed by recombinant expression of vesivirus 2117 VP1 and a chimeric sapovirus VP1, alongside an improved structure for FCV strain F9, all solved by cryogenic EM (cryo-EM) and three-dimensional (3D) image reconstruction. These data reveal that the vesivirus 2117 capsid structure more closely resembles that of sapoviruses, and surprisingly a lagovirus, than it does other known vesivirus structures. Phylogenetic analysis of capsid protein sequences supports the classification of vesivirus 2117 as a member of the genus milarity . Here weT=3 icosahedral capsid composed of VP1 dimers, giving rise to arch-like capsomeres. The VP1 dimers also appear to form intradimeric interactions between P2 domains and interdimeric interactions between P1 domains, similar to those seen in FCV and as previously described for SMSV [Vesivirus 2117 VLPs were prepared by baculovirus expression of VP1 in Hi5 insect cells and purified by differential centrifugation. VLP preparations were vitrified by plunge freezing in liquid ethane and imaged in a frozen-hydrated state by cryogenic transmission electron microscopy . Nine hufor SMSV . Viewed for SMSV . This moSapovirus at higher resolution, we calculated a 3D structure from images of VLPs produced by a chimeric VP1 construct that has been shown to yield high levels of expression [We observed that our 2117 structure closely resembled a published low-resolution structure of Parkville virus (a sapovirus) . To drawpression . Brieflypression . These pWe have extended the resolution of our structure for the vesivirus FCV strain F9 to 7 Å resolution using a new dataset. A total of 6965 particles were picked from 241 micrographs  and usedCaliciviridae. The structures of a lagovirus  were aligned and analysed to compute a phylogenetic tree  where CHO cells were used for the production of biopharmaceuticals. The contamination necessitated the shutdown and decontamination of bioreactors, causing a significant delay in the delivery of drugs to patients with Gaucher or Fabry disease  than other vesiviruses, including FCV. However, phylogenetic analysis based on VP1 sequences confirmed that vesivirus 2117 is correctly classified as a vesivirus, also showing that it resides in a clade distinct from FCV and VESV.The dimeric capsomeres formed of vesivirus 2117 capsid protein present two horn-like structures on the outer surface of the P2 domains. Similar structures are present on the outer faces of P domains of sapovirus VP1, although they are less pronounced. MuNV also exhibits prominent horn-like protrusions on the outer surface of the P2 domain of VP1 . ConversVesivirus.The outer face of the FCV capsomere incorporates the receptor-binding site. Two molecules of feline junctional adhesion molecule-A bind to the flat surface in a head-to-tail arrangement . ReceptoThere is a paucity of data concerning pathology caused by vesiviruses in the CCV and 2117 clades. Reports of enteritis associated with CCV suggest the possibility of sapovirus-like disease , whereasInterestingly, the first and second ORFs of vesivirus 2117 are separated by a stop codon, which is typical of the noroviruses and vesiviruses. However, the capsid protein is encoded within the first ORF along with the viral non-structural proteins, typical of the sapoviruses and lagoviruses . This haSapovirus. Structural analysis has been proposed to provide insights into the common ancestry of distantly related viruses following the discovery of fold conservation between viruses that infect highly divergent branches on the tree of life [Caliciviridae, VP1 proteins share a common topology. The fact that the outer face of the P domain presents the receptor-binding site and major immunodominant epitopes means that it is subject to significant evolutionary pressure from immune surveillance. Typically, receptor-binding sites of viruses are embedded in hyper-variable regions of capsid proteins. Thus, morphological differences/similarities in the P domain may be uncoupled from genetic relatedness but may provide important clues concerning critical aspects of virus biology, such as entry pathway.While phylogenetic analysis indicated a close genetic relatedness between vesivirus 2117 and canine vesivirus, the striking morphological similarities with sapoviruses led us to question the taxonomic classification of vesivirus 2117 as a vesivirus. However, based on sequence data alone, vesivirus 2117 is undoubtedly more closely related to FCV than it is to the genus  of life . Within g for 30 min. The supernatant was filtered (0.45 µm vacuum filter) and protein was precipitated by addition of polyethylene glycol to a final concentration of 10 % followed by incubation at 4 °C. The precipitate was centrifuged at 8000 g and 4 °C for 30 min. The VLP-containing pellet was then resuspended in boric acid buffer . VLPs were then centrifuged through a sucrose cushion . The VLP pellets were subsequently resuspended in boric acid buffer, centrifuged  and mixed with an equal volume of PBS containing 0.5 M NaCl and 4.51 M CsCl for ultracentrifugation using a Sw55 Ti rotor at 40 000 r.p.m. for 20 h at 4 °C. Purified VLPs were then collected and dialysed against PBS.Vesivirus 2117 VLPs were produced by baculovirus expression of the VP1 gene (lacking the predicted leader sequence) in Hi5 cells. Six days post-infection, cells were freeze–thawed and the lysate was clarified by centrifugation at 14 000 g, 10 min at 4 °C). The pellet was resuspended in TBS  and freeze–thawed at −80 °C prior to sonication with an equal volume of Vertrel XF (Sigma-Aldrich). The cell suspension was then clarified by low-speed centrifugation , and the aqueous phase was subjected to repeated sonication. Virus was then purified from the aqueous phase by centrifugation through a caesium chloride gradient (1.31–1.45 g ml−1) using a SW-41 Ti rotor at 28 000 r.p.m. for 8 h at 12 °C. The purified virus was collected from the gradient and dialysed into virion buffer .FCV strain F9 was propagated in Crandell Rees feline kidney cells for 8 h. The virus-infected cells were pelleted from the culture medium by centrifugation (1500 Sapovirus particles were made using a baculovirus expression system in Tn5 cells as previously described .Vesivirus 2117 VLPs and FCV virions (4 µl) were loaded on-to glow discharged C-flat holey carbon support films (R2/2 ProtoChips), blotted at 4 °C for 4 s at 100 % humidity and plunged into liquid-nitrogen-cooled liquid ethane using an FEI Mark IV Vitrobot. Vitrified samples were imaged in a JEOL 2200 FS cryo-microscope equipped with a Gatan 626 cryo-stage. Energy-filtered images were recorded (with a 20 eV slit width) on a Gatan Ultrascan US4000 charge-coupled device camera at a magnification of ×100 000, corresponding to a pixel size of 1.05 Å per pixel (vesivirus 2117) or a Direct Electron DE20 direct detection device at a magnification of ×40 000 with a pixel size of 1.39 Å per pixel (FCV). Sapovirus VLPs were plunged using the same method but using R1.2/1.3 Mo 200 mesh holey carbon grids (Quantifoil), imaging was performed in a JEOL 2200FS at a magnification of ×80 000 and micrographs were recorded on a TVIPS 4k×4k CCD camera, giving a pixel size of 1.6 Å per pixel.spider [eman program pdb2mrc. All density maps were then low pass filtered to a common resolution of 10 Å using eman [sd) and for calculation of correlation values between pairs of maps. Correlation values were calculated in UCSF Chimera using the ‘Fit in Map’ function [Two hundred and forty-two micrographs of vesivirus 2117 VLPs, 241 micrographs of FCV and 222 micrographs of SV VLPs were processed to calculate 3D reconstructions. Images of particles were contrast transfer function corrected and extracted from micrographs using the BSoft program Bshow . The vesspider . The mosspider . Resoluting eman . UCSF Chfunction .blastp search against the GenBank non-redundant database. The top hit was to an existing 2117 sequence , followed by three hits to other suspected 2117 sequences sampled in Geel, Belgium  and Allston, USA . The next most similar hits were canine vesivirus  and canine calicivirus  sequences. All of the above sequences had an E value of 0 and were selected for inclusion in the phylogenetic analysis. Representative sequences from other species within the genera Vesivirus, Sapovirus, Lagovirus and Norovirus of the family Caliciviridae were also selected from the blast hits for inclusion in the phylogenetic analysis. In total, 57 protein sequences were selected (including our 2117 capsid sequence) and aligned using clustalw within the mega software [Our 2117 capsid amino acid sequence was used as a query sequence in a software . The evo


West Nile virus infection in humans is mostly asymptomatic. Less than 1% of neuro-invasive cases show a fatality rate of around 10%. Acute flaccid paralysis of respiratory muscles leading to respiratory failure is the most common cause of death. Although the peripheral nervous system can be involved, isolated phrenic nerve palsy leading to respiratory failure is rare and described in only two cases in the English literature. We present another case of neurological respiratory failure due to West Nile virus-induced phrenic nerve palsy. Our case reiterates the rare, but lethal, consequences of West Nile virus infection, and the increase of its awareness among physicians. The incidence of West Nile virus (WNV) infection is increasing in the United States from its first description three decades ago . AlthougAn 80-year-old man complaining of drowsiness was brought to the emergency room by his family. He was in his usual state of health in the previous week and started having influenza-like (flu) symptoms with on-and-off fever, body ache, lethargy, and generalized weakness. His family noticed that, for the past two days, he was sleepier than usual and remained in bed. This was mentioned as very unusual because he maintains an active lifestyle for his age. His family continued that he had recently completed a successful hunting season. He was drowsy on presentation, but oriented to all the spheres without any focal neurological deficits. A chest X-ray showed possible right lower lobe infiltrates, and he was admitted with the initial diagnosis of aspiration pneumonia. Despite appropriate antibiotics, his mental status continued to decline and developed severe encephalopathy responding only to painful stimuli. A computed tomography (CT) scan of the head did not show any acute changes. Arterial blood gas showed evidence of hypercapnic respiratory failure .The patient was started on bilevel positive pressure ventilation (BIPAP) for carbon dioxide narcosis. A CT chest angiogram did not show any pulmonary embolism, nor any evidence of pneumonia. Despite meticulous titration of BIPAP settings, the patient continued to have persistent hypercapnia with compensated respiratory acidosis (Table1). Based on previous serum bicarbonate levels there was no evidence of chronic respiratory failure. The CT of the chest did not show any structural defect of the pulmonary system, and the patient’s wife denied any symptoms of sleep apnea. In view of persistent encephalopathy despite compensated acidosis, further evaluation was performed. An electroencephalogram did not show any seizure activity. A lumbar puncture showed elevated protein levels (367 G/L) in the cerebrospinal fluid (CSF) and a total cell count of 13/cc with lymphocyte predominance (92%) suggesting meningoencephalitis. With supportive care, his mental condition improved, but the patient could not be weaned from BIPAP. Any interruption of BIPAP would lead to hypoxic hypercapnia respiratory failure. Meanwhile, the CSF viral panel came back as positive for West Nile virus (WNV) antibodies, both IgM and IgG. Upon further questioning, the patient’s wife revealed their recent trip to El Salvador one month ago.As the patient was dependent of BIPAP, neurological respiratory failure due to WNV was considered. The patient did not show any evidence of acute flaccid palsy. In view of his overall condition, fluoroscopic examination for diaphragmatic movements, and detailed pulmonary functions tests to assess respiratory muscle strength were not performed. Manometry showed reduced maximum inspiratory pressure to 25 cm of water , and an ultrasound of the thorax showed absent movements of both diaphragms with deep inspiration (Video 1). Due to a lack of resources, electrophysiological studies of the phrenic nerve and diaphragm were not conducted. Based on these corroborative findings, neurological respiratory failure due to WNV encephalitis with isolated phrenic nerve involvement was diagnosed. The patient was continued on BIPAP therapy with improvement in their mental and respiratory condition, however, he succumbed to the terminal illness within two months of disease onset. Isolated phrenic nerve palsy due to WNV leading to respiratory failure is rare, and has only been reported in two previous cases in the literature ,2.Flaviviradae, which includes Japanese encephalitis virus, St. Louis encephalitis virus, Rocio virus, along with Murray valley encephalitis virus [West Nile virus (WNV) is a single-stranded RNA virus. It belongs to the family is virus . WNV is is virus .Most patients with WNV infections are asymptomatic ,5. ArounWNV meningitis presents with fever, headache, and classic meningeal signs . This foWest Nile Virus can also involve the peripheral nervous system leading to demyelinating neuropathies, motor axonopathy, axonal polyneuropathy, and brachial plexopathies. Involvement of the phrenic nerve leading to diaphragmatic palsy is rare . Our patWest Nile virus infection can cause respiratory failure due to acute flaccid paralysis of respiratory muscles. Physicians should be wary of the rare possibility of isolated phrenic nerve involvement leading to respiratory failure, as indicated in our patient.


African cassava mosaic virus Cameroon strain (ACMV-CM) and the Kenyan strain K201 of East African cassava mosaic virus (EACMV KE2 [K201]). Genotypes carrying the CMD1 (TMS 30572), CMD2  and CMD3 (TMS 97/0505) resistance mechanisms showed high levels of resistance to ACMV-CM, with viral DNA undetectable by PCR beyond 7 days post inoculation (dpi). In contrast, all genotypes initially developed severe CMD symptoms and accumulated high virus titers after inoculation with EACMV KE2 (K201). Resistant genotypes recovered to become asymptomatic by 65 dpi with no detectable virus in newly formed leaves. Genotype TMS 97/2205 showed highest resistance to EACMV KE2 (K201) with <30% of inoculated plants developing symptoms followed by complete recovery by 35 dpi. Deep sequencing of small RNAs confirmed production of 21–24 nt virus derived small RNAs (vsRNA) that mapped to cover the entire ACMV-CM and EACMV KE2 (K201) viral genomes in both polarities, with hotspots seen within gene coding regions. In resistant genotypes, total vsRNAs were most abundant at 20 and 35 dpi but reduced significantly upon recovery from CMD. In contrast, CMD susceptible genotypes displayed abundant vsRNAs throughout the experimental period. The percentage of vsRNAs reads ranked by class size were 21nt (45%), 22 nt (28%) and 24 nt (18%) in all genotypes studied. The number of vsRNA reads directly correlated with virus titer and CMD symptoms.Mitigation of cassava mosaic disease (CMD) focuses on the introgression of resistance imparted by the polygenic recessive (CMD1), dominant monogenic (CMD2) and CMD3 loci. The mechanism(s) of resistance they impart, however, remain unknown. Two CMD susceptible and nine CMD resistant cassava genotypes were inoculated by microparticle bombardment with infectious clones of  Under optimum conditions, maximum yield potential is 50 Mt (MT) of fresh storage roots per hectare  genomic components, namely DNA A and DNA B, ranging in size from 2.7–3.0 kb . DNA A eManihot glaziovii Muell. Arg. (ceara rubber) and has been reported to be polygenic and recessive in nature (Manihot esculenta (TME) series (Breeding has delivered high yielding cassava varieties that are resistant or tolerant to CMGs . Presentn nature . In cont) series . Breedin) series . Recentl) series . TMS 97/) series . Field r) series . Genetic) series .Under field conditions, genotypes containing CMD1, CMD2 and CMD3 loci develop moderate to severe CMD symptoms followed by complete recovery from disease . ImportaArabidopsis thaliana genome encodes four DCL and ten AGO families, many of which participate in antiviral defense , CMD2  and CMD3 (TMS 97/2205) type resistance. Analysis of viral nucleic acids and deep sequencing of siRNAs involved in symptomatic and recovered leaves is described along with their relationships with CMD resistance.22.1Cassava genotypes TME 3, Oko-iyawo, TME 7, TME 14 and TME 204 (CMD2 types), TMS 30572 and TMS 30001 (CMD1 types), TMS 97/2205 and TMS 98/0505 (CMD3 types), 60444 and Ebwanatereka (CMD susceptible) were selected for this study. Experimental plants were established in tissue culture and multiplied as described by 2.2African cassava mosaic virus Cameroon strain (ACMV-CM), NCBI accession numbers AF112352 and AF112353; and a Kenyan strain of East African cassava mosaic virus (EACMV-KE2) isolate K201, NCBI accession numbers AJ717541 and AJ704953, were used in the study. DNA A and DNA B genomic components were cloned from head to tail as partial tandem dimers, previously described by Escherichia coli (DH5α). Virus inoculation was performed according to the method described by Infectious clones of two cassava mosaic virus species: 2.3Inoculated plants were monitored for development of disease symptoms starting five days post inoculation (dpi) and thereafter once per week for nine weeks. The proportion of plants showing CMD symptoms on the three uppermost newly opened leaves was expressed as the percentage of total number of plants inoculated. Disease severity was scored visually using a scale of 0–5 whereby 0 represents no disease symptoms and 5 represents severe mosaic with complete leaf curling as described by 2.4Total nucleic acid was extracted from newly opened leaves using cetyltrimethylammonium bromide (CTAB) method . Samplin2.5Five micrograms of total genomic DNA were fractionated on a 1% (w/v) agarose gel at 34 V overnight (ca. 12 hrs). The DNA was transferred onto a positively charged nylon membrane  using 20× SSC for at least 8 hrs and UV crosslinked . Probes 2.6AC1) and movement protein (BC1) genes specific to each virus species. The PCR was performed as follows: 95 °C for 3 min, followed by 35 cycles at 95 °C for 40 s, 58 °C for 30 s, 68 °C for 1 min and final extension at 68 °C for 10 min. The fragment sizes were confirmed on a 1% (w/v) agarose gel. The gel image was acquired and analyzed using Molecular Imager® Gel Doc™ XR documentation system with Image Lab software .Detection of EACMV KE2 (K201) and ACMV-CM was performed in systemically infected leaves by PCR. Primers (Supplementary Table 1) were designed to target the virus replicase (2.7The virus titre of EACMV KE2 (K201) was assessed by real-time quantitative PCR (qRT-PCR) at full disease establishment (20 dpi) and the recovery phase (65 dpi). Sample DNA was standardized to a concentration of 4.5 ng/μl and 10 μl subjected to SYBR Green I chemistry as described by 2.8in vitro transcribed with DIG RNA labelling kit SP6/T7 .Low molecular weight RNA molecules were separated by Northern blotting of 30 μg total RNA on 15% Criterion TBE-Urea polyacrylamide gel  at 100 V for 2 h and vsRNAs were detected . Probes 2.9Double tagged cDNA libraries were prepared from 5 μg of total RNA obtained from EACMV KE2 (K201) infected and mock inoculated cassava plants at 20, 35 and 45 dpi. Small RNAs were sequentially ligated to adenylated NEBNext 3′ and 5′ SR adaptors for Illumina 5′-rAppAGATCGGAAGAGCACACGTCT-NH2-3′ and 5′- rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC-3′. Adaptor ligated small RNA libraries were reverse transcribed into cDNA with ProtoScript II reverse transcriptase followed by PCR amplification with Illumina index primers. Size selection was performed on 6% polyacrylamide gel , whereby 140–150 nucleotide bands corresponding to adapter-ligated small RNAs of sizes 21–30 nucleotides were isolated to generate fragments for Hiseq Illumina sequencing.2.10Samples were sent to Genome Technology Access Center (GTAC) at Washington University in St. Louis for quality check on Agilent Bioanalyzer and sequencing on Illumina HiSeq 2500 platform using 1 × 50 single-end read protocol. Raw sequencing data was demultiplexed by QIIME  and sequ2.11A. thaliana DCLs was retrieved from the cassava reference genome version 6.1 database . The nucleotide sequences were imported to MEGA6.0 and multiple alignments performed with respective DCL retrieved from A. thaliana. A phylogenetic tree was constructed using the near-neighbor joining method. Bootstrap analysis was performed with 500 replicates to test the strength of the nodes. Primers for DCL qRT-PCR are described in Supplementary Table 1. The transcript levels of DCLs were quantified using qRT-PCR as described by Cassava orthologous to 33.1Mosaic symptoms were first observed five days post inoculation (dpi) with the highest percentage of symptomatic plants across all genotypes recorded between 20 and 35 dpi , Fig. 2.Plants of all genotypes inoculated with EACMV KE2 K201) developed more severe CMD symptoms (severity score up to 4), compared to those challenged with ACMV-CM (severity score up to 3) at full disease establishment B and D.  developeIn order to verify whether recovery from CMD was a temporary or permanent phenomenon, stems of all inoculated and control plants were cut back to 10 cm above soil level at 75 dpi and all leaves removed. Shoot tissues were allowed to regrow and newly formed leaves assessed for presence and severity of CMD symptoms. A flush of severe symptoms was observed on new growth of plants of susceptible cassava genotypes challenged with ACMV-CM, reaching an average severity score of 4 D. Howeve3.2AC1 and BC1 virus genes  was detectable starting at 12 dpi with susceptible genotypes accumulating the highest viral DNA load . Between was dete3.3Virus infection in plants triggers production of virus derived small RNAs (vsRNAs) that potentially direct antiviral defense. Presence of vsRNAs within young leaves systemically infected with EACMV KE2 (K201) and ACMV-CM was assessed by Northern blot hybridization with virus specific probes derived from AC1/AC2. ACMV-CM derived vsRNA was not detected by Northern blot hybridization within tissues of the CMD resistant cultivars TME 3, TME 204, Oko-iyawo, TMS 30572 and TMS 97/2205 throughout the experimental period . Accumulation of vsRNA was detectable as early as 12 dpi in samples collected from symptomatic tissues, and corresponded with the onset of CMD symptoms for both virus species (CMD2-type and susceptible cultivars accumulated high amounts of EACMV KE2 (K201) derived vsRNAs at 20–35 dpi, which correlated with high disease severity at that time (3.4Profiles of vsRNAs produced in cassava genotypes during infection with EACMV KE2 (K201) and ACMV-CM were obtained by Illumina Solexa sequencing of total small RNAs isolated from systemically infected leaves. Cassava genotypes showed a differential response in the amount of vsRNAs accumulated in response to infection with the two virus species . The resCassava genotypes 60444, TME 204 and TME 3 were challenged with EACMV KE2 (K201) and three individual plants sampled at three time points, depicting disease establishment (20 dpi), maximum disease severity (35 dpi) and the recovery phase (45 dpi). EACMV KE2 (K201) derived siRNA was most abundant in 60444 and TME 3 ranging from 23% to 38% of total siRNA across the three different time points, whereas in TME 204, EACMV KE2 (K201) derived siRNA ranged from 18% to 22% . The proLeaf samples were collected only at 20 dpi from cultivars TMS 97/2205 and TMS 30572 and then challenged with EACMV KE2 (K201) for the study of siRNA. After this time point, these plants had fully recovered from virus infection A and gemEACMV KE2 (K201) vsRNAs exhibited bias in strand polarity, whereby >55% of total mapped reads originated from the sense orientation for both genomic components A. A shifIn all CMD infected cassava genotypes, small RNA size classes of vsRNAs 21, 22, 23 and 24 nt in size spanning the entire virus genome in sense and antisense orientations were identified B. The mo3.5The pattern of vsRNA distribution along the virus genome was assessed according to their dichotomy and genomic position. From the analysis of redundant sRNA reads, several sharp peaks were identified unevenly distributed across the virus genome . HotspotOn the ACMV-CM genome, the major small RNA peaks were observed in sense orientation on DNA A (nt-1361-1701) and DNA B (nt-1361-1701) and on antisense orientation on DNA B (nt-851-1021 and nt-2011) B. AlignmThe density of vsRNAs was evaluated to identify the abundance of small RNAs relative to ORF and/or overlapping regions. High abundance of small RNAs was identified in complementary and virion sense transcriptional units of both DNA A and DNA B, respectively for the two viruses, with positive and negative reads represented equally  contains 46% GC in DNA A and 43% in DNA B (Supplementary Table 2). For both virus species, AC1/AC2, AC4, AV2 and IR right; and, for EACMV KE2 (K201), BC1, BV1 and IR left, are GC rich at greater than 45% (Supplementary Table 2). Genome-wide distribution of vsRNAs along the virus genome identified various hotspots between nucleotide position 900–2000 for DNA A and 900–1801, corresponding to high GC regions A and B. 3.6To elucidate the potential interaction of EACMV KE2 (K201) and ACMV-CM vsRNAs with distinct cassava AGO complexes, the 5′-terminal nucleotide composition of different-size species was analyzed . Overall3.7Each of the four Arabidopsis DCL orthologs were identified from the cassava genome as follows: DCL1 (Manes.05G015200.1); DCL2 (Manes.12G002800.1 and Manes.12G003000.1); DCL3 (Manes.03G056500.1); and DCL4 (Manes.14G140300.1) A. Transc4M. glaziovii × M. esculenta has been reported in TMS 30001 and TMS 30572  whereas EACMV and ICMV AC2 potentially target both PTGS and transcriptional gene silencing (TGS)  induced disease in all cassava genotypes while ACMV-CM failed to induce symptoms in genotypes carrying innate CMD resistance mechanisms . Virus encoded suppressors of gene silencing (VSR) have been implicated in virus pathogenicity . Althougng (TGS) . ICMV isICMV-Dha . TargetiRecovery from CMD infection was associated with reduction in viral DNA titer and vsRNA quantity in CMD1, CMD2 and CMD3 resistant types in a manner that reflected the visually scored disease curve as evidence of activation of potent defense mechanisms that clear systemic infection . Onset aA. thaliana, four DCL proteins act hierarchically and exhibit functional redundancy to actuate sRNA biogenesis  (Cabbage leaf curl virus (CabLCV). Differences in the relative accumulation of vsRNAs among cassava genotypes studied here may indicate varying proportions of infected cells in the tissues analyzed whereby in susceptible plants, cassava-infecting geminiviruses replicate to high copy numbers while innate and/or adaptive antiviral defense mechanisms limit virus replication in resistant genotypes to few cells.In ogenesis . DCL1 progenesis , while Dogenesis . In the ogenesis . Despiteogenesis . Data pr(2.8 kb) , the lar(2.8 kb)  may reflN. benthamiana systemically infected with diverse species of CMGs. However, that study did not resolve vsRNAs based on their size classes. The highest proportion of EACMV KE2 (K201) vsRNAs originated from AV1, AC1, AC3, BC1 and BV1 whereas ACMV CM vsRNAs were mostly derived from AV1, AV2, AC1 and AC2, indicating unique processing of vsRNAs from different CMGs . Different patterns of CMG siRNA distribution along the genome have been reported using reverse Northern blot . Other studies have shown variation in size allocations of geminivirus-derived sRNAs in different host plants  followed by 22 nt (22–30%) and 24 nt (15–25%) , suggestt plants . All theWheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) (EACMV KE2 (K201) and ACMV-CM vsRNAs were mapped and found to cover the entire DNA A and DNA B genomes in both sense and antisense polarities , indicat (TriMV) . Accumul (TriMV) . Therefo (TriMV) . In agreAssociation of sRNAs and AGO proteins is mostly determined by 5′-terminal nucleotides and the structure of sRNA duplex . The majExploiting inherent resistance to CMGs remains the most important strategy to mitigate CMD pandemics. CMD1 and CMD2 types have been employed to mediate resistance to CMD for several decades . HoweverThe authors have no conflict of interest to declare.This research was supported by the Bill and Melinda Gates Foundation; the United States Agency for International Development from the American people; and the Monsanto Fund. The sponsors had no role in study design, in the collection, analysis and interpretation of data; in the writing of the report; nor in the decision to submit the article for publication.


There is limited population-level research on end-of-life care in Australia that considers health care use and costs across hospital and community sectors. The aim of this study was to quantify health care use and costs in the last 6 months of life in a cohort of elderly Australian decedents and to examine the factors associated with end-of-life resource use and costs.A retrospective cohort study using routinely collected health data from Australian Government Department of Veterans’ Affairs clients. The study included two cohorts of elderly Australians who died between 2005 and 2009; one cohort with a recorded cancer diagnosis and a comparison cohort with no evidence of a cancer history. We examined hospitalisations, emergency department (ED) visits, prescription drugs, clinician visits, pathology, and procedures and associated costs in the last 6 months of life. We used negative binominal regression to explore factors associated with health service use and costs.The cancer cohort had significantly higher rates of health service use and 27% higher total health care costs than the comparison cohort; in both cohorts, costs were driven primarily by hospitalisations. Older age was associated with lower costs and those who died in residential aged care incurred half the costs of those who died in hospital.The results suggest differences in end-of-life care pathways dependent on patient factors, with younger, community-dwelling patients and those with a history of cancer incurring significantly greater costs. There is a need to examine whether the investment in end-of-life care meets patient and societal needs.The online version of this article (doi:10.1186/s12904-017-0213-0) contains supplementary material, which is available to authorized users. Elderly populations continue to grow, with estimates that in Organisation for Economic Co-operation and Development (OECD) countries one in five people will be 65 years and older by 2030  and, in There is a growing literature dedicated to understanding patterns of health care use and costs at the end of life, much of which has been conducted using routinely collected health care data –9. The rThe aim of this study is to quantify health care use and costs in the last 6 months of life in the Australian setting and to examine the factors associated with end-of-life care in an elderly decedent population. We compare total health care use and costs in two elderly decedent cohorts; a cohort with a history of cancer and a cohort without a cancer history. We focused on comparing cohorts with and without a previous cancer diagnosis as cancer is the leading cause of death in Australia and other similar countries and cancer patients represent a significant proportion of patients receiving end-of-life care , 20. AddThe Australian Government Department of Veterans’ Affairs (DVA) funds the health care of eligible veterans, war widows, war widowers and their dependents. DVA clients have access to the universal health care arrangements provided to Australian permanent residents and citizens plus additional DVA-approved services and pharmaceutical items not subsidised for the general population; the types of services subsidised depends on the level of entitlement.This study used data from the cancer and comparison cohorts developed for the End of Life in Cancer Care (EOL-CC) study, the details of which have been published elsewhere . BrieflyThe data infrastructure comprises DVA data holdings linked with NSW Ministry of Health data collections. Data were linked by a third party under the custodianship of the DVA, the NSW Ministry of Health and the Cancer Institute NSW, using best practice, probabilistic, privacy preserving protocols . The linThe linked datasets also include information about all subsidised health services including hospital admissions, emergency department (ED) presentations and dispensed prescription medicines. Clinical consultations, pathology services and procedures; procedures include diagnostic services , therapeutic services  and surgery are also captured; unit costs were available for all these services, while costs for the hospital admissions and ED presentations were calculated per admission or presentation using the methodology described in the NSW Costs of Care Standards Report 2009/10. Full details of the costing methods are described elsewhere .Outcomes were health service use and associated costs in the last 6 months of life based on six constructed ‘months’ consisting of 30 days each; all individuals are observed for exactly 180 days to the date of death.We calculated person-level mean ) and median (with inter quartile range (IQR)) service use and costs for the entire six-month period, and by month until death.We allocated unit costs to each item of resource use to calculate mean (95% CI) and median (IQR) costs in 2009/10 Australian dollars. Total costs were calculated as the sum of costs of health services , pharmacy, ED and hospital costs ; more details are reported elsewhere , 24.We used negative binomial regression to determine factors associated with health service use and costs. The cost data can be thought of as counts (of dollars and cents). Cost data are non-negative and skewed so count data models can be applied to these data, and in our case this makes sense because it is consistent with the method used for utilization . Factorsp < 0.05 were used as a criterion for statistical significance.Separate models were estimated for utilisation and costs of each type of health service  and total health care costs. The strength of associations was represented by adjusted incident rate ratios with 95% CIs, and two-tailed We used SAS version 9.3 (SAS Institute) for data manipulation and performed statistical analyses using STATA version 12 (StataCorp).A total of 9862 decedents met the eligibility criteria for the cancer cohort and 15,483 for the comparison cohort. The cohorts were similar in relation to age, socioeconomic status and geographical location of residence. Comorbidity burden  was higher in the cancer cohort; 17% of cancer decedents versus 10% of the comparison cohort had a comorbidity score of three or more. However, comorbidity burden was similar for both cohorts when calculated based on prescription medicines dispensed [Table On average, decedents in the cancer cohort were dispensed 41 medicines (vs. 38 medicines in the comparison cohort), received 90 clinician visits/procedures (vs. 66 in the comparison cohort) and had approximately three hospital admissions (vs. two in the comparison cohort). The mean number of ED visits was similar in both cohorts at about one visit/decedent in the last 6 months of life [Additional file The mean total cost associated with health care in the last 6 months of life was higher in the cancer cohort  than the comparison cohort . Costs were driven primarily by hospitalisations, accounting for about 80% of total costs in both cohorts [Fig. The mean cost of medicines per person in the cancer cohort was $1840 compared with $1234 for the comparison cohort. [Additional file Differences in utilisation can be attributed mainly to higher numbers of medical specialist visits and pathology services in the cancer cohort. The greatest difference in health service costs related to diagnostic procedures at a mean cost of $1041 per person in the cancer cohort compared to $568 for the comparison cohort. The cost of therapeutic procedures also differed substantially between cohorts, with mean costs of $877 and $464 per person in the cancer and comparison cohorts respectively [Additional file The difference in hospitalisation costs was driven by the higher number of admissions in the cancer cohort  than the comparison cohort . [Additional file Rates of health service use and associated costs increased over the last 6 months of life and peaked in the last month of life. Total costs for both cohorts and in each month to death are driven by the cost of hospitalisations Fig. .Fig. 2Hep < 0.001); clinician visits and procedures ; hospitalisations  and ED visits  than the comparison cohort. Decedents aged over 90 at death had lower rates of prescription medicines ; clinician visits and procedures ; hospitalisations  and ED visits  than younger decedents [Fig. Multivariable analyses demonstrate that cancer decedents had significantly higher rates of prescription medicines  [Fig. p < 0.001), 32% higher costs for clinician visits and procedures  and 28% higher costs for hospitalisations  than the comparison cohort. There was no significant difference in costs associated with ED visits  [Fig. Overall, cancer decedents incurred 27% higher health care costs than non-cancer decedents,  than those aged85 years and older. Future research is required to understand which patterns of care are associated with improved patient-reported care and to understand which patterns of care represent the best value in terms of appropriateness, quality and patient preferences.


During 2015–2016, several outbreaks of tilapia lake virus infection occurred among tilapia in Thailand. Phylogenetic analysis showed that the virus from Thailand grouped with a tilapia virus (family Orthomyxoviridae) from Israel. This emerging virus is a threat to tilapia aquaculture in Asia and worldwide. Viral diseases are common causes of illness and death in cultured fish; such viruses include infectious salmon anemia virus, infectious hematopoietic necrosis virus, and viral hemorrhagic septicemia virus (Oreochromis niloticus) and red hybrid tilapia (Oreochromis spp.). The outbreaks occurred at fish farms in central, western, eastern, and northeastern Thailand : 10–30 moribund fish and 5–10 apparently healthy fish from the same culture areas. In total, 325 samples were collected and tested for etiologic agent(s)  Table 1.For our study, we selected a field sample positive for TiLV (designated TiLV/Tilapia/Thai/TV1/2016) and processed it for whole-genome sequencing. Another 6 TiLVs were selected for sequencing of the putative polymerase basic 1 (PB1) gene  Table 2.Orthomyxoviridae showed that TiLVs from Thailand possessed motifs preA, A, B, C, D, and E similar to those of Orthomyxoviridae viruses, including influenza A, B, and C viruses; infectious salmon anemia virus; Dhori virus; and Thogoto virus (Orthomyxoviridae but not Arenaviridae and Bunyaviridae (Comparison of the TiLVs from Thailand with those from Israel showed high nucleotide and amino acid identities (95.18%–99.10%). Among TiLVs from Thailand, nucleotide and amino acid identities for segment 1 or the putative PB1 gene of the virus were high (99.61%–100%)  Table 3.to virus  Table 4 aviridae . This reOur PCR and whole-genome findings demonstrate genetic homology between TiLV from Thailand and the etiologic agent of a novel RNA virus infection of tilapia in Israel and Ecuador  Figure 2Our results emphasize that the virus isolated from Thailand shares high sequence similarity with TiLV from Israel, suggesting that this virus spreads across continents. Given that tilapia are the main aquaculture species, control of TiLV will be improved by further efforts such as strict biosecurity, vaccine development, and selection of resistant tilapia breeds. Additional materials, methods, and results of study of tilapia lake virus outbreaks in Thailand, 2015–2016.