A rapid increase of Medicaid expenditures has been a serious concern, and housing stability has been discussed as a means to reduce Medicaid costs. A program evaluation of a New York City supportive housing program has assessed the association between supportive housing tenancy and Medicaid savings among New York City housing program applicants with serious mental illness and chronic homelessness or dual diagnoses of mental illness and substance use disorder, stratified by distinctive Medicaid expenditure patterns.n = 737) and people eligible but not placed (n = 2090), including very low Medicaid coverage, increasing Medicaid expenditure, low, middle, high, and very high Medicaid expenditure patterns. We assessed the impact of the program on Medicaid costs for 2 years post-baseline via propensity score matching and bootstrapping.The evaluation used matched data from administrative records for 2827 people. Sequence analysis identified 6 Medicaid expenditure patterns during 2 years prior to baseline among people placed in the program . Stratified by Medicaid expenditure patterns, Medicaid savings were found among those with very low Medicaid coverage , increasing Medicaid expenditures , and high Medicaid expenditure patterns . Savings were largely driven by shorter psychiatric hospitalizations in the post-baseline period among those placed.The supportive housing program was associated with Medicaid savings, particularly for individuals with very low Medicaid coverage, increasing Medicaid expenditures, and high Medicaid expenditures pre-baseline.The online version of this article (10.1186/s12913-017-2816-9) contains supplementary material, which is available to authorized users. Medicaid is a public health insurance program in the United States (US) that provides comprehensive healthcare services to vulnerable populations including individuals with disabilities, older adults, and low-income families . SeveralA recent review of US and Canadian studies found that the housing first model for homeless individuals with serious mental illness has led to decreases in emergency department (ED) visits and hospitalizations . HoweverThe population in this evaluation was composed of individuals aged 18 years or older who were eligible for a NYC supportive housing program because they met one the 2 following conditions: 1) chronic homelessness and serious mental illness or, 2) dual diagnosis of mental illness and a substance use disorder. This is one of 9 population groups in a NYC supportive housing program launched by NYC and New York State governments in 2007 to create 9000 supportive housing units for people who were homeless or at risk of homelessness . The pron = 20) because these continuous housing experiences might have confounded changes in outcomes associated with the program. Baseline  was defined as the first placement date (for the placed group) or the first eligibility date (for the unplaced group). For each individual, we examined the 2-year period prior to baseline and 2-year period post baseline. For example, for those who became eligible for the program on December 31, 2010, the evaluation period was January 1, 2008 through December 31, 2012. For placed people, a median of 95 days  passed between the first eligibility date and the first move-in date. This gap between the eligibility and placement dates reflected administrative processes to finalize placement .In this evaluation, we included individuals who were eligible for the program during 2007–10. Housing or program directors received multiple eligible applicants per 1 vacancy and determined a placement decision after interviewing a maximum of 3 candidates. We categorized these individuals into 2 groups: 1) applicants who were placed in the program for more than 7 days (“placed”) and 2) applicants who were not placed in the program during 2 years after eligibility (“unplaced”), and were not placed in any other government-subsidized housing tracked by the evaluation during the first 6 months after eligibility  to ensure lack of exposure to a supportive housing program during the initial treatment assignment. We excluded those who were housed in other government-subsidized housing programs immediately before becoming placed in the program  during the 2 years after placement. The final dataset contained 2827 people. This sample selection process was illustrated in Additional file In this program evaluation, the exposure variable was an indicator of supportive housing placement (defined above). The outcomes were aggregated Medicaid costs during 2 years after baseline, adjusted for medical inflation to 2012 dollars. The total Medicaid costs were further divided into costs from 1) outpatient care, 2) inpatient care, 3) emergency department visits, and 4) prescription drugs. The remaining costs (“other costs”) mainly consisted of those from home health agencies and personal care, and residential care. Capitation payments were classified in the other costs category and combined with fee-for-service costs because Medicaid savings in this evaluation were assessed using the cost-to-government perspective  rather than the societal cost perspective. Because capitation payments did not provide information about costs associated with specific use of services, service-specific costs in the remaining categories might have been underestimated; yet this potential bias is likely small because behavioral health-related claims, which represented most claims in this population, were carved out and reimbursed via a fee-for-service mechanism during the evaluation period.As an additional outcome, we calculated the percent of individuals who participated in managed care for at least 6 months during the 2 year pre- or 2 year post-baseline because pre- and post-changes in managed care enrollment by placement status, which is a cost containment effort, could help explain changes in Medicaid costs associated with the housing program.Lastly, to describe and account for differences between placed and unplaced people, we included covariates that captured the use of government services and benefits during 2 years prior to baseline using the other administrative data sources and monthly Medicaid coverage dates during 2 years prior to baseline and post-baseline using Medicaid data. To construct the Medicaid expenditure patterns via sequence analysis, we calculated monthly Medicaid expenditures during 2 years prior to baseline. We also obtained demographic and clinical descriptors at the time of program application using the NYC supportive housing application data  . This meWe used propensity score matching to minimize baseline differences between the placed and unplaced groups . For eacp < 0.05. For post-baseline mean comparisons, statistical significance was established using 95% confidence intervals via bootstrapping. Sequence analysis, optimal full matching, and bootstrapping were performed using TraMineR, cluster, optmatch, and boot packages in R 3.3.1 software  [We performed independent t-tests for continuous variables and chi-square tests for categorical variable to test whether placed people were different from unplaced people in baseline or pre-baseline characteristics before and after propensity score matching. In addition, we described baseline characteristics and pre-baseline Medicaid utilization  by 6 Medicaid expenditure groups. Similar to tests for difference between placed and unplaced people, we performed independent t-tests for continuous variables and chi-square tests for categorical variables as we tested differences across Medicaid expenditure groups. Statistical significance for these tests was established using 2-tailed Austria) . All othAustria) .Table very low coverage , low user , middle user , emerging user , second-highest user , and high user .The sequence analysis identified 6 distinct patterns Fig. . These rvery low coverage pattern was composed of individuals who were not covered by Medicaid during most of the 2-year period except for later months and had monthly Medicaid costs of $921 on average  when they were on Medicaid. These individuals exhibited a pattern of having persistent Medicaid coverage and low Medicaid expenditures . Middle user was composed of individuals who had a consistent pattern of monthly Medicaid costs of $755 on average  throughout the 2-year period. There were individuals with almost complete Medicaid coverage who experienced a sharp cost increase after an initial period of not being on Medicaid (emerging user). Lastly, there were 2 consistent patterns of high Medicaid expenditures: high user with Medicaid costs of $5290 on average per month , and second-highest user with monthly Medicaid costs of $1987 on average .The very low coverage and emerging user patterns were less likely than others to have been receiving supplemental security at the time of application. Lastly, for more than 80% of the 2-year period prior to baseline, the housing applicants were on Medicaid, except for the very low coverage and emerging user whose Medicaid coverage was less than 53%.In general, each expenditure pattern displayed a unique profile of baseline characteristics that was distinguishable from the others Table . Individhigh user , followed by second-highest user   or other expenses  was greater than that due to outpatient care. Other Medicaid costs for middle user were mostly due to managed care costs (73%), whereas those for low user were more evenly attributed to three major types of costs . Medicaid costs for second-highest user were largely attributed to inpatient (34%) and outpatient care types (29%). Emergency department visits contributed the smallest amount of Medicaid costs across all 6 Medicaid expenditure patterns.The total mean Medicaid costs over the 2 years prior to baseline was the highest among very low coverage pattern , emerging user , and second-highest user  after accounting for baseline characteristics via propensity score matching . Stratified by Medicaid expenditure patterns, this difference was statistically significant only among emerging user and second-highest user.Table very low coverage pattern, $9020  among those with the emerging user pattern, and $14,450  among those with the second-highest user pattern.In this evaluation, we found mean Medicaid savings of $9526  for 2 years post-baseline among the program applicants who were placed in the NYC supportive housing program. Stratified by pre-baseline Medicaid expenditure patterns, we observed mean Medicaid savings of $15,694  among those with the very low coverage, emerging user, and second-highest user patterns were mostly driven by reduced costs due to psychiatric hospitalizations. Whereas psychiatric inpatient costs more than doubled from pre-baseline to post-baseline among unplaced individuals with the very low coverage pattern ($2573 to $5731), slightly decreased among those with the emerging user pattern ($7920 to $5767), or were unchanged among those with the second-highest user pattern ($6596 to $6338), substantial reductions in psychiatric inpatient costs were observed among placed individuals . This implies that the supportive housing program may be particularly effective in preventing future inpatient events and reducing the length of hospitalization, which in turn contributes to decreased total Medicaid costs. Along with small cost savings from emergency department visits, these inpatient cost savings are consistent with previous findings that supportive housing is associated with a reduction in the number of emergency department visits and hospital admissions among high Medicaid users [Cost savings for those with id users , homelesid users , and thoemerging user and second-highest user pattern when stratified by Medicaid expenditure patterns, which supported this hypothesis. Future studies with detailed housing service data are warranted to better understand the association between housing placement and increased managed care enrollment. Additionally, estimates of the real cost of managed care beyond capitation costs are needed to explain contributions of managed care enrollment to cost savings.Another possible explanation for these cost savings was the increased managed care enrollment after placement. Among the applicants overall, placed people were more likely to enroll in managed care than unplaced people during the post-baseline period. Given that high Medicaid costs were likely to have been controlled under the managed care payment system at least for non-behavioral health-related claims, managed care enrollment among the placed group might have contributed to containing Medicaid costs during 2-year post baseline . Similarhigh user pattern , this estimate was not statistically significant, indicating high estimation uncertainty. This lack of statistically significant savings may reflect the fact that the high user group had the highest prevalence of pre-existing chronic diseases that would still require treatment after placement. Even if some medical costs for treatment inevitably occur after housing placement, previous studies highlight reduced numbers of emergency department and hospital admission events instead of evaluating total medical costs [Despite the fact that this evaluation, like other studies, found an association between living in a supportive housing program and mean Medicaid savings in inpatient and emergency department hospitalizations, the magnitude of the program impact on Medicaid savings in this evaluation is smaller than has been found in previous studies , 26, 27.al costs . The difal costs  or lack This evaluation has some limitations. First, because applicants were not randomly selected for housing placement, our results might be biased due to unobserved confounders. Despite thorough efforts to ensure comparability between placed and unplaced groups via propensity score matching and sequence analysis, two groups could be different in unobserved characteristics, which could affect Medicaid utilization post-baseline. Second, Medicaid expenditure patterns were identified based on the assumption that there were underlying groups of individuals who have distinct trajectories of Medicaid utilization. This assumption is consistent with health policy approaches of distinguishing high Medicaid users from low users , 4, 30. A main strength of this evaluation is that sequence analysis identified distinct groups of individuals based on 2 years of pre-baseline Medicaid records. Compared with conventional mean- or frequency-based methods, our method produced groups that likely more validly captured patterns of Medicaid utilization because they preserved sequences of claims over a sufficiently long time period , 15, 18.very low coverage, emerging user, or second-highest user of Medicaid-funded services prior to baseline. This evaluation found that tenancy in a supportive housing program among individuals at risk of homelessness and with serious mental illness had a significant impact on Medicaid expenditures, and that cost savings were mostly driven by reduced psychiatric hospitalizations. These findings call for further evaluation of this program to examine its long-term impact and inclusion of detailed data about housing services or referrals to identify mechanisms through which supportive housing placement influences Medicaid cost savings.In conclusion, in an NYC supportive housing program for people with serious mental illness and chronic homelessness or dual diagnoses of mental illness and substance use disorder, there were Medicaid savings overall as well as for tenants who had patterns of Additional file 1:A flow chart of sample selection process. This file illustrates how exclusion criteria were applied in order to generate a final evaluation sample. (DOCX 20 kb)Additional file 2:Covariates included in the propensity score models. This file contains a list of covariates that were included in the propensity score models. (DOCX 19 kb)Additional file 3:Summary of balance of baseline characteristics between placed and unplaced people before and after propensity score matching. This file shows performance of propensity score matching in balancing baseline characteristics between placed and unplaced people. (DOCX 13 kb)Additional file 4:Weighted mean psychiatric inpatient costs among placed versus unplaced persons with very low coverage, emerging user, and second-highest user patterns. This file shows weighted mean psychiatric inpatient costs among placed and unplaced persons, stratified by three trajectory groups, including very low coverage, emerging user, and second-highest user patterns. (DOCX 12 kb)


Zika virus RNA has been detected in semen collected several months after onset of symptoms of infection. Given the potential for sexual transmission of Zika virus and for serious fetal abnormalities resulting from infection during pregnancy, information regarding the persistence of Zika virus in semen is critical for advancing our understanding of potential risks. We tested serial semen samples from symptomatic male patients in the United Kingdom who had a diagnosis of imported Zika virus infection. Among the initial semen samples from 23 patients, Zika virus RNA was detected at high levels in 13 (56.5%) and was not detected in 9 (39.1%); detection was indeterminate in 1 sample (4.4%). After symptomatic infection, a substantial proportion of men have detectable Zika virus RNA at high copy numbers in semen during early convalescence, suggesting high risk for sexual transmission. Viral RNA clearance times are not consistent and can be prolonged. Zika virus is an emerging flavivirus currently causing a major outbreak of human disease in the Americas, the Caribbean, and Western Pacific regions; 71 countries and territories have reported mosquitoborne transmission since 2015  assay based on a published method targeting the nonstructural protein 1 (NS1) gene . In addition, 7 patients (patients 17–23) submitted semen samples after serologic diagnoses .Since December 2013, nearly 2,500 Zika virus diagnostic molecular detection tests have been performed on travelers returning to the United Kingdom with possible Zika virus infection. As of October 5, 2016, a total of 116 persons, 55 of whom were males, had detectable Zika virus RNA in serum, plasma, or urine  of the 16 patients with rRT-PCR–diagnosed infection and from 2 (28.6%) of the 7 patients with serologically diagnosed infection. For the 10 patients who had a positive initial blood or urine sample and a subsequent positive semen sample, the median cycle threshold (Ct) value was significantly lower for semen samples (Ct 27.3) than for acute-phase diagnostic serum, plasma, or urine samples . We detected Zika virus RNA in t values increased consistently over time . The day 167 semen sample of patient 8 repeatedly demonstrated a subthreshold curve in the rRT-PCR test, suggesting evidence of Zika virus RNA at the assay detection limit. This patient was taking immunosuppressive drug therapy when infected . No other patients were known to have immunosuppression.Further semen samples were provided by 8 of the 12 Zika virus–positive patients . A serieZika virus RNA was undetectable in semen samples from 5 of the 16 patients with rRT-PCR–diagnosed infection (31.3%); results were indeterminate (subthreshold) for 1 patient (6.3%). All 6 patients subsequently seroconverted for Zika virus IgG (data not shown). The indeterminate result occurred in the only patient who had undergone a vasectomy (day 16 sample). Zika virus RNA was also undetectable in the semen of 5 (71.4%) of the 7 patients with serologically diagnosed infection. Although Zika virus infection was not definitively confirmed by molecular testing in any of these 5 patients, laboratory tests excluded recent chikungunya or dengue infection  Table 2.Of the 15 patients with rRT-PCR–confirmed infection in whom previous dengue serostatus could be assessed at the time of their initial Zika virus diagnosis , only 3 had serologic evidence of previous dengue virus infection. Two of these patients (patients 3 and 6) did not have Zika virus RNA in subsequent semen samples  Table 1.t 24). We attempted virus isolation in Vero and C6/36 cells; we observed propagation only in C6/36 cells for the original sample, although C6/36 infected supernatant was capable of infecting Vero cells and produced a cytopathic effect. We deposited the complete genome for this isolate in GenBank (accession no. KX673530)  of the 23 patients we tested lacked detectable Zika virus RNA in semen on the first sample . Our detection of Zika virus RNA in early semen samples from 12 (52.2%) of 23 infected patients is similar to the detection of viral RNA in 50% of case-patients in small-number Belgian .One patient with an indeterminate rRT-PCR result for his initial (day 16) semen sample was reported to have had a vasectomy; ongoing viral replication might have been occurring in the urogenital tract. A recent publication reported Zika virus RNA in the ejaculate of a vasectomized man 96 days after onset of symptoms in addition to infectious virus cultured at 69 days  yielded replicating virus. Isolation from samples with similar Ct values was unsuccessful, highlighting the inconsistency for this technique at present. The inability to isolate virus cannot be taken to indicate the absence of infectious virus until more is known about the intricacies of virus isolation from semen. Although we have been unable to isolate virus in tissue culture consistently from semen samples, it is striking that all recognized transmission events to date have occurred relatively soon after infection in the male index patient  guidelines advise that male and female travelers should adopt safer sex practices or consider abstinence to reduce the risk for sexual transmission for 6 months after leaving a country with ongoing Zika virus transmission (>8 weeks after leaving a Zika-affected area, as suggested by WHO (Currently, we lack knowledge of viral persistence in semen. More data, especially from larger systematic studies, are urgently needed to support evidence-based policies to prevent sexual transmission of Zika virus. In the meantime, testing of semen from symptomatic men Characteristics of patients with real-time reverse transcription PCR–confirmed and serologically diagnosed Zika virus infection who were evaluated to determine presence and persistence of Zika virus RNA in semen, United Kingdom, 2016.


Postmortem examination of 7 neonates with congenital Zika virus infection in Brazil revealed microcephaly, ventriculomegaly, dystrophic calcifications, and severe cortical neuronal depletion in all and arthrogryposis in 6. Other findings were leptomeningeal and brain parenchymal inflammation and pulmonary hypoplasia and lymphocytic infiltration in liver and lungs. Findings confirmed virus neurotropism and multiple organ infection. From the discovery of Zika virus in Uganda in 1947 through 2007, when an outbreak occurred on Yap Island, Micronesia, only sporadic cases of human infection had been reported . Real-time reverse transcription PCR (RT-PCR) of cerebrospinal fluid and tissue, performed at the Central Public Health Laboratory (Ceará), confirmed congenital Zika virus infection . We reviOf the 7 mothers, all were HIV negative and 5 had had symptoms compatible with viral infection during the first trimester of pregnancy. Neonate survival times ranged from 30 minutes to 6 days after birth; 5 survived <1 hour, 1 died 48 hours after birth, and 1 survived for 6 days. Four neonates were male; for 1 of these in whom the genitalia were ambiguous (neonate 2), the sex was ascertained by identification of undescended testes at autopsy. Gestational ages ranged from 30 to 42 weeks (median 37 weeks) .Brain weight was decreased in all neonates; however, body weight was within reference range for gestational age in all except neonate 6 . For 6 neonates, microcephaly was present , panel EAll infants had thinning of the brain parenchyma with severe depletion of neuronal precursors , panel ALymphocytic inflammation was observed in the meninges of 5 neonates , panel DPulmonary hypoplasia was present in all neonates ; relative lung weight (lung weight/body weight) was 0.004–0.01 g , panel ILiver specimens were available from 6 neonates; moderate to severe hydropic degeneration was found in 5 of these specimens. Round eosinophilic cytoplasmic structures suggestive of megamitochondria were observed in 2 neonates, mild to moderate steatosis and Councilman bodies (apoptosis) in 3, hepatocyte necrosis in 1 (neonate 7) , and mild periportal lymphocytic inflammation in 1 (neonate 2).Kidneys were available from 6 of the neonates. Focal glomerular sclerosis was present in neonates 2 and 6. In neonate 2, tissue was positive for Zika virus by RT-PCR . ModeratOf the 7 Zika virus–infected neonates examined, 6 did not show intrauterine growth restriction, but all 7 had remarkably decreased brain weight , emphasizing the neurotropism of Zika virus. These findings are similar to those earlier reported for congenital Zika virus infection  differs from features seen in other common infections associated with congenital abnormalities  and should raise suspicion for congenital Zika virus infection, warranting further workup. For instance, the pattern of calcifications seen in tissue from patients with congenital cytomegalovirus infection and in toxoplasmosis are predominantly periventricular (<0.012 (1.2%), seemed to be a major factor determining death during the perinatal period , lungs , and bladder (lymphocytic cystitis). PCR detection of Zika virus in liver, lung, and kidney tissue also strengthened our hypothesis that Zika virus can infect multiple tissues.Postmortem findings for 7 neonates with congenital Zika virus infection.


To quantify and predict the economic burden of dementia in China for the periods 1990–2010 and 2020–2030, respectively, and discuss the potential implications for national public health policy.Using a societal, prevalence-based, gross cost-of-illness approach and data from multiple sources, we estimated or predicted total annual economic costs of dementia in China. We included direct medical costs in outpatient and inpatient settings, direct non-medical costs – e.g. the costs of transportation – and indirect costs due to loss of productivity. We excluded comorbidity-related costs.The estimated total annual costs of dementia in China increased from 0.9 billion United States dollars (US$) in 1990 to US$ 47.2 billion in 2010 and were predicted to reach US$ 69.0 billion in 2020 and US$ 114.2 billion in 2030. The costs of informal care accounted for 94.4%, 92.9% and 81.3% of the total estimated costs in 1990, 2000 and 2010, respectively. In China, population ageing and the increasing prevalence of dementia were the main drivers for the increasing predicted costs of dementia between 2010 and 2020, and population ageing was the major factor contributing to the growth of dementia costs between 2020 and 2030.In China, demographic and epidemiological transitions have driven the growth observed in the economic costs of dementia since the 1990s. If the future costs of dementia are to be reduced, China needs a nationwide dementia action plan to develop an integrated health and social care system and to promote primary and secondary prevention. It was hoped that, by quantifying the economic costs of dementia, Chinese policy-makers would be motivated to develop a nationwide action plan, prioritize policies on dementia-related care and research and reduce the economic and societal burdens of dementia in China.,In this cost-of-illness study, we used a prevalence-based, bottom-up approach to quantify or predict the costs of dementia in China between 1990 and 2030, from a societal perspective. We categorized all the costs into three classes:International statistical classification of diseases and related health problems, 10th revision.China Statistical Yearbook 2015.We used multiple data sources for all estimates. We used age-specific prevalence of dementia in China, for the period 1990–2010, derived from a comprehensive systematic review.,,For our estimates we included costs for hospitalization, formal care, informal care, loss of productivity, outpatient care and transportation. Our estimates of hospitalization costs, which included the costs of all medicines, clinical examinations, specialist consultations and bed care, were based on the mean values of the costs recorded in the electronic health records of the two study facilities and the values given in a published articleAll the estimated costs were converted to United States dollar (US$) values in January 2015, when US$ 1 was equivalent to about 6.2 Chinese yuan.We estimated the numbers of people with dementia in China in 1990, 2000 and 2010 by multiplying the age-specific prevalence of dementiaWe conducted multiple sensitivity analyses to assess the impact of variations in the key input parameters on our primary estimates. Specifically, we estimated the total costs of dementia by (i) using prevalence data derived from a different systematic reviewThe total number of people with dementia in China was estimated to be about 3.5 million in 1990, 5.1 million in 2000 and 9.6 million in 2010 . The oveBetween 1990 and 2010, the mean annual costs of formal and informal care for each dementia case increased more than 60-fold and more than 18-fold, respectively . Over thThe total costs of dementia were projected to reach US$ 69.0 billion in 2020 and US$ 114.2 billion in 2030 . The ageAccording to our estimates, the costs of informal care for dementia accounted for 94.4%, the total costs of dementia care in China in 1990, decreasing to 92.9% in 2000 and 81.3% in 2010 . In contThe results of our sensitivity analyses indicated that variations in informal care hours, prevalence of dementia and productivity weighting had a substantial impact on estimates of the total costs of dementia, whereas changes in medical prices, wages and the proportions of patients living at home or seeking professional care had relatively little impact on such estimates .,,In China, from an age of 60 years, the prevalence of dementia almost doubles every five years and, as elsewhere,,,,,,,Our analyses indicated that, in China, informal care accounted for more than 80% of the total dementia costs in 2010. This proportion is consistent with the global trend revealed by a systematic reviewIn China, public health policy needs to be tailored to address the economic burden posed by dementia. There needs to be greater focus on developing an integrated health and social-care system, including improvements in the efficiency of dementia care and improved health education and financial and social support for dementia cases and their caregivers. Dementia care might be integrated with the national programme for critical illness insurance. Informal caregivers could be taught knowledge and skills relevant to dementia care.–,Policy-makers also need to establish a strategic action plan designed to promote the primary and secondary prevention of dementia. In the past few decades, epidemiological studies have identified several modifiable risk factors for dementia, e.g. cardiovascular disease, that can be targeted for primary prevention.,,Our estimates were based on data from multiple sources. The cost data on the use of health resources by dementia cases were from itemized routine hospital records, prevalence data were from a comprehensive systematic reviewIn conclusion, demographic and epidemiological transitions in the past two decades have driven substantial growth in the economic costs of dementia in China. This trend is likely to continue over the next two decades. Given the huge economic burden of dementia, policy-makers in China are advised to make dementia a national health priority and to develop a strategic nationwide action plan. Failure to take appropriate action now will allow the economic burden of dementia to grow even further and could, in the long-term, cause dysfunction throughout China’s entire health-care system.


Flaviviridae is a family of small enveloped viruses with RNA genomes of 9000–13 000 bases. Most infect mammals and birds. Many flaviviruses are host-specific and pathogenic, such as hepatitis C virus in the genus Hepacivirus. The majority of known members in the genus Flavivirus are arthropod borne, and many are important human and veterinary pathogens . This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the Flaviviridae, which is available at www.ictv.global/report/flaviviridae.The  Flavivirus, Hepacivirus and Pegivirus) or three (genus Pestivirus) envelope proteins.Virions are typically spherical in shape with a lipid envelope , Fig. 1.Flavivirus, Pestivirus, Hepacivirus and Pegivirus, respectively  Report: http://talk.ictvonline.org/links/hcv/hcv-classification.html.Hepatitis C virus classification:


Pseudogymnoascus destructans. The fungus is widely distributed in North America and Europe and has recently been found in some parts of Asia, but interestingly, no mass mortality is observed in European or Asian bats. Here we report a novel double-stranded RNA virus found in North American isolates of the fungus and show that the virus can be used as a tool to study the epidemiology of White-nose syndrome. The virus, termed Pseudogymnoascus destructans partitivirus-pa, contains 2 genomic segments, dsRNA 1 and dsRNA 2 of 1.76 kbp and 1.59 kbp respectively, each possessing a single open reading frame, and forms isometric particles approximately 30 nm in diameter, characteristic of the genus Gammapartitivirus in the family Partitiviridae. Phylogenetic analysis revealed that the virus is closely related to Penicillium stoloniferum virus S. We were able to cure P. destructans of the virus by treating fungal cultures with polyethylene glycol. Examination of 62 isolates of P. destructans including 35 from United States, 10 from Canada and 17 from Europe showed virus infection only in North American isolates of the fungus. Bayesian phylogenetic analysis using nucleotide sequences of the viral coat protein geographically clustered North American isolates indicating fungal spread followed by local adaptation of P. destructans in different regions of the United States and Canada. This is the first demonstration that a mycovirus potentially can be used to study fungal disease epidemiology.White-nose syndrome is one of the most lethal wildlife diseases, killing over 5 million North American bats since it was first reported in 2006. The causal agent of the disease is a psychrophilic filamentous fungus,  Many species of bats in North America have been severely impacted by a fungal disease, white-nose syndrome, that has killed over 5 million bats since it was first identified in 2006. The fungus is believed to have been introduced into a cave in New York where bats hibernate, and has now spread to 29 states and 4 Canadian provinces. The fungus is nearly identical from all sites where it has been isolated; however, we discovered that the fungus harbors a virus, and the virus varies enough to be able to use it to understand how the fungus has been spreading. This study used samples from infected bats throughout Pennsylvania and New York, and New Brunswick, Canada, as well a few isolates from other northeastern states. The evolution of the virus recapitulates the spread of the virus across these geographical areas, and should be useful for studying the further spread of the fungus. Pseudogymnoascus destructans  is an emerging fungal pathogen responsible for a fatal disease, white-nose syndrome (WNS) in hibernating bats in North America 2–4 [PEG] and the value was adjusted to the Pd culture temperature of 10°C. An agar plug containing actively growing Pd was placed in 50 ml autoclaved modified SN liquid media with a targeted amount of PEG 8000  and grown as described above. After three weeks, pieces of newly growing mycelia of Pd were transferred to normal SBD routinely used to culture Pd and the fungus was harvested after a normal culture period. The fungi from different treatments were examined for PdPV-pa both by dsRNAs gel electrophoresis and RT-PCR with PdPV-pa specific primers. In all methods Pd isolate LB-01 was used.For PEG induced matric stress on water availability we used PEG 8000 (Fisher BioRegents) in a modified Spezieller Nährstoffarmer liquid media  of 10 approved species of Gammapartitivirus available in the GenBank. The sequences of Pepper cryptic virus 1, type member of genus Deltapartitivirus, which is the closest group to Gammapartitivirus in Partitiviridae family was used as outgroup. We used nucleotide sequences (CP) to study phylogenetic relationships of PdPV-pa in North American population. The nucleotide sequence of PsV-S CP was used as outgroup in the analysis. In Bayesian trees construction using amino acid sequence of the RdRp and CP ORFs, Jukes-Cantor substitution model was applied and for nucleotide sequences of CP General time-reversible (GTR) model with gamma rate variation was used based on the best model tested out of 28 models.Genetic variation in North American PdPV-pa isolates were determined by sequence analysis of RdRp and CP segments amplified in RT-PCR using specific primers. The primer pairs specific to RdRp  and CP  were designed from the consensus sequences of RNA 1 and RNA 2 assembled from LB-01 isolate cloned sequences. PCR products using RdRp and CP specific primers from 45 North American PdPV isolates were sequenced and aligned with MUSCLE default settings in the program Geneious 8.0 . The RdRS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


Hydrogenophaga intermedia strain PBC, a 4-aminobenzenesulfonate-degrading bacterium, by supplementing the assembly with Nanopore long reads which enabled the reconstruction of the genome as a single contig. From the complete genome, major genes responsible for the catabolism of 4-aminobenzenesulfonate in strain PBC are clustered in two distinct genomic regions. Although the catabolic genes for 4-sulfocatechol, the deaminated product of 4-aminobenzenesulfonate, are only found in H. intermedia, the sad operon responsible for the first deamination step of 4-aminobenzenesulfonate is conserved in various Hydrogenophaga strains. The absence of pabB gene in the complete genome of H. intermedia PBC is consistent with its p-aminobenzoic acid (pABA) auxotrophy but surprisingly comparative genomics analysis of 14 Hydrogenophaga genomes indicate that pABA auxotrophy is not an uncommon feature among members of this genus. Of even more interest, several Hydrogenophaga strains do not possess the genomic potential for hydrogen oxidation, calling for a revision to the taxonomic description of Hydrogenophaga as “hydrogen eating bacteria.”We improved upon the previously reported draft genome of  Hydrogenophaga consists of rod-shaped yellow-pigmented gram negative bacteria that are generally considered capable of using hydrogen as an energy source , a recalcitrant intermediate compound in the synthesis of colorants  were performed using Blast Ring Image Generator   . Mappingersion 3 . Generatersion 3 .Hydrogenophaga species were performed using JSpecies V1.2 (ANIm setting) (Pair-wise average nucleotide identity of strain PBC against the publicly available genome sequences of setting) . StrainsWhole proteome was predicted using Prodigal (-p meta setting) and piped into PhyloPhlan for the identification of conserved proteins . The con-5 setting) or TIGRFAM (-cut_nc or –E 1e-5 setting) HMM profiles  uniquely present in strain PBC, that were subsequently identified as prophage in origin by Phaster. Both phage regions were incomplete and only code for two to three major phage components. Phage components 1 and 2 on the contrary are complete, uniquely shared by both H. intermedia strains  and have the highest number of homologous proteins to Enterobacter phage Arya (NCBI Reference Sequence: NC_0310480) isolated from a termite gut.A total of 90,491 1D and 51,630 2D reads were generated from a full 48-h MinION run. After filtering for reads longer than 2,500 bp, 14,404 1D and 12, 959 2D reads were retained for genome assembly. The final data output used for assembly was 173 megabases representing approximately 33× genome coverage of strain PBC. The average read length was 6,350 bp and the longest read generated was 126,959 bp. Initial Spades hybrid assembly using both Illumina and MinION filtered reads generated 5 scaffolds  which were subsequently scaffolded and gap-closed into a single contig using Npscarf. The complete genome of strain PBC is 5,232,477 bp in length with a GC content of 68.4%. Based on NCBI annotation pipeline, the complete genome consists of 4,904 protein-coding genes, 43 tRNA and single-copy 5S, 16S, and 23S rRNA genes. Comparisons of strain PBC and other sad operon appears to be fairly conserved in all currently reported Hydrogenophaga genomes, suggesting that the ability to deaminate 4-aminobenzenesulfoate may be a defining metabolic trait of Hydrogenophaga strains. However, given the diverse isolation sources of Hydrogenophaga strains (Table 1) and their presumably limited exposure to xenobiotic compounds, it is likely that the original substrate of the 4-aminobenzenesulfonate dioxygenase is a naturally occurring compound with close structural homology to 4-aminobenzenesulfonate.Genes responsible for the catabolism of 4-aminobenzenesulfonate in strain PBC are clustered in two distinct genomic regions. Interestingly, the pcaH2G2, pcaB2 and 4-SLH involved in the conversion of 4-sulfocatechol to maleylacetate is conserved only in the Hydrogenophaga intermedia strains . In addition, it is also worth noting that this region was identified as a phage region and flanked by genes coding for IS6 transposable elements , an indication of the mobility of the pcaH2G2, pcaB2 and 4-SLH gene cluster presumably through phage transduction  due to the inability of short reads to resolve the repetitive regions .In contradistinction, the genomic region containing sduction . As expe regions . This shHydrogenophaga. Recently, the complete genome of Novosphingobium resinovorum SA1, another well-studied 4-aminobenzenesulfonate degrader, has been reported and comparison of this genomic region and that of the homologous region in Novosphingobium resinovorum SA1, revealed a comparable gene organization, e.g., pcaH2-pcaG2-tnp-pcaB2-4SLH-MAR in H. intermedia PBC  and pcaB2 (scaA)-4SLH (scaB)-MAR(scaC)-oxidoreductase-pcaH2(scaE)-pcaG2(scaF) in Novosphingobium resinovorum SA1 . A more in-depth analysis of the H. flava genome may be useful to identify its additional nutritional requirements for optimal and consistent growth on culture medium. Such a genomic approach has been successfully demonstrated in Clostridium tyrobutyricum and other Clostridia associated with butyric acid fermentation leading to the identification of key vitamins and several amino acids essential for growth , could be reclassified to the species H. pseudoflava given its high pairwise ANIm (97%) to H. pseudoflava NBRC 102511T .H. flava . In silir growth . FurtherHydrogenophaga strains with H. intermedia strains and H. sp. PML 113 being basal to the rest of Hydrogenophaga . The genus Hydrogenophaga clade shares a sister relationship with a group of highly alkaliphilic hydrogen-utilizing Comamonadacea bacteria isolated from an alkaline serpentinizing springs at The Cedars, California . The 7 Hydrogenophaga strains include the original strains that were initially used to describe the genus Hydrogenophaga, such as H. palleronii, H. taeniospralis, H. flava and H. pseudoflava, corroborating their previously demonstrated in vitro hydrogen oxidizing ability . However, the absence of genomic potential for hydrogen catabolism in nearly half of the currently sequenced Hydrogenophaga strains is unexpected and indicates that the description genus of Hydrogenophaga as “hydrogen eating bacteria” is not sustainable and warrants revision.The genus  ability . The repin vitro  correlatHydrogenophaga genome sequences to the contig of H. palleronii NBRC102513  containing pabB revealed that only 8 out of 14 genomes exhibit nucleotide similarity  to the pabB gene region . Of the eight genomes, only partial pabB hits were observed for strains H7, NBRC102514, Root209, NBRC102511 and A37, hinting divergence at the nucleotide level. Interestingly, only two proteins belonging to H. palleroni and H. sp. PML113 achieved an HMMscore that is above the domain noise cutoff score for bona fide PabB (TIGR00553) . On the contrary, strain IBVHS2 despite exhibiting nucleotide similarity to the pabB gene region, does not code for protein with HMMscore above the TIGR00553 noise cutoff. However, it is worth noting that multiple proteins with HMMscore slightly below the TIGR00553 noise cutoff could be identified from the annotated Hydrogenophaga genomes . These proteins appear to be more closely related to PabB than TrpE as evidenced by their extremely low HMMscore to TrpE (TIGR00564) in comparison with other putative TrpE proteins and their phylogenetic affiliation to the PabB clade . TrpE is involved in the synthesis of anthranilate from chorismate and ammonia. Functional characterization of these divergent PabB homologs will be useful to explore their role in pABA synthesis as of them belong to Hydrogenophaga pseudoflava that has been shown to grow on minimal medium without pABA supplementation . Surprisingly, strains LPB0072, RAC07, Root209 and IBVHS1 appear to also lack the genomic potential for pABA synthesis. A future study investigating the ability of these strains to grow on defined medium without pABA supplementation will be necessary to verify their predicted pABA auxotrophy as most Hydrogenophaga strains were isolated on nutrient rich medium, which will contain traced amount of pABA. For example, strains Root209 and RAC07 were maintained on tryptic soy agar and minimal medium with yeast extract supplementation, respectively (The absence for pABA . A recen to PabC , potentiectively .Hydrogenophaga intermedia PBC has been deposited under the accession number CP017311. Raw data for both Illumina and Nanopore data have been placed under the SRA under the SRA project ID SRP092076.The complete genome of HG performed gDNA extraction, and bioinformatics analysis and drafted the manuscript. YL performed library preparation and MINION sequencing. CA supervised the study.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.


The emergence of Zika virus has challenged outbreak surveillance systems in many at-risk, low-resource countries. As the virus has been linked with Guillain–Barré syndrome, routine data on the incidence of acute flaccid paralysis (AFP) may provide a useful early warning system for the emergence of Zika virus.We documented all Zika virus outbreaks and cases in 21 Pacific Islands and territories for the years 2007 to 2015. We extracted data from the Global Polio Eradication Initiative database on the reported and expected annual incidence of AFP in children younger than 15 years. Using a Poisson probability test, we tested the significance of unexpected increases in AFP in years correlating with Zika virus emergence. Data were analysed separately for each Pacific Island country and territory.In most Pacific Island countries, early warning surveillance for acute public health threats such as Zika virus is hampered by poor health infrastructure, insufficient human resources and geographical isolation.Only one example was found (Solomon Islands in 2015) of a significant increase in reported AFP cases correlating with Zika virus emergence. We found no conclusive evidence that routinely reported AFP incidence data in children were useful for detecting emergence of Zika virus in this setting. More evidence may be needed from adult populations, who are more likely to be affected by Guillain–Barré syndrome. Reporting of AFP may be deficient in regions certified as polio-free. Zika virus surveillance strategies need to be convenient, timely and cost-effective, ideally using routinely collected information. Data on the incidence of acute flaccid paralysis (AFP) in children younger than 15 years are routinely collected for polio surveillance by 177 of the 194 WHO Member States as part of the Global Polio Eradication Initiative. The most common cause of AFP is Guillain–Barré syndrome.The Pacific Islands, which fall within WHO’s Western Pacific Region, are home to several of the world’s smallest, least developed and most isolated populations. The collective population of these islands  is approximately 11.4 million people, of whom 8.2 million reside in Papua New Guinea and the rest are dispersed over the thousands of islands and atolls that make up the other 20 Pacific Island countries and territories. Most islands’ health authorities rely on simple syndromic surveillance and ad hoc event reporting by clinicians for disease outbreak detection. Their ability to enhance early warning surveillance in response to acute public health threats such as Zika virus is hampered by limited coverage and sensitivity of existing early warning surveillance; limited capacity to investigate outbreaks; geographic isolation and widely dispersed populations; poor communication infrastructure; and inadequately resourced health systems.We documented all Zika virus outbreaks and cases in the Pacific Islands for the years 2007–2015 and tested the significance of unexpected increases in AFP in years that correlated with Zika virus emergence.We obtained data on Zika virus outbreaks in the 21 Pacific Island countries and territories from published and unpublished information. For published information, we performed a literature search using the search terms “Zika” and “Zika virus” in the PubMed database; the identified papers were reviewed for relevance to the Pacific Islands. Unpublished event-relevant information was extracted from WHO’s weekly Pacific Surveillance Syndromic ReportsAFP surveillance for polio eradication purposes requires health workers to promptly report and investigate identified cases of AFP, including the results of testing for wild polio virus. We extracted data on the expected and reported annual incidence of AFP in children younger than 15 years for Pacific Island countries and territories from the Global Polio Eradication Initiative’s surveillance database.P ≤ 0.05) of unexpected increases in the incidence of AFP in children in the years when Zika virus emerged in each Pacific Island country or territory:We used the following Poisson probability formula to test the significance ; and λ is the expected number of events in the specified time period. Where ,,The first human outbreak of Zika virus was documented in the Pacific Islands in Yap State, Federated States of Micronesia in April 2007.,In 2016  three other Pacific Island countries and territories and Kosrae State, in the Federated States of Micronesia (on which Zika had previously not been detected) reported autochthonous transmission: Tonga , American Samoa , Marshall Islands  and Kosrae State . In November 2016, Palau reported a single confirmed Zika case.In addition, in March 2016 Papua New Guinea reported that six cases of Zika virus infection had been confirmed through retrospective testing of samples collected in May 2015 (one case), December 2015 (two cases) and February 2016 (three cases), indicating low levels of Zika virus transmission within the country.Based on Global Polio Eradication Initiative predictions, the total expected annual number of AFP cases for the year 2015 was 36 (26 for Papua New Guinea and 10 for the other 20 Pacific Island countries and territories). The aggregated number of AFP cases among children reported in each year were 38 (2007), 35 (2008), 30 (2009), 37 (2010), 26 (2011), 14 (2012), 18 (2013), 27 (2014) and 41 2015; .P ≤ 0.001). From February to May 2015, there were five confirmed cases of Zika virus infection out of 324 suspected cases in a population of about 651 700 people. In that year, nine cases of AFP were reported compared with the expected number of two. None of the seven other countries and territories reporting Zika virus cases from 2007 to 2015  showed significant rises in reported cases of AFP associated with emergence of Zika virus.Analysis of individual Pacific Island countries and territories found only one example – the Solomon Islands in 2015 – where a statistically significant increase in reported AFP cases correlated with the emergence of Zika virus  incidence in children were insufficient for the identification of Zika virus emergence in Pacific Island countries and territories.Data from adults, who are more affected by Guillain-Barré syndrome, may confirm whether AFP incidence is a suitable early warning surveillance strategy for emergence of Zika virus in low-resource settings.More evidence is needed that AFP reporting requirements are being met in remote areas and in regions certified as polio free.Populations in Pacific Island countries are small and it is likely that the capacity to conduct and ensure compliance with AFP reporting requirements varies. This may be influenced by a lack of awareness of polio surveillance and associated AFP reporting, given that the last indigenous case of polio virus in the Western Pacific Region was reported in Cambodia in 1997, and the Region has been certified as polio free since 2000.It should be noted that the Global Polio Eradication Initiative’s AFP surveillance targets paediatric populations, who are less likely than adults to be affected by Guillain–Barré syndrome.


A total of 300 1-day-old-broiler chicks were randomly divided into 3 groups  and supplemented 0.2, 0.5 and 0.0% MOS, respectively in NRC recommended diet for 36 days. On day 21 the groups were further split into two sub groups A+ve, A-ve, B+ve, B-ve, C+ve and C-ve with 5 replicates each. The positive groups were shifted to remote sheds and were challenged intranasally with 0.1 ml of reference virus  with EID50 = 10-6.66. Treatment reduces (P<0.05) cloacal virus shedding from day 24 to 26 and 28 to 32. Tracheal virus shedding was lower (P<0.05) on days 25-26 and 28-30 in treatment groups. Day 27 showed highest (P>0.05) virus shedding in all groups. However the reduction of viral shedding is faster in treatment groups and showed no virus shedding on day 32. Maternal antibody titer against AI showed a declining pattern but MOS influenced (P<0.05) the titer in treated groups. Hence the use of MOS may constitute a novel and effective plausible alternative that reduces the spread of disease by decreasing virus shedding and contamination of environment from AIV (H9N2) infection in poultry.Avian influenza (AI) is a highly contagious disease causing significant economic losses worldwide. The aim of this study is to evaluate the effect of mannan-oligosaccharide (MOS) on tracheal and cloacal virus shedding in AI challenged broilers and contamination of environment with H Avian influenza (AI) virus is a member of family Orthomyxoviridae. The infection with a highly virulent virus is characterized by sudden onset of high mortality together with respiratory signs, sinusitis, excessive lacrimation, rales, cessation of egg laying, cyanosis especially on comb and wattles, edema of head and face, diarrhea and weight loss with fatal outcome  into three equal groups  with ten replicates (n=20). The treated groups of A and B were supplemented with 0.2% and 0.5% MOS, respectively from commercially available product SAF-Mannan  in NRC (2001)9 using Haemagglutination inhibition assay as described by Alexander and Chettle (1977)A total of 310 commercial 1-day-old Arbor Acre broilers were procured from a reputable firm (Ani Chicks (Pvt.) Limited) and transferred to experimental shed of University of Veterinary and Animal Sciences, Lahore. Chicks were individually marked using cotton tape wrapped around the wing and was replaced thrice with increase in growth. Ten chicks were slaughtered on day 1 to collect blood serum for presence of maternal antibodies against Avian influenza H9 virus challenge9N2) for challenge was obtained from University Diagnostic Lab. Before challenge the groups A+ve, B+ve and C+ve were shifted to remote shed and challenged intranasally with 0.1 ml of reference virus  with mean egg infective dose (EID50) = 10-6.66.The reference stock AI virus EIDVirus sheddingTracheal and cloacal swabsThe tracheal and cloacal swabs of live chicks were collected using universal viral transport swabs  from days 22 to 28 and then at alternate days i.e., day 30, 32, 34 and 36 to detect levels of virus shedding. The swabs were put back into a vial containing 3 ml of 15% brain heart infusion medium containing antibiotics .Determining EID50 of the cloacal and tracheal swabs50 of virus suspension . Repeated measure analysis of variance was applied to analyze virus shedding across time in the same group . The results of our findings are in accordance with the study of Poorbaghi et al. 9N2 in respiratory tract and hence provide protection against horizontal spread of AIV H9N2 and environmental contamination. Most importantly, probiotics have been shown to regulate the expression of genes related to innate immune-mediated cytokine responses in the intestinal (Ganguli et al., 2013Several earlier experiments revealed that MOS influence antibody titer (Cotter et al., 2000 al. 2013 who inve9N2 and morbidity in broilers horizontally infected with H9N2 AIV. Hence the use of MOS may constitute a novel and effective plausible alternative that reduces environmental contamination and spread of H9N2 from infected birds. In conclusion, we demonstrated that the dietary supplementation of MOS markedly diminished rep-lication and shedding of H No conflict of interest declared.


In early 2014, abortions and death of ruminants were reported on farms in Maputo and Gaza Provinces, Mozambique. Serologic analysis and quantitative and conventional reverse transcription PCR confirmed the presence of Rift Valley fever virus. The viruses belonged to lineage C, which is prevalent among Rift Valley fever viruses in southern Africa. Bunyaviridae, genus Phlebovirus) is a mosquito-borne virus that affects ruminants and humans. The virion contains 3 single-stranded RNA genome segments, large, medium, and small. In ruminants, RVF virus infection is characterized by high rates of abortion and of death, particularly in newborn animals. In humans, the infection is usually asymptomatic, but in severe cases, hemorrhage, meningoencephalitis, retinopathy, and death can occur  virus  in Zambézia Province were attributed to RVF virus, but no virus was detected or isolated , informed the veterinary authorities that 16 of 88 goats aborted their fetuses and 5 newborn kids died. The veterinary authorities also received reports from 2 farms in Xai-Xai  and Chibuto  in Gaza Province, where 26 goats and 8 sheep aborted, respectively, and a total of 7 newborn animals died on both farms. According to the farmers, no animals had been purchased or brought into the herds for >3 years.Serum samples were collected from farms in Goba (n = 88), Xai-Xai (n = 26), and Chibuto (n = 13). On the Goba farm, liver and spleen tissue samples were also collected from 1 aborted fetus and from 1 dead newborn goat. All the serum samples were tested for the presence of RVF virus IgM by using the IDvet Screen RVF IgM ELISA . In addition, the serum samples collected in Goba were further tested for RVF virus IgG by using the RVF recN IgG ELISA kit .https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence data (480 nt) were imported into MEGA6  according to the manufacturer’s instructions. A quantitative real-time reverse transcription PCR was performed as described  of 127  sampled animals were positive for RVF virus IgM, and 49 (55.7%) of 88 animals from the Goba Province were positive for RVF virus IgG. Only 25 animals had RVF virus IgG but no RVF virus IgM . The datRVF virus IgM and the molecular detection of RVF virus confirmed the cause of abortions and deaths in sheep and goats in Maputo (Goba) and Gaza (Xai-Xai and Chibuto) Provinces of Mozambique in the first quarter of 2014. Outbreaks of RVF in eastern Africa are usually associated with the circulation of a local virus lineages, triggered by abnormal rainfall that favors the multiplication of the mosquito vectors or by the introduction of virus through animal movement. Sequence comparison and phylogenetic analyses indicated that the Maputo RVF viruses belonged to lineage C, which suggested that the outbreaks had close links to the 2007 and 2010 RVF outbreaks in Sudan. The farms where the outbreaks occurred are noncommercial, small- to middle-scale farming systems with basic management and with no reports of animal importation. Occasionally, animals are bought from other farms, and the animals known to be imported in the southern part of the country come from the neighboring countries . Animal movement is also reported to occur frequently on the border with adjoining countries. Because no new animals were introduced onto the above mentioned farms and no animal importation was indicated either by the farmers or by the veterinary authorities, we hypothesize that the virus might have been introduced in the past. Then, after a 6-fold increase in rainfall in Maputo , the conditions for an increase in the vector population, and therefore in virus circulation, favored the occurrence of the outbreaks. The high level of seroprevalence reported previously in districts close to the study site  virus isolates used in this study and phylogenetic tree generated from sequence data obtained from RVF viruses from Goba District, Mozambique, and RVF reference sequences available in GenBank.


All collected blood and urine specimens were shipped to the Hawaii Department of Health Laboratory for Zika virus testing and to CDC for confirmatory testing. Early in the response, collection and testing of specimens from pregnant women was prioritized over the collection from symptomatic nonpregnant patients because of limited testing and shipping capacity. The weekly numbers of suspected Zika virus disease cases declined from an average of six per week in January–February 2016 to one per week in May 2016. By August, the EHR-based syndromic surveillance  circulating in American Samoa, and the possible long duration of anti-Zika immunoglobulin M (IgM) antibody positivity, serology was considered a less specific method for detection of Zika virus and therefore a less reliable indicator of ongoing Zika virus transmission.During August 31, 2016–October 15, 2016 , routine testing of asymptomatic pregnant women who conceived within 8 weeks after the calculated transmission end date  was recommended  all persons with signs and symptoms consistent with Zika virus disease; 2) asymptomatic pregnant women with an estimated date of conception  on or before December 10, 2016; 3) pregnant women with prenatal findings suggesting congenital Zika virus syndrome (Because many Zika virus infections are asymptomatic (8) can have negative psychosocial repercussions on pregnant women and significantly burden the health care system with only limited benefit. Establishing a timeline for discontinuing screening of asymptomatic pregnant women allows ASDoH to allocate resources appropriately toward early interventions for children and families affected by Zika virus. The surveillance processes outlined and the timeline established for American Samoa might have implications for jurisdictions where small populations and similar population immunity following widespread Zika virus exposure can facilitate interruption of transmission.The increased likelihood of false positive test results that occurs as disease prevalence declines  for active mosquito-borne transmission of Zika virus and to propose a timeline for discontinuation of routine screening of asymptomatic pregnant women in American Samoa .The rationale described in this report might be adapted by similar jurisdictions with small populations and a potential for interruption of Zika virus transmission to help guide decisions about when to discontinue routine screening of asymptomatic pregnant women for Zika virus infection following the end of active mosquito-borne transmission.


Hytrosaviridae) are non-occluded dsDNA viruses that are pathogenic to some dipterans. SGHVs primarily replicate in salivary glands (SG), thereby inducing overt salivary gland hypertrophy (SGH) symptoms in their adult hosts. SGHV infection of non-SG tissues results in distinct pathobiologies, including reproductive dysfunctions in tsetse fly, Glossina pallidipes (Diptera: Glossinidae) and house fly. Infection with the G. pallidipes virus (GpSGHV) resulted in the collapse of several laboratory colonies, which hindered the implementation of area wide integrated pest management (AW-IPM) programs that had a sterile insect technique (SIT) component. Although the impact of GpSGHV infection has been studied in some detail in G. pallidipes, the impact of the virus infection on other tsetse species remains largely unknown. In the current study, we assessed the susceptibility of six Glossina species  to GpSGHV infections, and the impact of the viral infection on the fly pupation rate, adult emergence, and virus replication and transmission from the larval to adult stages. We also evaluated the ability of the virus to infect conspecific Glossina species through serial passages. The results indicate that the susceptibility of Glossina to GpSGHV varied widely amongst the tested species, with G. pallidipes and G. brevipalpis being the most susceptible and most refractory to the virus, respectively. Further, virus injection into the hemocoel of teneral flies led to increased viral copy number over time, while virus injection into the third instar larvae delayed adult eclosion. Except in G. pallidipes, virus injection either into the larvae or teneral adults did not induce any detectable SGH symptoms, although virus infections were PCR-detectable in the fly carcasses. Taken together, our results indicate that although GpSGHV may only cause minor damage in the mass-rearing of tsetse species other than G. pallidipes, preventive control measures are required to avoid viral contamination and transmission in the fly colonies, particularly in the facilities where multiple tsetse species are reared.Salivary gland hytrosaviruses (SGHVs, family  Glossina spp.) are responsible for transmission of African trypanosomoses, a group of anthropozoonotic neglected tropical diseases affecting humans and their livestock in most of sub-Saharan Africa  seriously hampers mass-production of G. pallidipes, a competent vector of several trypanosomes  that as a result become enlarged (SGH) (Musca domestica (infected with MdSGHV), and the phytophagous syrphid fly Merodon equestris  is the main route for MdSGHV infection, vertical transmission from mother to offspring seems to be the main route for GpSGHV infection in natural population (Members of the ed (SGH) . So far,questris . Unlike questris . The twoquestris . The obspulation .per os) and via vertical transmission from mother to offspring  but without either the onset of overt SGH symptoms or the release of detectable viral particles via fly saliva during in vitro membrane feeding  infection by feeding larvae of M. brassicae with Panolis flammea NPV, which is related to MabrNPV  were used to assess the induction of latent infection and the development of overt SGH symptoms in adults emerging from virus-injected third-instar larvae and in the F1 progeny produced by virus-injected mothers, and to assess the potential enhancement of latent SGHV infection in conspecific larvae of each of the Glossina species.In the current study, to investigate the host range of the GpSGHV and its impact on other tsetse species the susceptibility of six Glossina species  used in this study were obtained from colonies maintained at the Insect Pest Control Laboratory (IPCL) of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Seibersdorf, Austria. Unless otherwise stated, all experimental flies were fed on warm, defibrinated bovine blood for 10–15 min three times weekly, using an in vitro membrane feeding technique  and used to prepare the virus inoculum as described by G. pallidipes, SGs were dissected from 10-day old flies  that emerged from the larvae produced by mothers initially injected with the virus suspension derived from virus-infected G. pallidipes. Infection in the SGs was verified by the PCR protocol described by Salivary glands (SGs) with overt SGH symptoms were dissected from 1 progeny, teneral (non-fed) flies were immobilized (2–6°C for 5 min) 24 h post emergence, then maintained in a plastic Petri-dish on ice and inoculated in the thoracic cavity with either 2 μl of filter-sterilized phosphate buffered saline  or 2 μl of the virus suspension using a 1 ml Myjector U-40 Insulin type syringe . For each tsetse species, 40 male and 120 female flies were injected and placed in standard tsetse holding cages (20 cm diameter × 5 cm height) with a density of 80 flies per cage at a 1:3 mating ratio; 2–3 replications were carried out for each species. After the injections, 8 flies  were randomly sampled from each treatment at 0-, 1-, 3-, 5-, 7-, and 9-days post injection (dpi), and subsequently frozen at -20°C until further analyses.To monitor GpSGHV replication in adults, and the transmission of the virus from infected parents to their Fn = 112) were maintained under standard rearing conditions for 120 days and collected pupae were incubated until emergence of the F1 adults. All flies that remained alive at the end of the 120-day experimental period were dissected to assess for SGH symptoms. The F1 flies that emerged from the collected pupae were reared until they were 10 days old, after which 8 flies from each treatment were randomly selected and frozen at -20°C until further analyses. Total DNA of individual flies was extracted from the samples collected in the parental and F1 generations using the DNeasy tissue kit  following the manufacturer’s instructions. Viral copy numbers were estimated using pooled genomic DNA . For each DNA pool, the DNA concentration in each individual fly was determined using a spectrophotometer , followed by dilution to ensure that all individual samples contained equal final DNA concentrations. Then, 30 μl of each diluted DNA sample was pooled and used to quantify viral copy numbers by qPCR  that had emerged from larvae that were injected with GpSGHV and a virus suspension was prepared as described above. The virus suspension collected from each tsetse species, positive control , and negative control (PBS) were injected into third instar larvae of the same species. Larvae that pupated were maintained until adult emergence and adults were maintained for 10 days and then dissected to assess the SGH status and the SGs were tested by PCR as described above. The observed emergence rates in the GpSGHV or viremic SG homogenate injected larvae were corrected with Abbott’s formula  was determined by Tukey’s honestly significant difference (HSD) test. The analyses were done in R . The baseline viral copy number of the PBS-injected flies remained relatively stable over the 0–9 dpi period. Except in G. f. fuscipes, the viral copy number increased significantly in virus injected flies over the 0–9 dpi period for all tested Glossina species  . There was no significant difference in susceptibility to virus infection between female and male flies for all tsetse species  . Unlike in the other species, the viral copy number in G. brevipalpis increased between 0 to 5 dpi  . The virus copy number in G. pallidipes increased as of 5 dpi, , but in the case of G. m. centralis and G. m. morsitans, viral copy numbers only increased at 9 dpi. Comparing the overall increase in the viral copy number during the 0–9 dpi period of the six species, G. p. gambiensis males showed the highest increase in viral copy number, followed by G. m. morsitans males and G. p. gambiensis females. Despite this temporal increase in viral copy numbers, dissection of the SGs at the end of the experimental period (120 dpi) showed no evidence of overt SGH symptoms in any of the six Glossina species.The intra-hemocoelic virus injections into teneral female and male adults showed that all six tsetse species were susceptible to GpSGHV infection  . The highest and lowest virus-induced mortality was recorded in G. pallidipes (62% compared to 23% in the controls) , and in G. brevipalpis (23.8% compared to 4% in the controls) , respectively . The virus-induced mortality was similar in G. f. fuscipes, G. p. gambiensis and G. m. centralis . Injecting the virus in adult  flies significantly reduced  pupal production in G. f. fuscipes (p/f/10d) compared to non-injected control flies , but not in G. pallidipes , G. brevipalpis , G. p. gambiensis  and G. m. centralis . Virus injection significantly increased pupal production in G. m. morsitans , but this requires further investigation .Cumulative data over the 120-day experimental period revealed that injecting the virus in adults significantly increased mortality in both females and males compared with the untreated controls for all six F = 3028.7; P < < 0.001). Emergence of F1 descendants from pupae produced by virus-injected female parents was reduced significantly as compared with the PBS injected control flies across all six species  . The control groups showed high emergence rates (>75%) for all tested species, except for G. p. gambiensis (50%), which had lower emergence rates than the F1 adult emergence from virus-injected mothers of other species. Although virus injection in adult G. brevipalpis induced the lowest parental mortality rates as compared with other species , the injected virus had more impact on the emergence rate of the F1 progeny as compared with the untreated controls, i.e., 64.2% versus 95.2% in the control groups . Viral copy number was high enough to be measurable by qPCR in the F1 adults produced by the virus-injected G. pallidipes and G. m. morsitans flies, whereas only background viral copy numbers were detected in the control flies and in the progenies produced by virus-injected mothers of the other four tsetse species . There were no detectable SGH symptoms in any of the F1 progeny across all the species except G. pallidipes (41.9%).In general, a significant difference was observed in the adult emergence rate among different virus-injected tsetse species . The impact of virus injection on adult eclosion is presented in Table 1. Generally, adult emergence rates between the PBS and the virus injected larvae or between the different species injected with the virus were not significantly different . However, G. brevipalpis showed the largest difference in the average adult emergence rate between the PBS and virus-injected flies (74.7 and 24.4% respectively), whereas the differences in G. m. centralis and G. m. morsitans were smaller  (Table 1).The third instar larvae that successfully pupated after injection were incubated for adult emergence. Compared to the expected normal pupal period for Table 1). SGH symptoms were observed in 6.2 and 60.1% of adult G. pallidipes that developed from the PBS-injected and the virus-injected larvae, respectively. Dissection results for the other five species were negative for SGH symptoms. In addition to the fly dissections, PCR analyses were carried out on all dissected SGs and their corresponding carcasses to assess GpSGHV infections (Table 1). The frequency of virus infections detected by PCR of the SG homogenates of all species showed large variations and was significantly different  among different species with the highest virus infection rate in G. f. fuscipes and no virus detected in G. brevipalpis (Table 1). Virus was detectable by PCR in the fly carcasses of all the flies that emerged from the virus-injected larvae, but without overt SGH symptoms. The infections were generally higher in the carcasses than in the SG homogenates. For instance, whereas 100% of G. pallidipes carcasses were PCR- positive, only 60.1% of the dissected SGs homogenates were PCR positive for virus infection. Furthermore, 37.5% of the G. f. fuscipes carcasses of the adults that emerged from the PBS-injected larvae had detectable virus infections. Whether this result is a case of virus reactivation from a latent state requires further investigations.Salivary glands were dissected on day 10 post-emergence from males and females that had developed from PBS- and virus-injected larvae to assess the occurrence of the SGH symptoms , with the highest viral genome copy number recorded in G. pallidipes (∼105.9 copies) and the lowest in G. p. gambiensis (∼103.7 copies) . There were no detectable viral infections in the SGs dissected from 10-day-old G. brevipalpis that were used for the conspecific injections; G. brevipalpis was therefore not included in the conspecifics bioassays.We investigated the impact of GpSGHV infection on the induction of SGHV latent infection and the possibility of enhancing GpSGHV infection through passaging the virus in conspecific tsetse. For this we prepared SG homogenates dissected from 10-days-old adults that emerged from virus-injected larvae that were positive for virus by PCR, and re-injected the virus suspensions into conspecific third-instar larvae. The viral copy number in 10-days old adults emerged from virus injected larvae varied significantly among species , or between the emergence rate of the GpSGHV and conspecific injected groups  (Table 2).When the SG homogenates were re-injected into conspecific third instar larvae, the rates of adult emergence are presented in G. pallidipes flies that developed from larvae injected with virus homogenates prepared from hypertrophied SGs of G. pallidipes (Table 2). When the dissected SGs were subjected to conventional PCR, virus infections were detected in the glands dissected from flies injected with virus suspensions prepared from the hypertrophied SGs of G. pallidipes. Viral prevalence was highest (93.7%) in G. pallidipes and was lowest in G. m. centralis (9.2%) (Table 2). The difference in the viral prevalence amongst the tested species was significant  (Table 2). Following conspecific injection, virus infection was detectable only in the SGs dissected from G. m. centralis (2.3%). As it is possible that injected GpSGHV does not reach and replicate in fully differentiated tsetse SGs . The prevalence of virus infections varied significantly among the tested species  and was highest in G. pallidipes , but virus infections in the other species were also common; 66.6, 63.6, 50, and 28.5% in G. m. morsitans, G. m. centralis, G. p. gambiensis, and G. f. fuscipes, respectively. Following conspecific injection, virus infections were detectable in the carcasses of G. f. fuscipes (56.2%), G. m. morsitans (50%) and G. p. gambiensis (6.25%) (Table 2).When SGs were dissected from 10-day old flies, overt SGH symptoms were only detectable in etse SGs , we analG. pallidipes and not in the other tested species. Similar observations were made with the Musca domestica MdSGHV, which can infect stable flies  and black dump flies (Hydrotaea aenescens), but does not induce SGH symptoms in the heterologous hosts  that were virus-injected agrees with previous reports  from undifferentiated tissues to induce SGH symptoms  compared to the controls, the virus caused the greatest reduction in F1 adult emergence in this species. Virus infection could interfere with larval to pupal metamorphosis in several ways, including neuroendocrine regulation of hormonal synthesis, or transcriptional disruption of the expression of enzymes that are critical for metamorphosis. This has been demonstrated during virus infection in various Diptera such as the fruit fly, Drosophila melanogaster and the tobacco hornworm, Manduca sexta  will induce the expression of overt disease symptoms. This notwithstanding, the finding that all the Glossina species are susceptible to GpSGHV infection and that it reduces colony performance points to the need for the implementation of strict protocols to protect colonies from GpSGHV infection. We have already developed and implemented GpSGHV management protocols that are effective in the control of the virus in G. pallidipes colonies . Compared to injection, oral infection may significantly reduce the chances of productive virus infection. The implications of these facts are that since the intra-hemocoelic injection did not induce development of overt SGH in most of the colonies , 2014.GD-U, AP, HK, and AA-A conceived and designed the experiments and analyzed the data. GD-U performed the experiments. HK, GD-U, AA-A, AP, and MV wrote the paper. All the authors read and approved the final 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.