The exudative age-related macular degeneration (AMD) causes considerable healthcare costs for patients and healthcare system, which are expected to grow as the population ages. The objective of this study was to assess the incremental economic burden of exudative AMD by comparing total healthcare costs between the exudative AMD group and non-AMD group to understand economic burden related to exudative AMD.This retrospective cohort study used the National Health Insurance Service database including the entire Korean population. Exudative AMD group included individuals with at least one claim for ranibizumab and one claim using the registration code for exudative AMD (V201). Non-AMD group was defined as individuals without any claims regarding the diagnostic code of H35.3 or ranibizumab. The exudative AMD group and non-AMD group were matched using a propensity-score model. Incremental healthcare resource utilization and healthcare costs were measured during a one-year follow-up by employing econometric models: ordinary least squares (OLS) with log transformation and heteroscedastic retransformation; and generalized linear model (GLM) with a log link function and gamma distribution.P-value < 0.0001), while the length of hospitalization was shorter in exudative AMD group . Exudative AMD patients had total costs 2.13 times  greater than non-AMD group using OLS, and total costs 4.06 times  greater than non-AMD group using GLM. Annual incremental total costs were estimated as $5519 (OLS) and $3699 (GLM).A total of 7119 exudative AMD patients were matched to 7119 non-AMD patients. The number of outpatient visits was higher in the exudative AMD group (Exudative AMD was associated with significantly increased healthcare costs compared to the non-AMD group. Attention is needed to manage the socioeconomic burden of exudative AMD. Age-related macular degeneration (AMD) is a major cause of irreversible visual loss in the elderly, and can be classified as nonexudative or exudative . The majEarly intervention with anti-vascular endothelial growth factor (VEGF) therapy can reduce visual loss in patients with exudative AMD, whereas it causes considerable healthcare costs for patients and healthcare system due to its high price and needs for repetitive administration accompanied by frequent outpatient visits , 5. To eHowever, there is a paucity of data exploring economic burden of exudative AMD. A prospective, cross-sectional study conducted in Canada, France, Germany, Spain, and the UK estimated the annual societal cost per patient to be €5300–12,445 in 2005 . Among tTherefore, we aimed to compare the incremental burden, which encompasses all aspects of medical expenses, in the exudative AMD group and non-AMD reference group, whose set of comorbidities is similar to the exudative AMD group, by using a nationwide claims database covering the entire Korean population.We conducted a retrospective cohort study using data from the National Health Insurance Service (NHIS) database from 2006 through 2013, which was the total period of available dataset. The NHIS database includes records for the entire Korean population, since a single payer program covers the entire Korean population, about 50 million persons, through either the National Health Insurance system (97%) or Medical Aid (3%) . TherefoThe provided NHIS-customized database consisted of all patients with degeneration of macula having diagnostic code of H35.3 among the entire Korean population. Of these, we defined patients with incident exudative AMD when individuals with at least one claim for ranibizumab and one claim using the registration code for exudative AMD (V201) between August 1, 2010 and December 31, 2012. Korean registration program for rare intractable diseases was initiated in 2006 and covers 138 rare intractable diseases including exudative AMD for copayment reduction (up to 10%). To be registered for copayment reduction, ophthalmologist should confirm the diagnosis of exudative AMD according to the NHIS diagnostic criteria as follows: (1) a dilated fundus examination using indirect ophthalmoscopy, (2) optical coherence tomography, and (3) fluorescein angiography to confirm the exudative AMD-related macular pathologic features. The NHIS reviews the eligibility at the first registration and verifies the accuracy and reliability of the exudative AMD diagnosis based on the test results described above and doctor’s note. After registration, all exudative AMD-related claims contain the registration code V201 in addition to the diagnostic code for exudative AMD. Detailed information was reported elsewhere [We set the non-AMD group using the NHIS-NSC database. The non-AMD group was defined as individuals without any claims regarding the diagnostic code of H35.3 (‘degeneration of macula and posterior pole’), which includes H35.30 (‘nonexudative AMD’) and H35.31 (‘exudative AMD’) or regarding ranibizumab during the whole study period.Of these individuals in the both groups, we excluded individuals having a history or new diagnosis of malignant neoplasms (diagnostic codes of C00-C97) and stroke  during the entire study period, because these diseases generally involve enormous costs that can affect the healthcare costs of exudative AMD .P-values (≥0.05), which measure the differences between summary statistics of baseline covariates among two groups [c-statistic.We preliminarily selected 10 to 20 matching-candidates as the non-AMD group to each exudative AMD patient according to their age group. Each of the matching-candidates had to visit a healthcare facility on the same date with the index date of matched-exudative AMD patient. We assigned the same index dates to these matching-candidates as their matched-exudative AMD patient. Then, we computed the propensity scores in the exudative AMD group and their matching-candidates using a logistic regression model and included the following covariates: the index date; age group, sex, type of insurance, income level, residence and disability at the index date; Charlson Comorbidity Index (CCI) score, comorbidities , length of hospitalization, number of outpatient visits and number of emergency department visits during the 1 year before the index date. Square and interaction terms were also included. To calculate CCI score, all diagnostic codes during the 1 year before the index date were analyzed according to the Quan’s coding algorithms . Thereafo groups , 16. TheThe exudative AMD and non-AMD groups were followed for 1 year to measure healthcare resource utilization and healthcare costs. Annual healthcare resource utilization per patient was assessed including the total length of hospitalization, number of hospitalizations, number of outpatient visits, and number of emergency department visits. From the perspective of Korean payer, healthcare costs incorporated direct medical costs incurred by both the insurer and the patient. Using a gross-costing approach, annual healthcare cost per patient was calculated as the sum of all expenses incurred during the period from 3 months before the index date to 1 year after the index date. This analysis began 3 months prior to the index date so as to include pre-diagnosis costs, which occur with regard to a diagnosis of exudative AMD . Healthcare costs included all expenses identifiable in healthcare claims, such as outpatient visit, hospitalization, medication, medical devices, and laboratory test. We categorized healthcare costs into pre-diagnosis costs, AMD-related costs, ophthalmology costs, and non-ophthalmology costs. The total burden of exudative AMD for the Korean population was calculated according to the following formula:We projected the number of Korean patients with exudative AMD in 2014 using the direct standardization method based on the 2008–2012 Korean data estimated in the epidemiologic study for exudative AMD in Korea and the information of population structure provided by the Korean Statistical Information Service , 17. HeaWe compared baseline characteristics, including demographic and clinical characteristics, annual healthcare resource utilization, and annual healthcare costs between the exudative AMD and non-AMD groups. Continuous variables were tested using the paired t-test, while categorical variables were tested with McNemar’s test.As most cost data has a positively skewed distribution, it is difficult to obtain unbiased and precise estimates of incremental costs by calculating mean differences or running a simple ordinary least squares (OLS) . HistoriSAS version 9.4  and STATA SE 14.0 was used for statistical analysis. Institutional Review Board (IRB)/Ethics Committee approval was obtained by the Seoul National University Bundang Hospital (X-1710-429-907) and the study complied with the guidelines of the Declaration of Helsinki.P-values , while the total length of hospitalization was shorter in the exudative AMD group  during the follow-up period. Ophthalmology costs, especially AMD treatment costs, greatly contributed to the total healthcare costs in the exudative AMD group   higher in the exudative AMD group relative to the non-AMD group 95% CI 3.82–4.31). After retransformation, the annual incremental total cost per patient with exudative AMD was estimated as $5519 using the OLS model and $3699 using the GLM model, which were greater than the observed differences  * .This study found that patients with exudative AMD had significantly higher medical costs in comparison to carefully-matched non-AMD reference group in the era of anti-VEGF treatment. Understandably, treatment costs related to exudative AMD contributes the majority of the total healthcare costs in the exudative AMD group and, however, incremental ophthalmology-related cost observed in the exudative AMD group was partly compensated by incremental non-ophthalmology-related cost in the non-AMD group.Two studies reported the cost of exudative AMD before the era of anti-VEGF treatment. Cruess et al. reported that the annual incremental cost of a patient with bilateral exudative AMD relative to a control subject ranged from €2965 to €7887  in 2005 using the prospective data collected in five countries [In Korea, Jeong et al. previously estimated the annual costs per patient associated with exudative AMD to be $10,246 in 2014, which is 2–3 times higher than our estimates . The difConsidering that the general population also pays healthcare costs according to their own health needs, measuring costs without appropriate controls may exaggerate the true burden of the disease. By defining the control as the non-healthy population, with a similar severity of comorbidities to the patients with exudative AMD, we adopted a conservative approach in measuring the burden of exudative AMD. Moreover, we did not include uninsured treatments, which may have led to an underestimation of the burden of exudative AMD. In fact, optical coherence tomography (OCT) is one of the most important tests performed to assess pharmacological response and disease progress in patients with exudative AMD, but it was not insured until 2015 in Korea and was not considered in our analysis. Generally, OCT is performed at every AMD-related visit in Korea, costing about $50–$150 for each service.In most circumstances, cost data has some or all of the following properties: (1) nonnegative outcomes; (2) a nontrivial fraction of zero outcomes; and (3) positively skewed outcomes . If therTo our knowledge, this is the first study to explore the incremental economic burden of exudative AMD group relative to a non-AMD reference group using a nationwide database. We tried to minimize the effect of confounders by comparing the exudative AMD and non-AMD groups, which had similar distributions of baseline covariates after being matched by propensity score. Moreover, we used a previously developed algorithm to enable more elaborate methodology for cost analysis, addressing the widespread problem of skewed distributions in medical expenditure studies.A high economic burden of disease could be a threat to sustainability of healthcare system. Therefore, understanding the costs of disease is necessary to justify and prioritize healthcare policies, and can help decision makers find out an efficient way of allocating the limited resources. In this respect, this study may provide further evidence for the costs and resource utilization related with exudative AMD, which could be used for assessing impact of healthcare policies and cost-effectiveness of certain interventions.Several limitations of this study should be considered. Since we used claims data, uninsured examinations (e.g. OCT and fundus indocyanine green angiography), the use of treatments for an unapproved indication, or over-the-counter agents were not included in the cost analysis. These facts might have led to an underestimation of exudative AMD treatment costs and the incremental total costs of exudative AMD. Further, exudative AMD was defined based on diagnosis and ranibizumab codes, which may not have been specific enough to prevent any misclassifications in the study population. However, given that most patients are treated with ranibizumab once they are diagnosed with exudative AMD, this misclassification issue is likely to be negligible. Since ranibizumab began to be covered in 2009, the reimbursement criteria have changed to increase the maximum number of ranibizumab injections allowable per year. While this change may affect the utilization pattern of ranibizumab, we were unable to address this detail in this study. Further studies are warranted to explore the economic burden of exudative AMD in relation to change in reimbursement criteria by using more recent data. Finally, exudative AMD can cause an economic burden beyond medical costs. Therefore, non-medical costs, such as productivity loss, should be explored in future studies.Exudative AMD was associated with significantly increased healthcare costs relative to those of a non-AMD group with similar demographic and clinical characteristics. A GLM analysis showed that patients with exudative AMD are estimated to cost an additional $3699 annually. Since the prevalence of exudative AMD is expected to grow continuously, policy actions are needed to manage the socioeconomic burden of exudative AMD.Additional file 1: Table S1. Baseline characteristics of study population before propensity score matching. Summary of baseline characteristics of exudative AMD group and non-AMD matching-candidates before propensity score matching.


Equus caballus papillomavirus 9 (EcPV9). The complete 7656 bp genome of EcPV9 exhibited similar characteristics to those of other horse papillomaviruses. Phylogenetic analysis based on concatenated E1-E2-L2-L1 amino acid sequences revealed that EcPV9 clustered with EcPV2, EcPV4, and EcPV5, although was distinct enough to represent a new viral species within the genus Dyoiotapapillomavirus . In sum, we demonstrate the presence of a novel equine papillomavirus for which more detailed studies of disease association are merited.Papillomaviruses (PVs) have been identified in a wide range of animal species and are associated with a variety of disease syndromes including classical papillomatosis, aural plaques, and genital papillomas. In horses, 13 PVs have been described to date, falling into six genera. Using total RNA sequencing (meta-transcriptomics) we identified a novel equine papillomavirus in semen taken from a thoroughbred stallion suffering a genital lesion, which was confirmed by nested RT-PCR. We designate this novel virus  Bos taurus papillomavirus 1 (BPV1) [2 (BPV2) [13 (BPV13) [Equus caballus papillomaviruses 1–8 (EcPV1–8) [Equus asinus papillomaviruses 1–2 (EaPV1–2) [Equine papillomaviruses are small, non-enveloped viruses that comprise a circular and double-stranded DNA genome of up to approximately 8 kb in length. These viruses are associated with a variety of equine diseases including classical viral papillomatosis, genital papillomatosis, and aural and genital plaques. To date, 13 species of papillomavirus have been documented to infect the Equidae (horses and donkeys): EcPV1–8) ,5,6,7,8,EaPV1–2) . These vDuring the 2018 southern hemisphere serving season, the stallion experienced difficulty covering mares, primarily manifest as apparent pain on ejaculation. A wart-like lesion, 1 cm in circumference, was observed at the tip of the penis consistent with a genital papillomavirus lesion . FurtherTwo sets of urine and semen samples were collected from the stallion for microbiological investigation, one set placed into a standard specimen container and the other stored in RNA later. Because the nature of any causative pathogen was unknown, we employed a meta-transcriptomic approach as this is able to detect any microbial species  as long as sufficient expressed RNA is present. Accordingly, total RNA was extracted using the RNeasy plus universal kit , with RNA sequencing libraries then constructed with the SMARTer Stranded total RNA-seq kit . RNA sequencing of 100 bp pair-end libraries on the Illumina NovaSeq platform yielded 84.54 Gb of data . All seq−10 and 1 × 10−4 [de novo assembled using Megahit v1.1 [RNA sequencing reads were quality trimmed and horse reads were subsequently removed by mapping to the horse genome. To identify potential viral transcripts, non-horse reads from each library were compared against the non-redundant nucleotide (nt) and non-redundant protein (nr) databases using Blastn and Diamond blastx, respectively, with e-value thresholds of 1 × 10hit v1.1 ,12. Viruhit v1.1 , followehttps://www.ncbi.nlm.nih.gov/orffinder/). A conserved domain search  revealed that the L1 protein of the new virus exhibited the highest nucleotide and amino acid identities with EcPV2, at 69.35% and 70.44%, respectively, indicative of a novel papillomavirus. A novel Equus caballus papillomavirus 8 (EcPV8) associated with viral plaques, viral papillomas, and squamous cell carcinoma has been recently described [Equus caballus papillomavirus 9 , in accordance with current guidelines for the classification of papillomaviruses [A 7605 bp genome sequence of a papilloma-like virus was identified in one semen library. Prediction of open reading frames (ORFs) was performed using the ORF Finder tool at NCBI  and two late (L1 and L2) proteins, consistent with other PVs . The preTwo zinc-binding domain(s) (CXXC-X29-CXXC) were found in E6  and one in E7 , separated by 29 amino acids . No PDZ We next performed pairwise alignments based on both nucleotide (nt) and amino acid (aa) sequences for the seven viral ORFs. In the case of L1, which is currently used for classification, EcPV9 shared 69.35%, 59.25%, and 58.00% nucleotide similarity with EcPV2, EcPV4, and EcPV5, respectively . For E1,Dyoiota PVs—EcPV2, EcPV4, and EcPV5 . Amino acid sequences (concatenated E1-E2-L2-L1) of 13 equine PVs, as well as the type species of each of the 52 PV genera, were aligned using the E-INS-I algorithm in the MAFFT v7 package . A phylond EcPV5 . Hence, As no biopsy samples could be taken from this case, it is not possible to confidently determine its significance in the observed pathologies. Nevertheless, the novel EcPV described here was extracted from semen samples, collected when a wart-like lesion was visible on the tip of the penis , and henDyoiotapapillomavirus) in a thoroughbred Australian stallion suffering a genital papilloma (“wart”), highlighting the broad diversity of these viruses in horses. Further investigation of the clinical impact of this virus on horse health is clearly merited.In conclusion, we report the identification of a novel equine papillomavirus (genus


Experimental infections of insects with arboviruses are performed to achieve a variety of objectives but principally to draw inferences about the potential role of field populations in transmission or to explore the molecular basis of vector–pathogen interactions. The design of such studies determines both their reproducibility and the extent to which their results can be extrapolated to natural environments, and is constrained by the resources available. We discuss recent findings regarding the effects of nutrition, the microbiome, co-infecting agents and feeding methods on the outcome of such experiments, and identify resource-efficient ways to increase their relevance and reproducibility, including the development of community standards for reporting such studies and better standards for cell line and colony authentication. Current Opinion in Insect Science 2018, 28:105–112Vectors and medical and veterinary entomologyThis review comes from a themed issue on Jason L RasgonEdited by Issue and the EditorialFor a complete overview see the Available online 23rd May 2018https://doi.org/10.1016/j.cois.2018.05.007http://creativecommons.org/licenses/by/4.0/).1877-3435/© 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license , Rift Valley fever virus (RVFV) and bluetongue virus (BTV). The proportion of individuals in a population that develop a transmissible infection in response to exposure to an arbovirus is defined as its vector competence. Experimental infections of insects with arboviruses are mostly performed either to draw inferences about the potential role of field populations in transmission, which include but are not limited to their vector competence , or to rategies . Less corategies  and, incrategies  or via cbacteria .Reproducibility is the basis of the scientific method . DespiteThe critical factors determining the outcomes of experimental arbovirus-vector infection studies fall into four broad categories: first, the source of the vector population studied, second, how the vectors are maintained before and after infection, third, the method used to infect the vector, and fourth, how infection in the vector is characterised. These four stages are illustrated in Laboratory insect–arbovirus interaction studies generally use either colony-reared individuals ,13 or fiCulicoides-borne viruses in Northern Europe, where the females of some vector species are morphologically cryptic [Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle [Culicoides genus only two species are continuously maintained in colony globally: the North American BTV vector Culicoides sonorensis Wirth and Jones, and the European species Culicoides nubeculosus (Meigen) [Culicoides have been unsuccessful to date [Culicoides sonorensis is absent, are based largely or solely on data obtained from infection studies conducted on a North American vector species. If obtaining isolates of relevant virus strains is problematic, alternative virus strains may also be used, potentially introducing similar issues.As noted above, the increased potential for reproducibility afforded by the use of established insect colony lines may also come at the expense of reflecting the epidemiologically relevant diversity present in field populations. Natural ‘populations’ may be highly genetically heterogeneous, and even represent an assemblage of multiple cryptic species , 26, 27. cryptic  (e.g. Cud Kettle ), confoud Kettle ,30. The d Kettle , 32, 33.(Meigen) , which i to date . As a coR0 [Aedes and Anopheles mosquitoes as larvae have been shown to affect not only their rate of development but also their permissiveness to infection by arboviruses [Plasmodium [Leishmania infection studies [Studies can also be significantly affected by the methods used for insect maintenance, both before and after arbovirus exposure. The most important factors falling within this category include diet and temperature. The latter is the critical determinant of the duration of the extrinsic incubation period, and even small changes in temperature can have large consequences for R0  but expoR0 ,38. The oviruses  and Plasasmodium  respectiasmodium , which casmodium . After easmodium ••, a fin studies .Other potential modulators of vector competence include the vector microbiome and virome, which may vary significantly between field and colony populations. However, co-infecting agents such as insect-specific viruses may persist cryptically within colonies for extended periods ,46 leadi® system   ) or via Culicoides [To permit manipulation and prevent clotting, blood is normally defibrinated or mixed with an anti-coagulant such as heparin or EDTA. The influence of anti-coagulant on the outcome of vector–virus interactions is largely unknown, but some commonly used anticoagulants have been shown to significantly affect vector survival . The doslicoides ), they alicoides .Culicoides sonorensis [Orbivirus BTV has been shown to increase the infectivity of virus particles for insect cells [Other potential sources of variation include the origin of blood used in infection experiments. The species from which blood is sourced has been shown to strongly affect the outcome of vector–virus interactions, such as those between African horse sickness virus and norensis  via the ct cells . Other hct cells . The prect cells , howeverLow feeding rates for some vectors, particularly field-caught vectors, coupled with the low dissemination rates described above, make the design of studies guaranteeing large numbers of insects developing transmissible infections  challenging. One solution often used is intrathoracic inoculation, in which virus is introduced directly into the thorax of the vector. The mortality associated with such a method is typically outweighed by the quantitative benefit of avoiding the problems of low feeding rates and highly effective midgut infection and escape barriers ,65•, andet al. [C. sonorensis [The outcome of vector infection studies may be significantly affected by the virus source. When viral particles in the bloodmeal originate from cell culture, both the cell line used to propagate the virus and any secondary processing such as purification may impose selective pressures on the virus population, leading to significant effects on the genetic diversity of viral particles present in the bloodmeal. For example, Caporale et al.  found panorensis ). A siminorensis .In addition to the vectors microbiome and virome, one last factor to consider within experimental design is the presence of co-infecting viruses, parasites and other agents within the infectious bloodmeal. Evidence to date indicates that the presence of parasites such as microfilarial nematodes is likely to be of greater importance than the presence of other arboviruses in a bloodmeal, with studies of simultaneous co-ingestion of multiple arboviruses resulting in similar levels of infection, dissemination and transmission to those seen during single-virus studies , suggestIssues with inferring the infection status of vectors following exposure fall into two broad categories. The first is the decision to detect or quantify infectious virus or viral genome. The latter may detect not only infectious viral particles but also defective virus particles or RNA fragments, but the higher sensitivity of qPCR and related techniques may allow the detection of potentially transmissible infections which are below the detection threshold for cell culture based techniques . In addiCulicoides (body-size 1–3 mm), or to use a proxy such as the presence of virus in the salivary glands [The second is whether to collect saliva, which is time-consuming and problematic especially for small vector species, for example, y glands , in othey glands  or legs y glands ,80 assumy glands  or poolsy glands .The choice of techniques used in insect-infection studies is necessarily a balance between reproducibility, relevance and practicality , particuTo increase confidence in the findings of such studies, we make several recommendations. Firstly, we recommend the publication of open-access protocols and raw data. This is increasingly required by publishers and facilitated by the availability of open access data repositories. Secondly, we recommend the routine screening, regular authentication and characterisation of cell lines, virus isolates and insect colonies used in insect infection studies, and reporting of these findings in relevant publications. Thirdly, we advocate more consistent measurement and reporting of factors which are known to strongly influence the outcome of insect exposure to virus. Given the volume of publications in the field, we suggest the most realistic way to ensure this last recommendation would be via the development, including regular review, by the vector community of reporting standards similar to those developed for quantitative PCR . HoweverNothing declared.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as


Borrelia burgdorferisensu lato, in Australian ticks, the etiological basis of this disease syndrome remains controversial. To characterize the viruses associated with Australian ticks, particularly those that might be associated with mammalian infection, we performed unbiased RNA sequencing on 146 ticks collected across two locations along the coast of New South Wales, Australia. This revealed 19 novel RNA viruses from a diverse set of families. Notably, three of these viruses clustered with known mammalian viruses, including a novel coltivirus that was related to the human pathogen Colorado tick fever virus.Each year a growing number of individuals along the east coast of Australia experience debilitating disease following tick bites. As there is no evidence for the presence of the causative agent of Lyme disease,  Borrelia burgdorferisensu lato, are present in Australia. The diversity of bacteria infecting Australian ticks has been studied using both culture- and metagenomics-based techniques. However, little is known about the virome of Australian ticks, including whether this includes viruses with the potential to infect mammals. We used a meta-transcriptomics approach to reveal the diversity and evolution of viruses from Australian ticks collected from two locations on the central east coast of Australia, including metropolitan Sydney. From this we identified 19 novel RNA viruses belonging to 12 families, as well as 1 previously described RNA virus. The majority of these viruses were related to arthropod-associated viruses, suggesting that they do not utilize mammalian hosts. However, two novel viruses discovered in ticks feeding on bandicoot marsupials clustered closely within the mammal-associated hepacivirus and pestivirus groups (family Flaviviridae). Another bandicoot tick yielded a novel coltivirus (family Reoviridae), a group of largely tick-associated viruses containing the known human pathogen Colorado tick fever virus and its relative, Eyach virus. Importantly, our transcriptomic data provided no evidence for the presence of B. burgdorferisensu lato in any tick sample, providing further evidence against the presence of Lyme disease in Australia. In sum, this study reveals that Australian ticks harbor a diverse virome, including some viruses that merit additional screening in the context of emerging infectious disease.Understanding the microbiome of ticks in Australia is of considerable interest given the ongoing debate over whether Lyme disease and its causative agent, the bacterium IMPORTANCE Each year a growing number of individuals along the east coast of Australia experience debilitating disease following tick bites. As there is no evidence for the presence of the causative agent of Lyme disease, Borrelia burgdorferisensu lato, in Australian ticks, the etiological basis of this disease syndrome remains controversial. To characterize the viruses associated with Australian ticks, particularly those that might be associated with mammalian infection, we performed unbiased RNA sequencing on 146 ticks collected across two locations along the coast of New South Wales, Australia. This revealed 19 novel RNA viruses from a diverse set of families. Notably, three of these viruses clustered with known mammalian viruses, including a novel coltivirus that was related to the human pathogen Colorado tick fever virus. Ticks are one of the most important vectors of infectious disease in humans, wildlife, and domestic animals , in partBorrelia burgdorferisensu lato, is not present in Australian ticks  following experimental infection  is a powerful tool for the discovery of novel viruses and other microbial pathogens from both host- and tick-derived samples , 23–25. 23–(This article was submitted to an online preprint archive .)Ixodes holocyclus, Ixodes trichosuri, and Amblyomma moreliae , negative-sense single-stranded RNA (−ssRNA), and double-stranded RNA (dsRNA), segmented, and nonsegmented. It is unlikely that any of these viruses are endogenous viral elements present within the tick genome, as they were identified by open reading frames (ORFs) unbroken by stop codons, and none of the nucleotide sequences showed similarity to tick gene sequences within the NCBI BLAST database. All novel viruses identified in this study were named after locations and landmarks within the tick collection areas.Overall, the complete coding sequences of 16 viral genomes were identified within the tick data set collected here using a series of BLAST searches based on protein sequence similarity. An additional four partial viral genomes were also identified. Of these 20 complete or partial genomes, 19 contained previously undescribed viruses, although all fell within known viral families or orders, specifically: The housekeeping gene COX1 was used to measure the abundance of the invertebrate  transcripts in each library. Accordingly, the COX1 abundance ranged from 0.15% to 1.1% of the total number of reads  and the Picornaviridae (+ssRNA) were the most abundant, while viruses from the Flaviviridae (+ssRNA) were the least abundant. In libraries containing multiple viruses, the abundance of each species varied substantially. For example, in library 1, Manly virus represented 54% of the total number of virus reads, while Quarantine Head virus represented only 0.9% of the total virus reads in that library  and native bush rats (Rattus fuscipes)  . Similarndicoots .Luteoviridae. While Blue Fish Point virus, a bisegmented virus with 4 ORFs, was most closely related to mosquito-associated viruses, Store Beach virus appeared to comprise a single segment with two ORFs that grouped closely with other Ixodes-associated viruses . Th. Th23).  viruses . These ves ticks . Finallyes ticks  (23). A Viruses showing similarity to the narna-like virus group were identified only in ticks collected in southern NSW (Timbillica), and all three viruses were extremely divergent from previously described viruses. The amino acid sequence similarity to the most closely related virus species ranged from 24 to 32% for these three viruses across the RNA-dependent RNA polymerase (RdRp) region. The genomes of these viruses comprised a single genome segment with one predicted ORF containing an RdRp-like region, typical of viruses within this group, which have no known structural proteins . CompletI. holocyclus ticks using a viral RNA isolation method, was present in five libraries (libraries 4 to 8). This was the only previously described virus found in any of the 11 data sets generated here. This virus was identified only in I. holocyclus ticks collected in the Sydney region.The previously identified Ixodes holocyclus iflavirus , found iI. holocyclus ticks from the Sydney region  and Eyach virus  in Japan and the blood of free-tailed bats  in Côte d’lvoire . The most closely related viruses were also tick associated and were isolated from ticks in Norway and North America (Ixodes ticks rather than with Albatross Island virus (previously known as Hunter Island virus), identified in avian ticks collected from albatross in Tasmania , 34PhenuTasmania . HoweverTasmania . This coTasmania . InteresTasmania .Mononegavirales. Specifically, we identified two novel viruses clustering within the recently described Chuviridae family of arthropod-associated viruses  was present in library 1 . Howevertermedia . FinallyB. burgdorferisensu lato, the complex of bacterial species causing Lyme disease in parts of Europe and North America. Although a number of bacterial, fungal, and eukaryotic species were identified within our data, no B. burgdorferisensu lato bacteria were detected. This is consistent with the findings of recent bacterial 16S rRNA-based metagenomic studies performed with larger samples of Australian ticks , which currently comprises four virus species, two of which have been implicated in human tick-associated disease: Colorado tick fever virus and Eyach virus in North America and Europe, respectively  to Heartland virus that has been implicated in human disease in North America  in northwestern Tasmania, Australia  and by the Australian National University Animal Ethics Committee for the southern brown bandicoot sampled at the Timbillica state forests (A2015/26). Samples were collected under a scientific license provided by the NSW Office of Environment and Heritage (number SL100104).Perameles nasuta) in the north (Sydney) and southern brown bandicoots (Isoodon obesulus) in the south (Timbillica). As well as bandicoots, ticks were collected from the eastern blue-tongued lizard (Tiliqua scincoides scincoides) and both native Australian bush rats (Rattus fuscipes) and invasive black rats (Rattus rattus) rats unintentionally caught in treadle cage traps. Unfed I. holocyclus nymphs were collected at Katandra Bushland Sanctuary  by flagging during five collection trips between May and September 2017. All the collected ticks were taken to the laboratory and identified using tick taxonomic keys  and Timbillica state forests  south of Eden, NSW . Specifimic keys .Ticks were washed in 1× Dulbecco’s phosphate-buffered saline (DPBS) solution and homogenized in 800 μl lysis buffer using a TissueRuptor homogenizer (Qiagen). Total RNA was then extracted using an RNeasy Plus minikit (Qiagen) according to the manufacturer’s instructions. RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies). Samples were pooled for sequencing based on criteria including tick species, tick stage, sampling location, and, where possible, the tick host species . Host rRde novo assembled using Trinity (v.2.1.1) software (https://web.expasy.org/translate/). The predicted ORF structure of the contig was then compared to that of the genome of the closest BLAST hit to determine if the contig was potentially an endogenous virus element rather than a true exogenous virus.Sequencing reads were trimmed for quality using a Trimmomatic trimmer and software . The asssoftware , and conc oxidase subunit I (COX1) to confirm the species identification of the ticks in each library. To assess the bacterial, fungal, and single-celled eukaryotic composition of each library, including the presence of B. burgdorferisensu lato bacteria, all contigs showing sequence similarity to the sequences of mammalian or arthropod proteins were excluded from the Diamond BLAST results. The remaining hits were then filtered to include only those contigs showing more than 90% protein sequence similarity over a length of more than 200 amino acids with an E value of less than 1e−20. All results showing homology to ribosomal protein sequences, elongation factor 1 alpha, elongation factor Tu, or cytochrome c oxidase subunit 1 were then extracted, and the top BLAST hit was recorded.The assembled contigs were subjected to BLASTN and BLASTX (v.2.6.0)  searchesRibosomal reads were identified by selecting all contigs showing nucleotide sequence similarity of over 90% across a length of over 200 nucleotides and with an E value of less than 1e−20 to ribosomal reads within the NCBI nt database using a BLASTN (v.2.6.0)  search. To determine the evolutionary history of each virus, the RNA-dependent RNA polymerase (RdRp) or complete polyprotein of each virus was compared to previously described proteins from the relevant virus family. Accordingly, RdRp or polyprotein sequences representing the family of each virus discovered here were retrieved from the NCBI RefSeq database. The Mafft (v.7.3.0.0) program  was thenMK026564 to MK026605. All sequence reads are available in the BioProject database under BioProject accession number PRJNA494273.All consensus virus sequences produced as part of this study have been submitted to GenBank and assigned accession numbers


Hypertension in low‐ and middle‐income countries, including Kenya, is of economic importance due to its increasing prevalence and its potential to present an economic burden to households. In this study, we examined the patient costs associated with obtaining care for hypertension in public health care facilities in Kenya.We conducted a cross‐sectional study among adult respondents above 18 years of age, with at least 6 months of treatment in two counties. A total of 212 patients seeking hypertension care at five public facilities were interviewed, and information on care seeking and the associated costs was obtained. We computed both annual direct and indirect costs borne by these patients.Overall, the mean annual direct cost to patients was US$ 304.8 . Medicines , transport , and user charges  were the highest direct cost categories. Overall mean annual indirect cost was US$ 171.7 . The incidence of catastrophic health care costs was 43.3%  and increased to 59.0%  when transport costs were included.Hypertensive patients incur substantial direct and indirect costs. High rates of catastrophic costs illustrate the urgency of improving financial risk protection for these patients and strengthening primary care to ensure affordability of hypertension care. Kilifi is located on the coast of Kenya, and a high burden of stroke and heart failure has been described in this area.Six public health care facilities were purposively selected in consultation with county health officials to generate a sample of facilities with different workloads, plus the location of the clinics relative to the communities served. However, due to the nationwide nurses' strike at the time of data collection,2.2α/2Zis the critical value of the normal distribution at α/2 βZis the critical value of the normal distribution at β Pis the expected true proportion of hypertension in the population in Kenya of 15% (0.15)eis the desired standard size of standard error around the estimated proportion of 6% (+/− 0.06).The target enrolment was 278 patients for a sample size sufficient to obtain a precise estimate of hypertension patient costs based on the formulae by KirkwoodEvery hypertensive patient receiving treatment and available at participating facilities during data collection was approached to participate in this study. Patients were eligible if they self‐reported hypertension diagnosis, had received treatment for a minimum of 6 months after diagnosis, and were more than 18 years of age. Consenting patients were selected based on meeting the eligibility criteria and the order of arrival at the clinic. Respondents were asked to report on their health service use, associated costs, income, and coping mechanisms if they undertook any of the following to meet hypertension care costs: borrowing (having taken a loan), selling household items or assets , and use of savings.2.3Interviews were conducted using a structured questionnaire. Three trained interviewers collected the data. Interviews were conducted primarily in Kiswahili, with local languages  used to clarify questions where necessary. Respondents were asked about costs incurred for different care seeking episodes described in Table To annuitize sick visit costs, we summed up costs incurred during current care visit and any reported outpatient visit costs that occurred due to hypertension in the last 4 weeks and then multiplied by 13 (assuming there are 52 weeks in a year). On the other hand, to annuitize costs in other care seeking episodes described in Table For each of the care seeking episode, two broad costs categories were estimated: direct OOP costs and indirect (productivity losses) costs for both patients and their caregivers. Direct health care costs included any charges levied for medicines and user fees, ie, registration, consultation, and laboratory services. Direct non–health care costs included transport costs to and from a health provider and any costs associated with food and accommodation while seeking care. For this descriptive analysis, OOP costs were defined as the sum of direct health care and direct non–health care costs. Analysis was restricted to patients who reported any OOP costs for each care seeking episode.Indirect costs were estimated based on the total hours lost while seeking care as well as the cost of illness‐related to lost home productivity for both patients and their caregivers, assuming that these hours would have been used for productive activity in the absence of hypertension.Income was estimated by asking detailed questions about income categories, including patient income, income for household members, welfare payments, and government assistance. As a measure of financial risk protection, we compared total direct costs incurred against annual household income and total direct costs excluding transport costs and defined costs as catastrophic if they exceeded 10% of household income.2.4http://www.oanda.com/currency/converter/).Data were double‐entered by two different individuals to enhance data quality. The two data sets were compared to eliminate data entry errors. Consistency and range checks were used to ensure completeness of data. Data were analysed using STATA 14.0 . Frequency counts and percentages were used to describe patient demographic and socio‐economic variables. Due to skewed nature of costs data, mean and median values were reported for all cost estimates as a measure of central tendency, and 95% confidence intervals (CI) and interquartile range (IQR) were reported. Cost information  from Kilifi. Less than half of the sampled patients had secondary level education, were formally employed, or were subscribed to a health insurance scheme (Table 3.23.2.1Thirty‐six percent of the patients sought care outside of their scheduled clinic appointments. Transport costs during a sick visit represented the highest cost category (42.2%) of all direct OOP costs. Patients incurred substantial indirect costs  for such outpatient sick visits . Of the total direct costs, user charges—especially during an inpatient admission—were a substantial cost compared with other care seeking episodes, with respondents spending an average of US$ 50.8 per annum. This was followed by food and/or accommodation costs  . Diagnostic costs were likely low given that blood pressure and weight are checked for free at public health care facilities. Other reported tests were blood pressure  and urinalysis and echocardiogram . The mean annual direct cost for seeking diagnostic or laboratory test services was US$ 31.8  with transport costs accounting for 74.2% of total OOP costs  of the respondents attended their routine clinics monthly. The highest direct cost category during scheduled clinic appointments was medicines accounting for 45.8% of total OOP costs followed by transport 34%), user charges (17.9%), and food (2.3%) . Under direct costs, medicines  attracted the largest share of costs (42.4%) followed by transport , which represented 38.4% of total patient costs. User charges , food , and accommodation  accounted for 16.7%, 2.2%, and 0.3% of total direct costs, respectively  of the households if all direct costs were considered. Alternatively, 43.3%  of households experienced catastrophe if transport costs were excluded. Among respondents experiencing catastrophic costs, the poorest group of patients incurred higher direct costs with fewer resources to pay for it  working days. Forty‐two percent of respondents reported disrupted social life due to hypertension, affecting sexual life (n = 27), job loss (n = 46), divorce (n = 5), and separation from spouse (n = 10).4This study has documented patient costs for adults with hypertension that sought care in five public health care facilities in Kenya. Our findings show that patients cost for hypertension are driven by medicines. This is particularly worrisome given that unaffordability of hypertensive medicines is a likely cause of uncontrolled hypertension.Patients reported a median time of 30 minutes to access the clinics either by public means of transport or by walking; the cost of travelling to the medical clinics for different care seeking episodes was found to be a significant driver of patient costs. For instance, while the incidence of catastrophic health care costs was 43.3% , it increased to 59.0%  when transport costs were included. This could have negative access effects especially on poor patients as high transport costs and long distance to health facilities have been shown to hinder the poor from benefiting in pro‐poor health financing reforms in Kenya. A high number of patients (36%) reported a sick visit out of their scheduled clinic appointment. This indicates that poor management of hypertension impacts both the health and the economic burden for patients as evidenced by high costs on transport  and medication . In addition, high user charges seen in all care seeking episodes, particularly during an inpatient admission, are potential barriers to care seeking given that the negative impacts of user charges in low resource settings, which have been well documented.Patients had to sell their property in order to meet associated costs of hypertension management in keeping with other assessment of the impact of health expenditures on households.High indirect costs (in terms of waiting time and the resultant productivity hours lost) were reported in this study during care seeking episodes. This is common in public health facilities and has been identified as a major disincentive of seeking care in public facilities among employed hypertensive patients in South Africa.4.1This study had several limitations. First, the study was not nationally representative as it was carried out in five health facilities in two rural counties. Secondly, the use of an official minimum wage to estimate productivity loss for all patients could have potentially overestimated indirect costs among patients who were unemployed prior to their illness or underestimated indirect costs among those who were employed.5The study demonstrates that hypertension places a considerable economic burden on patients in Kenya. As Kenya reforms its health system to prioritize the attainment of UHC, our results suggest that there is need for interventions to provide financial risk protection to individuals with a chronic disease such as hypertension. Given that medicines are a key cost driver for patient out‐of‐pocket costs, one approach would be to explicitly include hypertension medicines in the universal health care benefit package that Kenyan citizens are entitled to.The scientific and ethics review unit at Kenya Medical Research Institute (KEMRI/SERU/CGMR‐C/041/3270) granted ethical approval for the study. County department of health officials and facility managers in respective counties provided permission to conduct the study in the selected facilities, and written informed consent was obtained from respondents.All the data for this study are available, and it can be accessed at the KEMRI‐Wellcome Trust Research Programme, subject to institutional data governance committee policies.Edwine Barasa, Anthony Etyang, Kenneth Munge, Jane Mbui, Zipporah Bukania, Fredrick Kirui, and Andrew Obala conceived the study. Robinson Oyando, Martin Njoroge, Antipa Sigilai, Kenneth Munge, Peter Nguhiu, and Edwine Barasa contributed to the development of data collection tools. Robinson Oyando collected the data. Robinson Oyando and Edwine Barasa analysed the data. Robinson Oyando developed the first draft of the manuscript. All authors contributed to writing subsequent versions of the manuscript


Zika virus infection during pregnancy causes serious birth defects and might be associated with neurodevelopmental abnormalities in children. Early identification of and intervention for neurodevelopmental problems can improve cognitive, social, and behavioral functioning.Pregnancies with laboratory evidence of confirmed or possible Zika virus infection and infants resulting from these pregnancies are included in the U.S. Zika Pregnancy and Infant Registry (USZPIR) and followed through active surveillance methods. This report includes data on children aged ≥1 year born in U.S. territories and freely associated states. Receipt of reported follow-up care was assessed, and data were reviewed to identify Zika-associated birth defects and neurodevelopmental abnormalities possibly associated with congenital Zika virus infection.Among 1,450 children of mothers with laboratory evidence of confirmed or possible Zika virus infection during pregnancy and with reported follow-up care, 76% had developmental screening or evaluation, 60% had postnatal neuroimaging, 48% had automated auditory brainstem response-based hearing screen or evaluation, and 36% had an ophthalmologic evaluation. Among evaluated children, 6% had at least one Zika-associated birth defect identified, 9% had at least one neurodevelopmental abnormality possibly associated with congenital Zika virus infection identified, and 1% had both.One in seven evaluated children had a Zika-associated birth defect, a neurodevelopmental abnormality possibly associated with congenital Zika virus infection, or both reported to the USZPIR. Given that most children did not have evidence of all recommended evaluations, additional anomalies might not have been identified. Careful monitoring and evaluation of children born to mothers with evidence of Zika virus infection during pregnancy is essential for ensuring early detection of possible disabilities and early referral to intervention services. Zika virus infection during pregnancy can cause serious birth defects, including structural abnormalities of the brain and eye . The USZPIR currently monitors outcomes of approximately 7,300 pregnancies, over 4,800 of which are reported from the U.S. territories and freely associated stateshttps://www.cdc.gov/pregnancy/zika/data/pregwomen-uscases.html). This report is the first to provide data on Zika-associated birth defects and neurodevelopmental abnormalities possibly associated with congenital Zika virus infection identified during infancy and early childhood among children aged ≥1 year who were born in the U.S. territories and freely associated states.To better understand the effects of Zika virus infection during pregnancy on mothers and children from a national surveillance perspective, CDC collaborated with state, territorial, and local health departments on the U.S. Zika Pregnancy and Infant Registry (USZPIR)https://www.cdc.gov/pregnancy/zika/research/lhdi.html) and the Epidemiology and Laboratory Capacity for Infectious Diseases Cooperative Agreement (https://www.cdc.gov/ncezid/dpei/epidemiology-laboratory-capacity.html). This report includes children who, among pregnancies reported to the USZPIR, 1) were born in U.S. territories or freely associated states; 2) had a date of birth on or before February 1, 2017, and reached age 1 year on or before February 1, 2018; and 3) had follow-up care reported to the USZPIR by June 1, 2018. For the purpose of this analysis, follow-up care was defined as clinical care at age >14 days reported to the USZPIR. Children from multiple gestation pregnancies were counted separately; infants who died during the first year of life were excluded.Pregnancies with laboratory evidence of confirmed or possible Zika virus infectionAmong children who met the definition for reported follow-up care, the percentages who were reported to have received each of the following types of clinical care or evaluations, recommended in CDC clinical guidance, were calculated: 1) neuroimaging  any time after birth; 2) hearing screen by automated auditory brainstem response (ABR) or audiologic evaluation by diagnostic ABR  any time after birth; 3) ophthalmologic evaluation any time after birth; 4) developmental screening or evaluation at age >14 days; and 5) physical examination, as indicated by reported growth parameters  at age >14 days.Data were reviewed by clinical subject matter experts to identify Zika-associated birth defects and neurodevelopmental abnormalities possibly associated with congenital Zika virus infection . Data foBOXA sensitivity analysis to address the concern about possible misclassification of microcephaly at birthThe U.S. territories and freely associated states reported 4,816 pregnancies with laboratory evidence of confirmed or possible Zika virus infection by June 1, 2018, including 4,320 (90%) completed on or before February 1, 2018; 4,165 (96%) pregnancies resulted in 4,199 live-born infants, and 155 (4%) resulted in a pregnancy loss . Seven iAmong these 1,450 children 1,376 (95%) had at least one physical examination reported after 14 days of life, 1,106 (76%) had at least one developmental screening or evaluation, 864 (60%) had postnatal neuroimaging, and 695 (48%) had at least one ABR-based hearing screen or evaluation. An ophthalmologic evaluation was reported for 522 (36%) children .Among all 1,450 children with reported follow-up care, 203 (14%) had a Zika-associated birth defect, neurodevelopmental abnormality possibly associated with congenital Zika virus infection identified, or both: 87 (6%) had at least one Zika-associated birth defect, 136 (9%) had at least one neurodevelopmental abnormality possibly associated with congenital Zika virus infection, and 20 (1%) had both . Among tThe sensitivity analysis to assess possible misclassification of microcephaly at birth identified 84 (6%) children with microcephaly among the 1,450 children who had follow-up care reported: five infants had microcephaly at birth with brain or eye anomalies identified at birth; 59 had microcephaly at birth with no brain or eye anomalies identified at birth; and 20 infants did not have microcephaly identified at birth but had postnatal identification of microcephaly. The 59 infants with only microcephaly at birth included 15 who had no other Zika-associated birth defects identified during follow-up care, had normal neuroimaging, and had at least two postnatal measurements with a head circumference above the tenth percentile for the infant’s sex and age. Excluding these 15 infants from the 87 with Zika-associated birth defects results in a decrease in the estimated percentage of affected children from 6% to 5%.Among the 1,450 children whose mothers had laboratory evidence of confirmed or possible Zika virus infection during pregnancy and who had follow-up care reported, 136 (9%) had neurodevelopmental abnormalities possibly associated with congenital Zika virus infection identified (Table). One hundred sixteen (8%) had one or more neurodevelopmental abnormalities possibly associated with congenital Zika virus infection identified, but no Zika-associated birth defects; of these 116 children, 58 (50%) had only possible developmental delay identified, and 25 (22%) had possible developmental delay with at least one other neurodevelopmental abnormality possibly associated with congenital Zika virus infection identified.Among 943 pregnancies with NAT-confirmed Zika virus infection, 144 (15%) had a Zika-associated birth defect, neurodevelopmental abnormality possibly associated with congenital Zika virus infection identified, or both: 62 (7%) had at least one Zika-associated birth defect identified. Ninety-nine (10%) had at least one neurodevelopmental abnormality possibly associated with congenital Zika virus infection identified, and 17 (2%) had both. Among the 1,450 children in this analysis, 607 (42%) did not receive testing for Zika virus infection in serum, urine, or cerebrospinal fluid. Among the 843 (58%) who did receive testing, 32 (4%) tested positive by either NAT or IgM . Zika-associated birth defects or neurodevelopmental abnormalities possibly associated with congenital Zika virus infection were identified in children with positive Zika virus IgM or NAT, negative IgM and NAT, and in those who did not receive testing.A total of  1,450 children aged ≥1 year were born to mothers with laboratory evidence of confirmed or possible Zika virus infection during pregnancy in the U.S. territories and freely associated states and were reported to the USZPIR. Among these children, approximately one in seven (14%) were identified during infancy or early childhood as having either a Zika-associated birth defect, a neurodevelopmental abnormality possibly associated with congenital Zika virus infection, or both.The 6% with Zika-associated birth defects in this report can be viewed in the context of the previously published baseline frequency of brain and eye abnormalities potentially related to Zika virus infection. Before the introduction of Zika in the Region of the Americas the baseline frequency of brain and eye abnormalities potentially related to Zika virus infection among live-born infants was approximately 0.16%  had an ABR-based hearing screen or evaluation, and approximately one third of the children (36%) had an ophthalmologic evaluation reported to the USZPIR. Because the full spectrum of adverse outcomes related to congenital Zika virus infection is not yet known, careful monitoring and evaluation of children born to mothers with laboratory evidence of confirmed or possible Zika virus infection during pregnancy is essential for ensuring early detection of possible disabilities and early referral to intervention services that might improve outcomes. For example, with early identification of vision problems, a prescription for corrective eyeglasses might be beneficial to a child’s development ; however, information about this cohort of children can inform and guide future responses to outbreaks of Zika virus that will inevitably occur among susceptible populations and disproportionately affect pregnant women and their children.It is essential that health care providers who care for children have access to information regarding maternal exposure to Zika virus infection during pregnancy. This will improve the identification of children born to mothers with laboratory evidence of confirmed or possible Zika virus infection during pregnancy so that they can receive recommended postnatal evaluations. Zika virus transmission is far less prevalent in the Americas in 2018 than during 2015–2017 (Zika virus infection during pregnancy can cause serious birth defects and might be associated with neurodevelopmental abnormalities.Among children aged ≥1 year born in U.S. territories and freely associated states to mothers with laboratory evidence of confirmed or possible Zika virus infection during pregnancy and who had follow-up care reported, 6% had a Zika-associated birth defect, 9% had ≥1 neurodevelopmental abnormality possibly associated with congenital Zika virus infection, and 1% had both.Given the potential benefits from interventions during early critical periods of infant development, health care providers should share information on maternal Zika virus exposure and closely monitor child health and development.


Examination of the virus populations present in the male genital tissues of the macaques revealed compartmentalized SIV populations in testis, epididymis, vas deferens, seminal vesicles, and urethra. We found genetic similarities between the viral strains present in semen and those in epididymis, vas deferens, and seminal vesicles. The contribution of male genital organs to virus shedding in semen varied among individuals and could not be predicted based on their infection or proinflammatory cytokine mRNA levels. These data indicate that rather than a single source, multiple genital organs are involved in the release of free virus and infected cells into semen. These findings have important implications for our understanding of systemic virus shedding and persistence in semen and for the design of eradication strategies to access viral reservoirs.The sexual transmission of viruses is responsible for the spread of multiple infectious diseases. Although the human immunodeficiency virus (HIV)/AIDS pandemic remains fueled by sexual contacts with infected semen, the origin of virus in semen is still unknown. In a substantial number of HIV-infected men, viral strains present in semen differ from the ones in blood, suggesting that HIV is locally produced within the genital tract. Such local production may be responsible for the persistence of HIV in semen despite effective antiretroviral therapy. In this study, we used single-genome amplification, amplicon sequencing  of virus in semen, however, remains unknown. To elucidate the anatomic origin(s) of the virus released in semen, we compared viral populations present in semen with those in the male genital organs and blood of the Asian macaque model, using single-genome amplification, amplicon sequencing (env gene), and phylogenetic analysis. Our results show that multiple genital tissues harbor compartmentalized strains, some of them  displaying genetic similarities with the viral populations present in semen. This study is the first to uncover local genital sources of viral populations in semen, providing a new basis for innovative targeted strategies to prevent and eradicate HIV in the male genital tract. Semen plays a key role in the sexual transmission of viruses. It is the foremost vector of dissemination of human immunodeficiency virus type 1 (HIV-1), the causative agent of the AIDS pandemic. About 37 million people are infected by HIV-1 worldwide, and every year an average of 2 million people become infected, most commonly through sexual contact. Although antiretroviral treatments drastically decrease HIV transmission, HIV RNA and infected cells persist in semen despite undetectable blood viral load in about 8% of men receiving suppressive antiretroviral therapy for more than 6 months, suggesting the existence of local sources of virus within the male genital tract. Indeed, multiple phylogenetic studies have demonstrated that HIV strains in semen are distinct from those in the blood  in over 60% of HIV-infected individuals (reviewed in reference env gene), and phylogenetic and population genetic analyses. Our results revealed that viral compartmentalization occurs in different male genital organs, indicative of sustained local replication. Most importantly, we uncovered for the first time several male genital organs involved in the release of viral particles and infected cells into semen. These findings highlight how virus replication within the male genital tract can influence virus populations in semen and impact virus transmission.Semen is the end product of excretions from multiple genital organs, which combine and travel through specific ducts. Our laboratory –10 and o–11–5 copies/ml (H1 [21362] and H2 [30855]), three had moderate (M) seminal viral loads, i.e., between 103 and 5 × 104 copies/ml  and three had low (L) (1.4 × 102 copies/ml) to undetectable viral loads  ; suggesting that, at least in some animals, the virus present in the semen could be from a local origin.We screened 8 adult cynomolgus macaques intravenously inoculated with SIVmac251, in the chronic stage of infection (6 to 15 months postinfection), displaying blood viral loads between 4 and 5 log copies/ml. Two animals had high (H) viral loads in seminal plasma, i.e., ≥10[29965]) . To facigag RNA, we measured infection levels in testis, epididymis, vas deferens, seminal vesicle, prostate, and urethra  for SIV  urethra . Viral R urethra .r = 0.90; P = 0.005)  . In summGenetic divergence between blood and semen virus populations has been shown to occur in about 60% of the over 100 HIV-infected men analyzed in multiple studies (reviewed in reference Fst) to evaluate virus compartmentalization between blood and semen in the five animals in which full-length env was amplified from semen . Fst is a measure of population differentiation due to genetic structure, with values ranging from 0  to 1 . Three additional methods  were used to validate compartmentalization. Viral populations were considered compartmentalized when all four tests converged. Semen/blood compartmentalization was statistically significant in 2/2 animals with high viral loads (H1 and H2) and in 1/3 animals with moderate viral loads (M1) and with a low to medium genetic structuring level   . The gento 0.36)  to D. Into 0.36)  to D. Into 0.36) . In agreto 0.36)  and F. Oto 0.36) . Populatto 0.36) . Naturalulations . In summenv sequences across the 3 macaques analyzed  harboring a specific virus genotype in semen. Overall, SGA and amplicon sequencing yielded a total of 429 analyzed  with no analyzed . All malanalyzed . Except analyzed . Similaranalyzed . Additioanalyzed  and D.Organ compartmentalization was assessed to uncover tissue-specific viral populations. In macaque H1, significant evidence of isolation between organ and blood virus was observed for all 6 male genital organs, with higher levels of population structuring associated with testis, epididymis body, vas deferens, and urethra  and lower levels associated with seminal vesicles and prostate  . Within We further extended the genital organs' compartmentalization analysis to determine whether their viral populations were distinct from each other . In macaWe next compared semen and male genital organ viral populations to determine the source of seminal SIV. First, population structuring analysis was conducted to identify the genital organs displaying the lowest divergence with seminal plasma and semen cell viral populations . In macaIn macaque H2, an absence of significant structuring was observed between seminal plasma and vas deferens, epididymis head, and epididymis tail populations. Seminal cell populations were not significantly different from epididymis head populations but differed from populations I all the other organs ; thus, aIn macaque M1, high levels of viral population differentiation were observed between seminal cells and genital organs, and therefore, none of the tissues analyzed could be identified as the source of infected cells . PhylogeInterestingly, whereas phylogenetic and population structuring results were not compatible with the urethra as a source of virus in semen, the short distance between semen and urethra sequence clusters in the phylogenetic tree of animal H1, together with the presence of specific sequence motifs in those 2 virus populations, suggested a link between these compartments . The samWe aimed to identify the sources of the AIDS virus in semen. To this end, we first examined the infection level and inflammation status of all of the 4 organs  involved in semen elaboration, as well as of 2 ducts involved in semen transport (vas deferens and urethra) from 8 chronically infected macaques. Numerous studies reported a significant correlation between HIV shedding and elevated cytokine concentrations in semen, along with specific pattern of cytokine network in semen compared with blood, suggesting that inflammation favors the burst/release of virus from local sources 1721, 25–3025–We next compared the genetic diversity and structure of viral populations in male organs, as well as in semen and blood cells and plasma for three animals with compartmentalized semen strains. Our data revealed that a number of male genital organs harbor viral strains phylogenically distinct from those in blood and other genital organs, resulting in the presence of multiple distinct viral populations within the male genital tract. These results were then confirmed by our compartmentalization analyses  and 10. Our analyses were performed in macaques infected by the intravenous route. This route of infection differs from the most common route of HIV acquisition in men, i.e., sexual transmission. Men can sexually acquire HIV through either the rectal or penile mucosa –38. Duri–+ men  and of a cell fraction containing primarily spermatozoa (produced in the testis before being matured and stored in the epididymis), together with leukocytes of undetermined origin. During ejaculation, the sperm is transported from the epididymis through the vas deferens ducts to the seminal vesicles, where it combines with its secretions, and then from the seminal vesicles through the ejaculatory ducts to the prostate, to finally travel through the urethra before its release through the meatus (urethra opening). We demonstrated that 100% of the semen viral quasispecies match viral populations from the seminal vesicles in macaque H1, whereas in macaque H2, around 60% of semen strains match viral populations from the epididymis and the vas deferens . Early s+ men . They co+ men . Thus, a+ men , other l+ men , along w+ men , 12, 13.+ men , 9, 12, When comparing RNA sequences of viruses produced by cells and tissues with RNA from the viral particles released in semen and blood, we did not observe any difference between cell-free and cell-associated semen virus populations, indicating common sources. Whereas differences between the cell-free and cell-associated virus have been reported , 48, our49–+ men over relatively short periods is likely to reflect the natural variation of semen composition rather than an extreme instability of the compartmentalization status of genital organs.Compartmentalization in semen has been reported to be a transient phenomenon, with variations sometimes as short as within few days . This coThis study is the first to identify specific male organs that release SIV into semen and to uncover the viral populations present in male genital tissues. Deciphering the origin of viruses in semen is crucial to improve our understanding of the persistence of HIV and other emerging viruses in this body fluid. We showed that rather than a single source, a number of genital organs are involved and that the nature of the shedding male organs varies among individuals. Importantly, we found that previously overlooked genital organs, such as the seminal vesicles and the epididymis, release both infected cells and free virions into semen. These results pave the way for further studies to determine the viral and host-specific characteristics associated with male organs' compartmentalization and virus excretion into semen.Macaca fascicularis) imported from Mauritius were housed in the facilities of the Commissariat à l'Energie Atomique et aux Energies Alternatives . Nonhuman primates (NHP) are used at the CEA in accordance with French national regulations (CEA permit number A 92-032-02). The CEA is also in compliance with Standards for Human Care and Use of Laboratory of the Office for Laboratory Animal Welfare   by the ethics committee of the CEA Comité d'Ethique en Expérimentation Animale, registered by the French Research Ministry under statement number 44. The animals were used under the supervision of the veterinarians in charge of the animal facility. Experimental procedures were conducted after animal sedation with ketamine chlorydrate  as previously described   under OLescribed . Tissuesescribed , 56; thu50) of pathogenic cell-free SIVmac251 in 1 ml of phosphate-buffered saline (PBS). Of note is that one of the macaques  was in the AIDS stage. The generation and titration of the SIVmac251 virus stock have been described elsewhere  and assembled using the Contig Editor program from GeneStudio.In order to maximize the chances of detecting viral sequences in cells and organs matching those of free virus in semen, we chose to focus on the viral strains being currently produced and therefore used viral RNA in tissues and cells for sequencing rather than viral DNA, which can represent archived/defective viruses. Given that both cell-associated and cell-free viruses are present in semen, and that it is currently not clear which of these initiates a new infection, we chose to amplify both cell-associated and free virus in semen. Total RNA was isolated from blood and semen plasma and infected cells using TRIzol-LS and TRIzol, respectively, according to the manufacturer's instructions (Invitrogen). Genital tract tissue samples were homogenized with a TissuLyser II system (Qiagen), and total RNA was extracted using TRIzol (Invitrogen) following the manufacturer's suggested protocol. RNA samples were DNase treated with RQ1 RNase-Free DNase (Promega) for 30 min at 37°C and reverse transcribed into cDNA as previously described , with miescribed , 57, witescribed . Brieflyenv sequences of semen and blood from 5 animals and genital tract tissues from 3 animals were selected (24 to 187 sequences per macaque). Nucleotide sequences were translated into amino acids and aligned in MAFFT  to synonymous (dS) substitution rates (ω) and estimated using the model M0 (one ratio) and the mixed-effects model of evolution (MEME) in HyPhy , while accommodating phylogenetic uncertainty, was inferred between body compartments using asymmetric discrete phylogeographic diffusion models (http://tree.bio.ed.ac.uk/software/figtree). Parameter uncertainty was summarized in the 95% highest posterior density (HPD) intervals. All output generated by BEAST was analyzed in Tracer (http://tree.bio.ed.ac.uk/software/tracer/) to test for convergence and mixing.Genetic diversity (θ)  and popuin HyPhy . Recombiin BEAST  using thn models . ResultsFST and nearest neighbor [Snn]) and tree-based  estimators implemented in HyPhy (see references therein). Viral populations were considered compartmentalized when all tests converged.Compartmentalization among body compartments in each macaque was assessed using several approaches, including distance-based   . When mu


Turdus merula) population. It is transmitted by the mosquito species Culex pipiens in Europe and Africa. Vector competence studies indicate that European strains of USUV are readily transmitted by indigenous Cx. pipiens. However, there is limited information on the ability of an African strain to infect European mosquitoes.Usutu virus (USUV) is an emerging zoonotic virus originally from sub-Saharan Africa. It has been introduced into Europe on multiple occasions, causing substantial mortality within the Eurasian blackbird (Cx. pipiens colonised within the United Kingdom (UK). Mosquitoes were fed blood meals containing this virus and maintained at 25 °C for up to 21 days. Individual mosquitoes were tested for the presence of virus in the body, legs and an expectorate saliva sample. Changes to the consensus of the virus genome were monitored in samples derived from infected mosquitoes using amplicon based next generation sequencing.We evaluated the ability of African strain SAAR-1776 to infect two lines of Cx. pipiens for this strain of USUV. When comparing the genome of input virus within the blood meal with USUV recovered from an infected mosquito, we observed limited changes in the consensus genome sequence.Infection, dissemination and the presence of virus in saliva in one mosquito line was observed, but no evidence for dissemination in the second mosquito line. This suggests a strong barrier to infection in UK Cx. pipiens for Usutu virus suggests a limited susceptibility to infection with USUV strain SAAR-1776 of African origin. However, within a single mosquito there was complete dissemination and expectoration of USUV, indicating that infection, and potentially transmission, is possible. Sequence changes were observed that may represent early adaption to the mosquito host and could reflect the early events of USUV establishment in European mosquito populations.The evaluation of vector competence of UK populations of The online version of this article (10.1186/s13071-018-2959-5) contains supplementary material, which is available to authorized users. Culex with birds acting as amplifying hosts. Human infection is common [Culex neavei collected near the Usutu River, Natal, South Africa [Cx. perfuscus, Cx. univitattus and Cx. quinquefasciatus, throughout sub-Saharan countries [Turdus merula) in 1996, in Italy [Strix nebulosa) [Usutu virus (USUV) is an emerging, mosquito-borne flavivirus that belongs to the Japanese encephalitis virus (JEV) antigenic complex , 2. Likes common –6, but ds common . The virh Africa . Subsequountries . Migratoountries . The firin Italy . It was in Italy . The virin Italy . In 2016ebulosa) .Culex pipiens (s.l.), one of the most abundant species in the northern hemisphere. In addition, USUV has been isolated from Cx. modestus [Aedes albopictus [Cx. pipiens following its introduction. The vector competence of Cx. pipiens (s.l.) colonised in the Netherlands has been investigated and demonstrated that this species is highly susceptible to infection with a European-derived strain of USUV [Cx. pipiens mosquitoes. This would be an early event in the introduction of African mosquito-borne virus in Europe, but one that has presumably occurred on multiple occasions.Surveillance of European mosquito species  has detemodestus  and Aedebopictus . Phylogebopictus . Genetic of USUV . In thisAedes (Ochlerotatus) detritus is a potential vector for JEV at 23 °C and 28 °C [Cx. pipiens derived from the UK. In addition, we monitored the genomic sequence of the infecting virus to evaluate virus adaptation.In the United Kingdom there has been no evidence for autochthonous vector transmission of a mosquito-borne arbovirus since the 19th century , althougnd 28 °C  and WNV nd 28 °C . We asseCulex pipiens species has two ecological forms, Cx. pipiens form pipiens, and Cx. pipiens form molestus, which are morphologically indistinguishable. They are found sympatrically and can hybridize along their distribution range [Cx. pipiens typical form  and Cx. pipiens hybrid form (Brookwood: BKW) were obtained from The Pirbright Institute [on range , with hyon range , 25. Botnstitute . DetailsCx. neavi in South Africa , was passaged three times in Vero C1008 cells to a titre of 4.0 × 106 PFU/ml. Vero cells were maintained in Dulbecco Modified Eagle’s Medium (DMEM)  containing 10% heat-inactivated Foetal Calf Serum (FCS), 2 mM L-glutamine and 50 μg/ml Penicillin/Streptomycin (Sigma-Aldrich).The USUV strain SAAR-1776 originally isolated from Culex pipiens (CBK and BKW) were tested for their vector competence for the SAAR-1776 strain of USUV at 25°C. Mosquitoes were provided an infectious blood meal composed of defibrinated horse blood, adenosine 5’-triphosphate  and virus stock to give a final virus concentration of 1.0 × 106 PFU/ml using a Hemotek membrane feeding system . Five to ten day-old adult female mosquitoes were allowed to feed for a minimum of 16 h at room temperature. Blood-fed and non-blood-fed specimens were anaesthetized with Triethylamine (TEA) FlyNap®  and separated in groups of 10–20 mosquitoes, which were placed in microhabitat pots of 118 × 73 mm in dimension (www.bugzarre.co.uk). Once fully recovered, the mosquitoes were maintained at 25 °C at a relative humidity of between 55–65% with a light cycle of 12:12 light:dark cycle. At 0, 7, 14 and 21 days post-infection (dpi) mosquito groups were processed to provide body, legs, head and saliva samples. Further details of mosquito infections are provided in Additional file Both UK lines of To estimate vector competence, we used previous methods –30. InfeCx. pipiens were homogenized individually in flat-cap homogenization tubes  containing 300 μl of EMEM media containing 10% foetal bovine serum (FBS), penicillin/streptomycin 1% and amphotericin-B 1% and one 3 mm stainless steel bead; beads were not added to the tubes containing the saliva. Homogenisation was undertaken using a Qiagen Tissue Lyser (model Retsch MM301) at 25 MHz for 3 min. All tubes were centrifuged for 5 min at 14,000× rpm and 30 μl of supernatant was collected for virus isolation assays. The remainder of the homogenate was used for RNA extraction using TriZol (see www.tools.thermofisher.com). The RNA pellet was eluted in 20 μl of nuclease-free water.Legs/wings, head, and body (thorax/abdomen) of female USUV RNA was detected using the primers and probe targeting a 91 base pair region of the NS1 as previously published . The rea2 for 3 h. After incubation, 1–1.5 ml overlay of warmed 3% carboxymethylcellulose (CMC) in 250 ml 2× EMEM mix , and 10× MEME-E) were added to each well. After 7 days incubation, 1 ml of 10% neutral buffered formalin solution was added to each well and the plates left for least 3 h to complete virus inactivation. Wells were stained with 200 μl of 2.3% crystal violet solution.Plaque assays were performed on those samples positive by PCR. A ten-fold dilution series of supernatant of the homogenized body parts or salivary secretions (30 μl) was prepared, and dispensed onto a confluent monolayer of Vero cells on a 12-well plate . The plates were incubated at 37 °C in an atmosphere of 5% COMultiplex PCR that generates amplicons spanning the whole genome in two reactions was achieved following the methodology of Quick and co-workers . A set oAmplified products were sequenced using the MiSeq  sequencing platform at the Animal and Plant Health Sequencing Unit. After excluding primer regions using Trimmomatic  and 100 female Cx. pipiens hybrid (BKW) were offered an infectious blood meal. In addition, 70 females for the CBK were used as non-infected controls. Examination of specimens showed that over 95% took a full blood meal. The exposure of engorged specimens to FlyNap for immobilization did not have a deleterious effect for their recovery as shown at dpi 0 and 3. However, a slightly higher mortality was seen after dpi 14  or BKW (P = 0.9962) (log-rank test).Two In both mosquito lines, USUV was detected by PCR in the blood meal (abdomen) at dpi 0. However, the infection rates and transmission efficiency were low, when examined at later time points. Only one CBK specimen had detectable USUV RNA in the body at dpi 14 and one at dpi 21 , while a single specimen from BKW was positive at dpi 21 (Infection rate: 5.5 %)  was originally estimated at 6.3 × 106 PFU/ml, the assessment of virus in the blood meal before and after the blood-feeding yielded a titre of 2 × 104 PFU/ml, equating to a 2-log drop in viral titre. Infectious viral titres (in PFU/ml) in the CBK specimen with positive saliva were: saliva 4.0 × 101, leg/wings 8.3 × 103, head 8.0 × 104, and abdomen 6.8 × 105. All other USUV RNA positive samples detected by RT PCR did not induce cytopathic effect to the Vero cell monolayer.Although the titre of the frozen virus was estimated to be 4 × 10USUV sequence was obtained covering > 97% of the genome from the input virus  as well as the abdomen  of the USUV positive CBK mosquito at day 14 post-infection  in birds in the UK , 34, theaponicus –37. USUVaponicus , 38. Allnstrated , 16, 39,assessed . The autCx. pipiens form pipiens was positive for USUV RNA in the saliva 14 days after taking a blood meal. A range of factors could influence the competency of a particular mosquito to vector a virus. These include the genetic variability of the virus strains, virus titres used during oral infection, the chosen temperature and selective pressures during laboratory colonization that may change susceptibility to infection [In contrast to high infection/transmission rates from experiments with a European USUV isolate, our results showed that only a single specimen of UK CBK nfection , 40–42. in situ evolution with host-specific mutations also being detected. Their observed mutations at the amino acid level in the NS2A protein (V91A) was suggested to be involved in the formation of the USUV European lineages 1 to 3. Nonetheless, it was concluded that the pathogenicity of specific USUV lineages has been poorly studied and more work is needed to determine the biological characteristics in each lineage.The effect of two nucleotide substitutions in the non-structural protein NS2A on the phenotype of the progeny virus is unknown, although it is interesting to note that a change with a deep coverage was only observed in the mosquitoes’ abdomen, where bottlenecks and selective pressures are said to occur while the virus overcome the midgut barrier Additional file 2:Text S2. Membrane-based infection studies in mosquitoes. (DOCX 17 kb)Additional file 3:Table S1. Primers used to amplify USUV genomes. (DOCX 22 kb)


Prolonged detection of IgM and neutralizing antibody cross-reactivity make it difficult to determine the timing of Zika virus infection and differentiate between related flaviviruses.Data on the duration of detectable Zika virus–specific IgM in infected persons are limited. Neutralizing antibody cross-reactivity occurs between Zika virus and related flaviviruses, but the degree to which this confounds diagnosis is uncertain. We tested serum specimens collected 12–19 months after illness onset from patients with confirmed Zika virus disease for Zika virus IgM and Zika virus and dengue virus neutralizing antibodies. Among 62 participants, 45 (73%) had detectable Zika virus IgM and 12 (19%) had an equivocal result. Although all patients tested had Zika virus neutralizing antibodies, 39 (63%) also had neutralizing antibodies against dengue virus; of those, 12 (19%) had <4-fold difference between Zika virus and dengue virus titers, and 5 (8%) had dengue virus titer  Persons with asymptomatic infection, pregnant women, and infants with congenital infection were excluded from enrollment. We enrolled participants during October 16, 2017–February 1, 2018. We obtained written consent from study participants or their guardians.Serum specimens were tested at the Centers for Disease Control and Prevention  by IgM antibody capture ELISA (MAC-ELISA) for detection of Zika virus and dengue virus IgM and by plaque reduction neutralization test (PRNT) to detect Zika virus and dengue virus neutralizing antibodies . This study was approved by the Florida Department of Health Institutional Review Board.We obtained descriptive and clinical data for case-patients, including age, gender, race/ethnicity, reported symptoms, symptom onset, and origin of infection, from Merlin, the Florida Department of Health surveillance system. We used Pearson χOf 352 eligible PCR-confirmed Zika virus disease case-patients, 62 (18%) were enrolled and provided follow-up serum specimens. The 62 enrolled participants and 290 eligible case-patients who were not enrolled were similar with regard to age, sex, race/ethnicity, and clinical manifestations; however, 55% of enrolled participants acquired their infections in Florida, compared with 45% of the unenrolled cases .>18 years of age  provided a specimen at 12 months after initial symptom onset, 1 (2%) at 13 months, 13 (21%) at 14 months, 21 (34%) at 15 months, 11 (18%) at 16 months, 3 (5%) at 17 months, 3 (5%) at 18 months, and 2 (3%) at 19 months. The median age of participants was 47 years (range 8–70 years); 60 (97%) were adults s of age . OverallAt follow-up, 45 (73%) patients had detectable Zika virus IgM, 12 (19%) had an equivocal result, and 5 (8%) were negative  patients had a <4-fold difference between Zika virus and dengue virus titers, and 5 (8%) had dengue virus titers that were >4-fold higher than Zika virus titers.All participants had Zika virus neutralizing antibodies at 12–19 months after their acute illness, and 39 (63%) had dengue virus neutralizing antibody titers at follow-up. Using a definition of positive or equivocal Zika virus IgM with confirmatory Zika virus neutralizing antibodies, 57 (92%) would have had a diagnosis of recent Zika virus or flavivirus infection on the basis of results from their follow-up specimens . OverallThese findings demonstrate that 73% of persons with PCR-confirmed symptomatic Zika virus disease still had positive IgM test results 12–19 months after their initial illness, and another 19% had equivocal results. Because all participants had Zika virus neutralizing antibodies, a high proportion (92%) would have had a recent Zika virus or flavivirus infection diagnosis on the basis of serologic testing performed at the follow-up time point. Current Zika virus testing guidance recommends Zika virus serologic diagnosis only for symptomatic patients with a clinically compatible Zika virus illness (The prolonged detection of IgM after Zika virus infection is consistent with previous findings for related flaviviruses (>1 year after symptom onset.Serologic cross-reactivity has been demonstrated between Zika virus and related flaviviruses, including dengue virus, but the degree to which neutralizing antibody cross-reactivity limits the ability to identify the specific virus responsible for the current infection is unclear (These findings are subject to several limitations. This report presents data from a single follow-up specimen but how long IgM may persist after this timeframe remains unknown. We cannot exclude the possibility that some participants may have been reexposed to Zika virus or another flavivirus between their initial illness and follow-up testing. The timing of the follow-up specimen varied among the participants and was limited to 12–19 months following onset of Zika virus symptoms. This, coupled with the small number of specimens at some time points, prevented us from assessing possible trends in IgM persistence over time. The small sample size and lack of specimens from the acute illness also limited our ability to detect factors that may be associated with prolonged detection of IgM, including possible differences between primary and secondary infections. Our findings of prolonged IgM seropositivity are specific to the Centers for Disease Control and Prevention MAC-ELISA, which targets Zika virus premembrane and envelope glycoproteins; other IgM serologic assays targeting other proteins are currently available and may not produce comparable findings. Finally, because we enrolled only symptomatic disease case-patients, it is uncertain whether persons with asymptomatic infections would exhibit similar IgM persistence.These findings support data for other flaviviruses and suggest that a substantial proportion of persons with Zika virus disease will still have detectable IgM 1–2 years after their initial infection. The results highlight the complexity of using serologic diagnosis to determine the specific timing of a recent infection, which is particularly important for pregnant women and challenging for residents of areas with previous or ongoing Zika virus activity. As such, the findings further support the current recommendations to use nucleic acid amplification for screening asymptomatic pregnant women with ongoing possible Zika virus exposure (Diagnostic test results for participants with PCR-confirmed Zika virus disease in Miami–Dade County, Florida, USA, 12–19 months after onset.


A series of experiments described in a recent study showed that the copy number of EBV in a cell during latency is strongly associated with oncogenicity . SpecifiThe results of these experiments, and the conclusions by Zuo et al., are consistent with the Microcompetition Model first described in the book entitled ‘Microcompetition with Foreign DNA and the Origin of Chronic Disease’ , 3.The model concentrates on certain viruses, such as the Herpes Simplex Virus 1 (HSV-1), Epstein- Barr virus (EBV), Cytomegalovirus (CMV), Human T-cell lymphotropic virus (HTLV), and Human Immunodeficiency Virus (HIV), that include a strong cis-regulatory element in their promoters/enhancers, called the N-box. This cis-element binds the cellular GABP·p300 transcription complex during the latent phase. Since the level of the GABP·p300 is limiting, the viral N-boxes reduce the availability of the complex for to the cellular genes. The lower pool of GABP·p300 causes the cellular genes to express abnormal levels of their proteins. The genes that are transactivated by the GABP·p300 complex synthesize fewer proteins. The genes that are suppressed by the complex synthesize more proteins. The abnormal levels of these cellular proteins cause many common chronic diseases, including cancer and diabetes.For example, the breast cancer associated gene type 1 (BRCA1) promoter has three N-boxes in its -200 to -178 region. GABP has been shown to interact with the N-boxes and stimulate transcription of the gene .TranscriIt is well known that there is a balance between the efficiency of the immune system and the copy number of latent viruses in the host. It is also known that many events can decrease the efficiency of the immune system, including, aging, certain medications, surgery, chemotherapy, radiation, and stress . When suTo summarize, Zuo et al. conclude: “It has been noticed that EBV load in tumor tissues or blood is associated with the clinical progression and prognosis in both lymphoma and NPC. Our result verifies this association. We also emphasize the importance to measure the level of gene expression or copy number in the virus study instead of only concerning ‘with and without’.” In this paper we show that our interpretation of the Microcompetition Model supports this conclusion. We also believe that the copy number of the latent viruses that include an N-box in their promoter/enhancer, including EBV, and not the ‘infected or not infected’ status is an important factor that determines the pathogenesis of cancer, and therefore should be tested in clinical practice.