However, there was variability in susceptibility between the individual RM with 1–>8 vaginal inoculations required to establish infection. After treatment with Depoprovera, a widely used contraceptive progestin, two RM that initially resisted 8 vaginal ZIKV inoculations became infected after one ZIKV inoculation. Thus, Depoprovera seemed to enhance susceptibility to vaginal ZIKV transmission. Unexpectedly, the kinetics of virus replication and dissemination after intravaginal ZIKV inoculation were markedly different from RM infected with ZIKV by subcutaneous (SQ) virus inoculation. Several groups have reported that after SQ ZIKV inoculation vRNA is rapidly detected in blood plasma with vRNA less common in urine and saliva and only rarely detected in female reproductive tract (FRT) secretions. In contrast, in vaginally inoculated RM, plasma vRNA is delayed for several days and ZIKV replication in, and vRNA shedding from, the FRT was found in all 6 animals. Further, after intravaginal transmission ZIKV RNA shedding from FRT secretions was detected before or simultaneously with plasma vRNA, and persisted for at least as long. Thus, ZIKV replication in the FRT was independent of, and often preceded virus replication in the tissues contributing to plasma vRNA. These results support the conclusion that ZIKV preferentially replicates in the FRT after vaginal transmission, but not after SQ transmission, and raise the possibility that there is enhanced fetal infection and pathology after vaginal ZIKV transmission compared to a mosquito transmitted ZIKV.Zika virus (ZIKV) is a mosquito-transmitted virus that can cause severe defects in an infected fetus. ZIKV is also transmitted by sexual contact, although the relative importance of sexual transmission is unclear. To better understand the role of sexual transmission in ZIKV pathogenesis, a nonhuman primate (NHP) model of vaginal transmission was developed. ZIKV was readily transmitted to mature cycling female rhesus macaque (RM) by vaginal inoculation with 10 Zika virus was introduced to Brazil in 2015 and it rapidly spread to all of tropical America. Although Zika virus infection is usually mild in adults, it can cause severe birth defects in the developing fetus that makes it critical to prevent ZIKV infection in women who are pregnant or who could become pregnant. Although Zika virus is transmitted primarily by mosquito bite, it can also be transmitted by sex. To understand the role of sexual transmission in Zika virus disease, we inoculated rhesus monkeys intravaginally with the virus and monitored virus in blood and reproductive tract secretions. ZIKV was detected in the female reproductive tract before it was detected in plasma and replication levels in the female reproductive tract did not reflect ZIKV levels in other parts of the body. Thus ZIKV prefers the reproductive tract after vaginal transmission suggesting that fetal disease could be more common or severe after vaginal ZIKV transmission compared to a mosquito transmitted ZIKV infection. Zika virus (ZIKV) was first isolated in the Zika forest of Uganda in 1947 21, 22, 30) and the first descriptions of human disease were reported a few years later 2, 53). ZIKV has a positive-sense RNA genome and belongs to the genus Flavivirus, which also includes dengue virus (DENV), Yellow Fever virus, Japanese encephalitis virus, and West Nile virus (WNV) (30). In approximately 20% of infected humans, ZIKV causes a febrile illness that can include rash, arthralgia and conjunctivitis. In addition, ZIKV has been associated with the development of microcephaly and lissencephaly and ocular lesions in infants born to women who acquired the infection during early pregnancy. In adults, ZIKV infection has also been associated with Guillan-Barré syndrome and other neurological complications including hearing loss and tinnitus. Although ZIKV is a mosquito-transmitted virus, sexual transmission of ZIKV in humans has been documented in several settings . After r1, 22, 303. ZIKV hIn 2007, an Asian lineage ZIKV outbreak from mosquito transmission was reported in Yap Island with 185 clinical cases and an estimated 5000 infections (75% of the population) in just 3 months ,18. Six The World Health Organization declared the ZIKV pandemic a public health emergency on February 1, 2016, and in November 2016, WHO declared Zika virus endemic in the Americas. As of May 2017, more than 5,109 cases of ZIKV infection have been reported in the United States, excluding those in Puerto Rico, Virgin Islands and Guam. Most infections are in travelers returning from affected areas, but 266 ZIKV infections were acquired in the continental US. Of these US acquired infections, 221 infections (83%) were transmitted through mosquito bites in Florida and Texas, while 45 infections (17%) were sexually transmitted . It is nThe captive-bred mature (> 5year old) parous, cycling female rhesus macaques (Macaca mulatta) used in this study were from the California National Primate Research Center. All animals were negative for antibodies to WNV, HIV-2, SIV, type-D retrovirus, and simian T cell lymphotropic virus type 1 at the time the study was initiated. The animals were housed in accordance with the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care International Standards and with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Institutional Animal Use and Care Committee of the University of California, Davis, approved these experiments (Protocol # 19471). When immobilization was necessary, the animals were injected intramuscularly with 10 mg/kg of ketamine HCl . All efforts were made to minimize suffering. Details of animal welfare and steps taken to ameliorate suffering were in accordance with the recommendations of the Weatherall report, "The use of non-human primates in research". Animals were housed in an air-conditioned facility with an ambient temperature of 21–25°C, a relative humidity of 40%-60% and a 12 h light/dark cycle. Animals were individually housed in suspended stainless steel wire-bottomed cages and provided with a commercial primate diet. Fresh fruit was provided once daily and water was freely available at all times. A variety of environmental enrichment strategies were employed including housing of animals in pairs, providing toys to manipulate and playing entertainment videos in the animal rooms. In addition, the animals were observed twice daily and any signs of disease or discomfort were reported to the veterinary staff for evaluation. The menstrual cycles were assessed on the basis of menstrual bleeding, with the first day of menses designated day 0 of the cycle. For sample collection, animals were anesthetized with 10 mg/kg ketamine HCl  or 0.7mg/kg tiletamine HCl and zolazepan  injected intramuscularly. The animals were sacrificed by intravenous administration of barbiturates.Plasma from a ZIKV infected blood donor was used to produce the ZIKV stock for these studies. The donated blood was collected at the Hematology and Transfusion Center, Hospital of Clinics, Universidade Estadual de Campinas-UNICAMP, Campinas, SP, Brazil, and after it was found to positive for ZIKV by RT-PCR, the fresh frozen plasma was released for research. However, no donor personal identification information accompanied the sample and thus, the donor is anonymous  and IRB 7 PFU/ml of infectious virus when titrated by Vero cell plaque assay and approximately 6x109 vRNA copies/ml by the Taqman RT-PCR described below. The atraumatic virus inoculation procedure consisted of inserting a 1 CC needless tuberculin syringe containing 1 ml of the ZIKV stock into the vagina until the tip touched the cervix. Then the syringe was gently withdrawn while the viral inoculum was expelled. This procedure was repeated weekly until an animal was plasma ZIKV RNA+ on 2 consecutive time points . Variant nucleotides were then called using SNPeff, using a 5% cutoff. The output.vcf and.bam files could be interrogated in Geneious and differences between the inocula and reference strains could be determined.The Zika virus inoculum was sequenced in duplicate using a method adapted from Quick et. al. . BrieflyBlood was collected from the femoral vein by venipuncture 3–4 times a week, on the day of ZIKV inoculation and, 2, 4, and often 6, days later. Urine samples were collected from pans placed under the animals’ cages on the days that blood samples were collected. Cervicovaginal lavages (CVL) were also collected on the days blood samples were collected by vigorously infusing 1–2 ml of sterile PBS into the vaginal canal and aspirating as much of the instilled volume as possible. Care was taken to insure that the cervical mucus was included in the lavage fluid and that no trauma to the mucosa occurred during the procedure. One half of the CVL sample was snap frozen on dry ice and stored at −80°C until analysis. The remainder was spun and the resulting cell pellet was used RNA isolation. The supernatant was treated with 10× Protease Inhibitor  and subsequently used for cytokine and chemokine quantitation. The lavage for sample collection and the preparation procedure resulted in at least a 10-fold dilution of the cervicovaginal secretions. RNA was isolated from 1 ml urine, EDTA blood plasma, or CVL by QIAamp UltraSens Virus Kit  following the manufacturer’s protocol.Genital tract tissues  and genital lymph nodes , gut tissues , oral tissues  distal lymphoid tissues , urinary tract , CNS , cerebrospinal fluid (CSF) and blood were collected at the time of necropsy and analyzed for ZIKV RNA levels. Tissues were stored in RNAlater  and kept at -20°C until preparation of RNA. Tissues stored at -20°C thawed and removed from RNAlater were diced with a razor blade in a sterile petri dish as small as possible. Tissues fragments were then placed into 2.0ml screw cap Sarstedt tubes with 1 x 7mm stainless beads  added per tube with 600ul RLT Buffer and shaken 5 mins in bead beater to homogenize. The homogenates were processed using the Qiagen RNeasy Mini Kit  to extract total RNA with optional DNase treatment on column per manufacturer’s instructions. Skin and fibrous tissues were treated with additional proteinase K digestion as described in Appendix C of the kit handbook. Brain samples required 5ul of Reagent DX to prevent excessive foaming.7,tissue mononuclear cells isolated from tissues were added to a confluent monolayer of Vero cells in 6-well plates  for tissues yielding <106 cells), or T25 flasks (Costar Inc.) for tissues yielding > 106 cells. The co-cultures were incubated at 37°C and culture supernatants were harvested at 2, 4 and 7 days after initiation. The supernatants were assayed for the presence of ZIKV RNA by qRT-PCR (described below). A sample was considered to be positive for infectious virus if the vRNA levels steadily increased in supernatants of the corresponding co-culture. No effort was made to titer the levels of infectious virus in samples.We used monolayers of Vero cells  to isolate infectious virus from selected tissue samples collected from the ZIKV-inoculated animals at necropsy. Briefly, up to 107 copies/well to 1 copy/well were run to generate the line equation used to analyze all qPCR assays. For each 96 well plate, 11 wells of each dilution were run including positive and no-template controls. When the dilution of this fragment is done correctly, we generate 7–10 positive wells out of 10 at the 10-copy range and 2–3 positive wells out of 10 in the single copy range. Thus, the assay can detect a single copy of ZIKV env cDNA per well. To determine the sensitivity of the assay in actual samples, 10-fold serial dilutions of vRNA from the ZIKV stock were added to plasma or RNA extracted from a mesenteric LN collected from a ZIKV negative RM. The assay was negative when 10–15 copies of ZIKV RNA were added to the cDNA synthesis reaction, which results in about 1 vRNA copy in each well. However, 6 of 6 wells were positive when 100–150 copies of ZIKV RNA were added to the cDNA synthesis reaction, which is equivalent to 10–13 copies of vRNA in each well. There was no amplification of ZIKV E glycoprotein sequences from the RNA isolated from any plasma or tissue samples from ZIKV negative animals. Thus, we estimate that the limit of quantitation in this ZIKV E glycoprotein PCR assay is 120 vRNA copies/ml of CVL, plasma or urine. While in tissue samples, the limit of quantitation is 33 vRNA copies/ug of total tissue RNA analyzed. Viral load data from plasma, urine, and CVL are expressed as vRNA copies/ml. Viral load data from tissues are expressed as vRNA copies/ug total RNA.For urine, plasma, and CVL samples, 25ul of eluted RNA was converted to cDNA with Superscript III  using random primers in a 60ul reaction and quantified in quadruplicate by qPCR on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System using 2x Universal Taqman Master Mix  with published primers and probe that target the ZIKV E glycoprotein from Lanciotti et al   uses a Ugandan ZIKV NS1 protein as the capture antigen. There is about 97.5% amino acid identity between the Ugandan virus and contemporary circulating Asian ZIKV virus strains in the NS1 region. The kit was used as directed by the manufacture to test plasma samples. CVL samples, processed as described above, were diluted 1:1 and 1:2 and tested with the same kit.Twenty-nine cytokines, chemokines and growth factors were measured in plasma and CVL samples using the Monkey Cytokine Magnetic 29-Plex Panel for the Luminex  according to the manufacturer's instructions. The analytes measured included IL-1β, IL-1RA, IL-2, IL-6, IFN-γ, IL-12, CCL3, CCL5, CCL11, CXCL8, CXCL9, CXCL10, CXCL11, and MIF. EDTA-plasma samples were diluted up to four fold with assay diluent and CVL samples were diluted up to 4 fold with a 1:1 mixture of PBS and assay diluent. Samples were incubated with antibody-coupled beads for 2 hours at room temperature, followed by incubation with a biotinylated detection antibody for 1 hour and streptavidin-phycoerythrin for 30 minutes. Each sample was assayed in duplicate, and cytokine standards supplied by the manufacturer were run on each plate. Multianalyte profiling was performed using a Luminex-100 system, and data were analyzed using Miliplex analyst software, version 5.1 . The median level of each analyte in a sample is reported. For these analytes, the sensitivity of the assay ranges from 0.5–20 pg/ml plasma according to the manufacturer.GraphPad Prism version 5 for Apple OSX10.4  and Macintosh computers  were used for statistical analysis and graphing the data.Zika virus strain PRVABC59; genbank accession number KU501215Zika virus strain/H.sapiens-tc/BRA/2015/Brazil_SPH2015; genbank accession number KU321639.17 PFU/ml/6x109 vRNA copies/ml) ZIKV stock by culturing the plasma of a Brazilian blood donor . Ob. ObR0) opulation  [39]. Topulation –46 and Rpulation ,36,47–49pulation ,36,47,48Perhaps, the most striking finding in this study is that the kinetics of virus replication and dissemination in RM after intravaginal ZIKV inoculation are markedly different than after SQ virus inoculation ,36,49. AIn addition to the delay in plasma vRNA in ZIKV vaginally inoculated RM compared to SQ infected RM, virus dissemination to tissues was slower and stepwise in the vaginally inoculated animals. Four days after vaginal inoculation, ZIKV RNA was present at low to moderate levels in the urinary tract, FRT, draining lymph nodes distal lymph nodes, spleen. However, at 8 days PI, vRNA levels were 100–1000 fold higher in all tissues, with the highest levels in salivary glands and lymphoid tissues indicating that the virus was still disseminating more than 1 week after infection. At 30 and 35 days PI, the vaginally infected RM were plasma ZIKV RNA negative but had low to moderate ZIKV RNA levels in all lymphoid tissues tested. In addition, low level ZIKV RNA was detected in the uterus of one of these 4 RM . SimilarIn the RM infected by vaginal ZIKV inoculation, the levels ZIKV RNA in CVL was similar to plasma vRNA levels. Given the 10–100 fold dilution of cervicovaginal secretions that occurs during the CVL collection process, vRNA levels in CVL were at least equal to, and often higher than, plasma vRNA levels . Thus thThere was substantial variability between the individual RM in susceptibility to infection after vaginal ZIKV inoculation in this study. It has been reported that the stage of the menstrual cycle at vaginal inoculation effects susceptibility to infection with SHIV and SIV in RM ,55SodoraDepo-Provera, a brand of the injectable hormonal contraceptive depot-medroxyprogesterone acetate (DMPA), is the most widely used injectable contraceptive in the world. We chose to test the effects of Depoprovera on vaginal ZIKV transmission because DMPA treatment enhances infectivity of viruses in various rodent and nonhuman primate models of female genital tract infection –61. In fSeveral mechanisms have been proposed to explain enhanced STI acquisition with Depo-Provera including mucosal epithelium thinning, enhanced tissue inflammation, suppressed cell-mediated immune responses, and altered vaginal microbiota. However, none of these putative biological mechanisms are experimentally proven ,67. It wAlthough our ZIKV inoculum was delivered to monkeys as cell-free virions suspended in tissue culture fluid, women are exposed to ZIKV virions in semen, which may affect virus transmission. Seminal plasma (SP) has a basic pH that neutralizes the acidic pH of the vagina, thus seminal plasma may limit the inactivation of ZIKV deposited into the vagina. In fact, it has been shown that SP boosts SIV and HIV-1 infection in vitro and semen amyloid proteins contribute to this activity –74. HoweRM infected by SQ inoculation with ZIKV during the first trimester of pregnancy have persistent plasma vRNA, leading to the hypothesis that the fetus or placenta may be the source of persistent virus replication in the immune suppressed pregnant female . This coAlthough we detected anti-ZIKV IgG antibodies in plasma of all 4 animals infected for more than 10 days with ZIKV. We did not detect anti-ZIKV IgG antibodies in CVL of any animals. This is unexpected as antiviral antibodies are routinely found in CVL of RM and women –82. It iThe systemic cytokine response is minimal after SQ ZIKV inoculation of RM , and it The pattern of inflammation in the FRT and systemic compartments also provides considerable insight into ZIKV pathogenesis. MIF, the only cytokine that was elevated in both plasma and CVL samples of all 4 animals. DENV infection induces MIF production and secretion and secreted MIF can enhance DENV replication and increase vascular leakage through autophagy . Thus MI+/+ mice, ZIKV replicates in the FRT but not in systemic tissues [The findings that ZIKV shedding in CVL is not related to plasma vRNA levels and that a local inflammatory response develops in the FRT that is distinct from the systemic response is consistent with the conclusion that ZIKV replicates, and persists, in the FRT independent of the systemic ZIKV infection. This conclusion is also supported by observation that after vaginal ZIKV inoculation of IFNR tissues . Thus, d


Based on our analysis, we expect that the NGS test cost concerns will be limited. In the current situation, NGS can indeed result in higher diagnostic test costs, which is mainly related to required additional tests besides the small TGP. However, in the future, we expect that the use of whole-genome sequencing (WGS) will increase, for which it is expected that additional tests can be (partly) avoided. Although the current clinical benefits are expected to be limited, the research potentials of NGS are already an important advantage.Next-generation sequencing (NGS) has reached the molecular diagnostic laboratories. Although the NGS technology aims to improve the effectiveness of therapies by selecting the most promising therapy, concerns are that NGS testing is expensive and that the ‘benefits’ are not yet in relation to these costs. In this study, we give an estimation of the costs and an institutional and national budget impact of various types of NGS tests in non-small-cell lung cancer (NSCLC) and melanoma patients within The Netherlands. First, an activity-based costing (ABC) analysis has been conducted on the costs of two examples of NGS panels ) based on data of The Netherlands Cancer Institute (NKI). Second, we performed a budget impact analysis (BIA) to estimate the current (2015) and future (2020) budget impact of NGS on molecular diagnostics for NSCLC and melanoma patients in The Netherlands. Literature, expert opinions, and a data set of patients within the NKI ( Next-generation sequencing (NGS) has reached the molecular diagnostic laboratories and is expected to (slowly) replace single-gene molecular diagnostic tests for the detection of DNA mutations in tumour tissues . Within There are high expectations around NGS and targeted therapies, they can improve patient survival and can reduce unnecessary harms and costs by preventing sequential single-gene testing and ineffective treatments –5. For rCurrently, molecular diagnostics and targeted therapies are mostly recommended for patients in an advanced stage of disease. Especially, for stage-IV non-small-cell lung cancer (NSCLC) and stage-IV melanoma patients, NGS can be very beneficial due to their heterogeneous nature, high mutation frequency, and the availability of targeted therapies. For both patient groups, the prognosis is poor, stage-IV NSCLC patients have a 5-year survival rate of 1% and stage-IV melanoma patients of 15–20% –9. SinceAlthough the NGS technology aims to improve the effectiveness of therapies, concerns are that NGS testing is expensive and that the clinical benefits are not yet in relation to these costs . CurrentTM and the Illumina HiseqTM, and per run a maximum of 48 samples can be included, except for a run with the medium TGP on the MiseqTM which is restricted to four samples per run. These maximums of four or 48 samples per run are related to the desired minimum depth of coverage and pipetting time needed per sample.The total costs of performing two types of NGS tests for diagnostics have been analysed for a small TGP (48 genes) and a medium TGP (178 genes). For this, we used activity-based costing (ABC), a cost calculation technique that links resource costs with products using a multistep allocation procedure on the basis of activity consumption . This prTM/HiseqTM), the numbers of samples per run, the number of runs per week and the completeness of the run. Variation in these parameters will result in different molecular diagnostic test costs and turnaround times of the diagnostic process.The test costs consisted of the following elements: personnel, material, equipment, and an estimation of the overhead costs. Personnel costs were based on the estimated minutes spent per process step, both per sample and run. Hour rates per personnel type were based on the NKI employers’ costs of 2014 and only the direct productive hours were included in this calculation. Real ‘timing activity’ was used to calculate the time required for the small TGP currently in clinical practice. Since the medium TGP was not used in a diagnostic setting, timing estimations were made by means of interviews with the executers of the process. Material costs were based on laboratory protocols and purchase prices within the NKI. Laboratory protocols were also used to calculate the equipment costs; realistic redemption durations and yearly service costs were taken into account. All costs were divided into fixed yearly costs, fixed costs per run and variable costs per sample. The cost consequences of important parameters of the NGS procedure were explicitly investigated; the type of sequence technology  was performed to estimate the yearly budget impact of NGS tests in NSCLC and melanoma patients from a Dutch perspective and an institutional perspective (NKI). A BIA focuses on the expected changes in the expenditure of a health care system after the adoption of a new intervention over a time period of 5 years in this case. Time frames 2012, 2015, and 2020 have been selected to show an estimation of the old, current and future budget impact. In 2012, mostly (a combination of) single tests and methods were used in clinical diagnostics. In 2015, a variety of NGS tests were represented from single-gene tests in the general hospitals, till small-medium TGPs in the academic centres. In the future time frame of 2020, it was assumed that WGS will be the clinical standard in academic/specialised hospitals by that time. The latter assumption is in the framework of the CPCT, their mission is to perform WGS in all specialised centres, for both diagnostics and research.Within our BIA, six sources of input are included: three test cost sources, literature for diagnostic guidelines and patient numbers, patient data for the case study and expert opinions. The BIA has been performed according to the BIA framework of the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) . This frThe NSCLC and melanoma incidence within The Netherlands and the stage distribution at diagnosis were used to calculate the size of the target population, stage IV NSCLC and melanoma patients within The Netherlands. The patients were divided into two groups: peripheral or specialised healthcare , according to the number of patients diagnosed per hospital type. Different molecular diagnostic test standards have been taken into account for these two types of hospitals. We assumed that for each new stage-IV patient, molecular diagnostics is executed once. Although repeated testing over time is often required in case of progression, among others caused by tumour heterogeneity within a patient, we assumed no influence by NGS on this frequency.The resource utilisation within this BIA reflects the use of molecular diagnostic tests  within the Dutch health care system. Based on the Dutch diagnostic guidelines –15, the Three different sources have been used to estimate the molecular diagnostic test costs. (1) The costs for single-gene tests were estimated within the NKI and it was assumed that these costs are comparable in other hospitals. (2) The small- and medium-TGP costs were based on the ABC analysis explained in the “Activity-based costing: test costs” section. (3) The WGS costs estimation was based on the public data of the National Human Genome Research Institute, showing the most recent sequence costs per DNA sequence or genome , and theConsidering the fact that many included parameters for the BIA were based on hypothetical values and extrapolations, plausible ranges for the model’s parameters were explored. We conducted two one-way sensitivity analyses, one for the test costs in the current NGS situation of NSCLC and one for the influence of future test costs on the budget impact. We varied key parameters one-by-one, using several uncertainty ranges. For the test costs, four parameters have been included: (1) Number of NGS samples per week, (2) Number of NGS runs per week, (3) Completeness of the NGS run, and (4) Required additional tests besides NGS. All base values and ranges were based on realistic possibilities within the NKI. Three treatment parameters were included in the sensitivity analysis for the future budget impact: (1) Price of TGP using NGS, (2) Price of WGS, and (3) Percentage of patients that will receive specialised care (using WGS testing). For these parameters, a range of 20% has been used, as seen in other comparable BIA’s .In order to analyse the budget impact of NGS in clinical practice, the NKI was used as a case study environment. Stage-IV NSCLC and melanoma patients for who molecular diagnostics were requested in a period before NGS implementation (November–December 2014) and in a period after NGS implementation (January–February 2015) were included. A data set was created with the diagnosis, number, and type of molecular diagnostic tests per patient and the observed mutations per test. Both actionable and non-actionable mutations were counted and the percentages of patients with an actionable mutation for which hypothetically a targeted therapy is available  were calculated. Treatment consequences were manually checked via the electronic health records of the patients and four categories were added to the data set: (1) whether there was a treatment option for the patient, (2) which treatment was selected, (3) whether the selected treatment was based on the test result, and (4) whether the patient received a first dose of the treatment. The molecular diagnostic tests, test results, and treatment consequences have been compared for the two periods. Based on the molecular tests executed per patient, the test costs per patient and for the total NKI population were calculated, including a budget impact of the NGS implementation. We assumed that the number of included patients in these 3.5 months was representative for the rest of the year, in order to estimate the yearly NKI population of NSCLC and melanoma patients for who molecular diagnostic tests are requested.t-test for the continuous variables with a normal distribution, an independent samples Mann–Whitney U-test for the continuous variables without a normal distribution and a Fisher’s exact test for the binary variables have been used to analyse differences between the subgroups of the NKI case study. P values below 0.05 were considered to indicate a statistically significant difference.Microsoft Excel and SPSS 23.0 have been used to perform the sensitivity analyses and to create descriptive statistics of the results. An independent two-sample TM and €956  on the HiseqTM. A run with the medium TGP on the MiseqTM is restricted to four samples per run, for €3009 per sample, and costs per sample on the HiseqTM were €1,137 . These costs are inclusive VAT, overhead (30%) and the costs of control samples, in a run with ≤8 samples one control sample is included and in a run with ≥9 samples two control samples are included. A general overview of the costs is shown in Within The Netherlands, the current incidence of stage-IV NSCLC and melanoma patients are, respectively, 4,045 and 800 patients per year –25. In tin situ hybridisation ) and one single-gene test was assumed for 50% of the melanoma patients (Sanger Sequencing (KIT)). In the future (2020), we assumed in our model that NGS with a small TGP will be clinical practice in peripheral hospitals for both NSCLC and melanoma patients. In specialised hospitals, we assumed WGS to be clinical practice, following the developments of the CPCT. For both NGS and WGS, we assumed in the future no required additional tests caused by inefficiencies.An overview of the specific test types per time frame and hospital type, including the costs per unit as discussed in the next part, is given in In peripheral hospitals, the average diagnostic test costs per NSCLC patient in the old, current, and future situation were, respectively, €295; €590, and €606. In the specialised hospitals, the test costs were, respectively, €1103; €1504, and €1100. For melanoma patients in peripheral hospitals, these costs were, respectively, €295, €295, and €606 and in specialised hospitals €559, €753, and €1100. In The yearly budget impact for NSCLC patients was as follows: the current situation will result in a test cost increase of €327 per patient, followed by a cost decrease of €110 in the future situation. For the total population in The Netherlands, this will be, respectively, an annual increase in €1,321,243 and annual decrease in €120,473. For melanoma patients, an increase of €136 and €336, respectively, in test costs per patient is expected, resulting in a yearly budget impact of €108,526 and €351,799 for the total population. A summary of the BIA results is given in The sensitivity analysis of the test costs in the current NGS situation of NSLCC in specialised hospitals showed a range of €606 to €2614. For the base value, €1504 as shown in the ‘costs per unit’ section, it is assumed that per NGS run 24 samples are included, per week one run is executed, on average 85% of the run is filled and three additional single gene tests are required. The biggest reduction in test costs can be achieved by a reduction in additional tests besides NGS. The base value, range, and the effect on the test costs are shown for each parameter in the tornado plot in In total 172 patients were included, all stage-IV NSCLC and melanoma patients within the chosen time periods have been selected. The test-related measures, mutation, and treatment characteristics for the period before and after NGS implementation are shown in As far as we know, this is one of the first published cost analysis and budget impact analysis regarding NGS. As expected, our results showed that the more samples are included and runs are performed, the less costly NGS will become. Regarding budget impact, we showed that currently NGS resulted in an increase in test costs, from 2012 to 2015 yearly €1.32 million for the total NSCLC population and €0.11 million for the total melanoma population. The finding of these current budget impacts was confirmed by the NKI case study. From the current to future situation, our BIA showed for the total NSCLC population an expected annual decrease of €0.12 and for the total melanoma population an increase of €0.35 million. This decrease in NSCLC patients was related to the more efficient use of testing by WGS in specialised hospitals, without the need of additional tests. The increase in future test costs for melanoma patients was related to both the implementation of NGS in peripheral hospitals and the implementation of WGS in specialised hospitals, while no additional test costs were involved. Our sensitivity analyses produced widely varying budget impact estimates in the future; especially the price of the small TGP in NSCLC and the price of WGS in melanoma patients had a high impact.The ABC analysis produced a wide range for the estimate of NGS test costs depending on the number of samples per run, number of runs per week, device, and type of TGP. These calculated test costs are comparable with other recently published cost analysis of NGS techniques. For example, in a situation with six samples per run 600–900 USD was calculated for a similar small TGP and 2000 USD for a medium TGP . Policy Our analyses have some limitations. Firstly, we made several assumptions in the BIA. In the future situation, we assumed no inefficiencies of NGS or WGS that require additional tests and no findings of new clinical relevant modifications not traceable with WGS. We also assumed that the current costs of molecular diagnostic tests in clinical practice are representative for the future and we have made an estimation for the WGS costs reduction in five years. The second limitation of our BIA is the fact that the consequences of NGS are not taken into account; in particular, treatments costs can have a major impact on the total budget. By identifying more targetable targets, NGS can result in an increased use of targeted therapies . HoweverNevertheless, the NKI case study showed interesting and comparable results with our main Dutch BIA. Between the period before and after NGS implementation, only differences in average number of tests and associated costs were found. No significant differences in mutation and treatment characteristics were found, except for the number of observed mutations in melanoma patients. We expected beforehand that no difference in EMA approved targeted therapy use would be observed, since the number of druggable targets is low and within the time period no additional targeted therapies were EMA approved . HoweverBased on our analysis, we expect that the concerns, of NGS resulting in high additional test costs, will be limited. Our BIA does show that NGS currently results in higher costs, which is mainly related to the required additional tests besides the small TGP. However, it is expected that these additional tests become (partly) superfluous in the future. Furthermore, the direct clinical benefits of NGS are currently limited, since no treatment consequences are yet expected due to the low number of druggable targets. Nonetheless, the research potentials of NGS are already an important advantage.Our cost analysis of NGS and the NKI case study can assist hospitals in the decision which panel or device is most beneficial for them. Based on our analysis, we would suggest a small TGP as currently the best NGS option for diagnostics and we recommend NGS process optimisation to focus on the reduction of additional tests. Our research provides useful input for a health technology assessment of NGS. Interesting follow-up steps would be an extension of our BIA approach to the total cancer population within The Netherlands and the inclusion of clinical data including treatment costs. Furthermore, the inclusion of additional future scenarios with liquid biopsies or a centralised NGS test option within The Netherlands, as proposed by the CPCT/MHF, would be of interest as well.NGSNext-generation sequencingWGSWhole-genome sequencingWESWhole-exome sequencingNSCLCNon-small-cell lung cancerABCActivity-based costingBIABudget impact analysisTGPTargeted gene panelNKINetherlands Cancer InstituteDNADeoxyribonucleid acidCPCTCentre for Personalised Cancer TreatmentHMFHartwig Medical FoundationSTZDutch teaching hospitalsUMCUniversity Medical CentreEMAEuropean Medicines AgencyEGFREpidermal growth factor receptorALKAnaplastic lymphoma kinaseBRAFRapid accelerating fibrosarcoma type B oncogene B1HER2Human epidermal growth factor receptor 2KRASKirsten-rat sarcoma 2 viral oncogene homologROSreceptor tyrosine kinaseRETrearranged during transfectionMETMNNG-HOS transforming geneHTAHealth technology assessmentTSACPTruSeq Amplicon Cancer PanelISPORInternational Society for Pharmacoeconomics and Outcomes ResearchAuthors declare no conflict of interest regarding the work described in this manuscript.RVA held in-house interviews, collected the input for the cost analysis and BIA, processed the data, analysed the results, and drafted the manuscript. VR held in-house interviews, coordinated the input collection, analysed the results, and co-drafted the manuscript. VC helped to set up the BIA, advised on the results analysis, and critically revised the manuscript. PN provided information on the test activities, process costs, and patient data, gave insight in future expectations and critically revised the manuscript. MJ provided information on the test activities, gave insight in future expectations, and critically revised the manuscript. WVH conceived the work, coordinated the project and helped drafting the manuscript. All authors read, revised, and approved the manuscript.


Data from human clinical trials have shown that the hepatitis B virus (HBV) follows complex profiles, such as bi-phasic, tri-phasic, stepwise decay and rebound. We utilized a deterministic model of HBV kinetics following antiviral therapy to uncover the mechanistic interactions behind HBV dynamics. Analytical investigation of the model was used to separate the parameter space describing virus decay and rebound. Monte Carlo sampling of the parameter space was used to determine the virological, pharmacological and immunological factors that separate the bi-phasic and tri-phasic virus profiles. We found that the level of liver infection at the start of therapy best separates the decay patterns. Moreover, drug efficacy, ratio between division of uninfected and infected cells, and the strength of cytotoxic immune response are important in assessing the amount of liver damage experienced over time and in quantifying the duration of therapy leading to virus resolution in each of the observed profiles. Hepatitis B virus (HBV) infection affects 250–350 million people worldwide . UniversTwo types of drug therapies have been approved for treating HBV chronic disease: interferon (standard and PEGylated) and nucleos(t)ide analogs . These m effects ,6. The n effects . Combina effects . The lim effects  and how  usually 2 months  effects .Insight into the trade-off between the duration and strength of therapy, the role of immune responses, and the therapy outcome can be provided through mathematical modeling ,11,12. TThe paper is structured as follows. In T, the productively infected hepatocytes I, and the hepatitis B virus V. Briefly, both uninfected and infected hepatocytes are maintained by homeostatic proliferation described by a logistic equation, with carrying capacity K and maximal per capita division rates p virions and virus is cleared at rate c. The combination therapy  under what conditions model ( under whun when:R=(1−η) will beOur goal is to determine the parameter regions for which the solution of by model  follows Note that all parameters in model  have clen values . The remn ranges . AlthougMC.1The initial condition  exists aMC.2The components of initial condition  are bounMC.3The drug-dependent virus asymptotic removal condition  holds, iMC.4t. This condition guarantees that the total liver cell population never decays below K [The total hepatocyte population is bound as follows ration K .In this study we numerically investigate the dynamics of model  with posgiven by  under thIn order to find model parameters which lead to bi-phasic and tri-phasic Using this statistical method, we aim to determine which parameter regions are predominantly leading to bi-phasic and tri-phasic Initially, we investigate the entire parameter space by randomly generating L have a darker red shade. Moreover, we compare the length of the flat phase L and its frequency among the samples, and find it to average at 10 days, with a range of 1 to 30 days  either a large ratio between uninfected and infected cell division rate oval see B,C; (2) erns see C; (3) 30oval see B,D; and ≤0.8 see D.To better determine the relative effects of erns see A, while py ϵ see A. If we ions see .In the subcase S2, we assumed that the uninfected and infected cell division rates wise see B. This ioval see . Moreoveerns see .Lastly, in case S3, we keep the the uninfected and infected cell division rates  studies . For thierns see C. When wered see .In all considered cases, we were unable to properly distinguish the parameter regions that completely separate the bi-phasic and tri-phasic Since we could not properly classify the samples, giving bi-phasic and tri-phasic To test this intuition, we grouped the samples obtained in the three subcases S1–S3  for the first ve model , and sepgiven by , and theIn subcase S4, we find that the amount of liver turnover due to immune response-induced killing is higher for tri-phasic than for bi-phasic erns see A. In parerns see  predictserns see B. There atio see . Indeed,Experimental and modeling studies have estimated the liver turnover when the infection is resolved to range between and 100% ,27,28,29The prediction that the samples explaining tri-phasic g rate δ . Since tTo determine the relationship between the initial conditions and the given by , over threatment C. This sIn this study, we investigate the mechanisms of virus decay in hepatitis B infections following the initiation of drug therapy. Data from drug clinical studies have shown that HBV DNA follows either a bi-phasic or a tri-phasic decay pattern ,16,17,18Using the mathematical model developed in , we showTo determine the relative contributions of these parameters and mechanisms we employ a statistical approach. By using rejection sampling methods we eliminate the parameter samples which do not lead to feasible virus dynamics. The distribution of the remaining parameters reveal how the virus dynamics are influenced by the sizes and correlation of parameters.We find that high drug efficacy is needed for any type of virus decay. Moreover, high uninfected to infected division rate combined with high immune response induced cytotoxic killing of infected cells result in bi-phasic viral decay patterns. This result indicates that bi-phasic decay patterns are better for a patient prognosis. We show that activation of the immune responses following the start of therapy is beneficial for the patient prognosis. This finding is supported by other experimental ,32 and mFor therapy that is and 100% ,27,28,29Our investigations could not find significant differences between the pattern of virus decay when either the uninfected to infected cell division rate, the immune response killing rate, or both have small or intermediate values. For such ranges, both bi-phasic and tri-phasic viral decay patterns can be observed. Moreover, for these small to intermediate On the other hand, in the tri-phasic samples, we find a correlation between high level of hepatocyte infection at the start of the treatment (greater than odchucks . The exaOur model does not consider non-cytotoxic immune responses, nor did it consider the relation between the liver resolution and the patient outcome. Data on the loss of other virological markers, such as the covalently closed circular DNA and surface antigens, are needed to further assess the relationship between the pattern of virus decay and the success of treatment.In conclusion, we have investigated the mechanisms behind two types of hepatitis B virus decay patterns following the start of drug therapy and found that high uninfected to infected division rate combined with high cytotoxic killing of infected cells results in bi-phasic virus decay, while infection of a large percentage of liver at the beginning of treatment results in tri-phasic virus decay.


We conducted an external quality assessment of Zika virus molecular diagnostic tests in Brazil using a new Zika virus standard. Of 15 laboratories, 73% showed limited sensitivity and specificity. Viral load estimates varied significantly. Continuous quality assurance is required for adequate estimates of Zika virus–associated disease and determination of patient care. The catastrophic Zika virus outbreak in the Americas has affected millions of persons. Brazil was the most affected country and reported ≈95% of all cases of suspected Zika virus–associated congenital disease  in Europe revealed that 60% of laboratories need to improve molecular Zika virus detection . This finding suggests a potential lack of sensitivity that may be problematic given that viral loads of 103–104 copies/mL are commonly observed in Zika virus–infected patients  reported correct results for all samples. Five (33%) reported 1 or 2 false-negative results from samples with low Zika virus concentrations , panel A>3 false results, including at least 2 false-positive detections of Zika virus–negative specimens. No heterologous flavivirus was particularly affected by false-positive detection, suggesting that false-positive results did not result from unspecific binding of assay oligonucleotides (Six (40%) laboratories reported leotides . InsteadEQA performance varied according to the way viral RNA was prepared. The 8 laboratories conducting Zika virus detection using automated platforms performed generally superior  compared with the 7 laboratories conducting manual RNA extraction  . This fiAs previously reported , panel B10 median deviation between results (p = 0.429 by Wilcoxon signed rank test). This observation suggests usability of the armored RNA for Zika virus quantification in tropical regions. Irrespective of the standard, viral load determinations among laboratories were comparable with 0.12–0.88 log10 median deviations of viral load estimates among laboratories for individual Zika virus specimens. However, we also observed drastic deviations of up to 6 orders of magnitude (Quantification of Zika virus loads did not differ significantly between use of the armored RNA and the WHO Zika virus standard, with only 0.76 logagnitude , suggestSome laboratories in Brazil showed suboptimal sensitivity and specificity of Zika virus diagnostic testing. However, these laboratories performed comparably to those in Europe (Independently of the challenges of Zika virus molecular detection, because of taxation and distributor margins, RT-PCR reagents in Latin America are usually 100%–200% more expensive than in affluent countries (Finally, lack of sensitivity directly affects estimates of the absolute risk for Zika virus–induced congenital disease upon maternal infection during pregnancy (


Pteromalus puparum). The complete viral genome is 12,230 nucleotides in length, containing five non-overlapping, linearly arranged open reading frames. Phylogenetically, the virus clusters with and is a novel member of the mononegaviral family Nyamiviridae, here designated as Pteromalus puparum negative-strand RNA virus 1 (PpNSRV-1). PpNSRV-1 is present in various tissues and life stages of the parasitoid wasp, and is transmitted vertically through infected females and males. Virus infections in field populations of P. puparum wasps ranged from 16.7 to 37.5%, without linearly correlating with temperature. PpNSRV-1 increased adult longevity and impaired several fitness parameters of the wasp, but had no influence on successful parasitism. Strikingly, PpNSRV-1 mediated the offspring sex ratio by decreasing female offspring numbers. RNA interference knockdown of virus open reading frame I eliminated these PpNSRV-1-induced effects. Thus, we infer that PpNSRV-1 has complex effects on its insect host including sex ratio distortion towards males, as well as possible mutualistic benefits through increasing wasp longevity.Parasitoid wasps are important natural enemies of arthropod hosts in natural and agricultural ecosystems and are often associated with viruses or virion-like particles. Here, we report a novel negative-stranded RNA virus from a parasitoid wasp ssRNA] viruses in parasitoids wasps have not been described. In this report, we identify a novel (-)ssRNA virus from a parasitoid wasp, verify the presence of virion-like particles in various wasp tissues, and characterize the viral genomic structure, transcription strategy, phylogenetic relationship, transmission strategy, and biological impacts on its host. Most importantly, we reveal that this novel virus mediates secondary sex ratio of its host by decreasing female offspring numbers, and can be transmitted both by male and female to offspring. Decrease in female offspring could be due to increased female mortality or to alterations in the primary sex ratio. The reduced female offspring number per female could therefore be a fecundity cost or a tradeoff for increased longevity of the wasp. However, it remains to be determined whether increased longevity under laboratory conditions translates into increased lifetime fecundity for the wasp in nature. Increased longevity could be beneficial to the virus by promoting its transmission to more hosts. The secondary sex-ratio distortion induced by the virus may also increase its transmission in host populations, because males transmit the virus to their offspring and can mate with multiple females, thus further spreading the virus. Ascoviridae, Polydnaviridae, Poxviridae), positive-sense, single-stranded RNA [(+)ssRNA] viruses , and segmented double-stranded RNA [dsRNA] viruses (Reoviridae) ) probably belong to the family Iflaviridae in the order Picornavirales; whereas the third virus (NvitV-3) is most similar to the unclassified Picornavirales member Nora virus, which infects fruit flies  and ds-ORF1-2(+): , t = 1.6558, df = 111, p = 0.1006] and ds-ORF1-2(+): , t = 2.3281, df = 58, p = 0.0234] and ds-ORF1-2(+): , t = 1.9638, df = 185, p = 0.0511). The longevity of both female and male wasps in the PpNSRV-1 dsRNA treated colonies was shorter than that of the ds-eGFP(+) colony, but longer than that of the ds-eGFP(-) colony P. puparum) and verified the presence of PpNSRV-1 particles in various wasp tissues. In addition, we characterized the PpNSRV-1 genome organization, its phylogenetic placement, transmission strategy, and biological impacts on its host. Our results represent the first unambiguous detection of a (-)ssRNA virus in insect parasitoids. Intriguingly, we revealed that PpNSRV-1 mediates the secondary sex ratio of its host wasp by decreasing female offspring numbers.We detected and sequenced the genome of a novel (-)ssRNA virus, PpNSRV-1, from parasitoid wasps  of mononegaviral genomes . Indeed,Nyamiviridae. This is a relatively novel family, which currently includes two genera, Nyavirus  and Socyvirus (soybean cyst nematode virus 1 [SbCNV-1])  from injected wasp pupae were individually placed into tubes and reared as described above for determination of longevity.To further confirm the effect of PpNSRV-1 infection on P. puparum wasps, we utilized RNAi knockdown of PpNSRV-1 ORF I to reduce the abundance of PpNSRV-1, and then evaluated effect of this knockdown on wasp fitness parameters. The ORF I-specific primers and primers targeting enhanced green fluorescent protein gene  were designed with added T7 promoter adaptors  into each yellow wasp pupa from the PpNSRV-1(+) or PpNSRV-1(-) colony. The PpNSRV-1 titer in the wasps emerging from injected wasp pupae was quantified by qPCR. Then, 30 injected female wasps that had mated with injected male wasps for 48 h were selected to investigate the successful parasitism rate, adult emergence proportion, and offspring number. Newly emerged wasps were collected to measure longevity as described above.We injected 50 nl of dsRNA (2 × 10p <0.05). The means on each same measured biological parameter were compared between two colonies with and without carrying PpNSRV-1 using Student’s t-test . Linear regression analysis was constructed between viral detection rate and mean temperature from the five different cities in October, 2012, and the three various cities in June, 2016. Temperature was calculated based upon data from the Weather Underground website (https://www.wunderground.com). All statistical calculations were performed using Data Processing System (DPS) software (version 14.50) [All values were expressed as mean ± standard error (S.E.M.). For qPCR and RNAi results, data were analyzed using a one-way ANOVA or two-way ANOVA followed by Tukey’s multiple comparison test (n 14.50) .S1 Fig(-) means virus-free and (+) means virus-infected.(TIF)Click here for additional data file.S2 Fign = 3). Differences in viral load were not statistically significant (t-test). (C) Comparison on adult longevity between PpNSRV-1-free and PpNRSV-1-infected wasp colonies naturally reared at 35°C. (-) means virus-free and (+) means virus-infected. The number in column is the sample size for each treatment. Data represent means ± standard error. Statistical significance (t-tests) is indicated by asterisks: *, p < 0.05; **, p < 0.01.(A) and (B) Viral load of PpNSRV-1 in adult wasps at 1, 3, 5, and 7 day post-eclosion reared at 25°C and 35°C respectively. Data represent means ± standard error ((TIF)Click here for additional data file.S3 Fig(A) Electron micrographs of purified PpNSRV-1 virions. (B) and (C) are the magnification pictures. Red arrows indicated viral particles.(TIF)Click here for additional data file.S4 Fig(A) Detection of PpNSRV-1 in individuals of different generations from virus-free and virus-infected wasp colonies. (-) means virus-free and (+) means virus-infected. The Arabic numbers represent the generation number. Vertical transmission of PpNSRV-1: (B) ♀+/♂+; (C) ♀−/♂−; (D) ♀+/♂− and (E) ♀−/♂+. M = marker .(TIF)Click here for additional data file.S5 Figt-test).(-) means virus-free and (+) means virus-infected. The number in the columns is the sample size for each treatment. Data represent means ± standard error. Differences in oocyte number in the two colonies were not statistically significant ((TIF)Click here for additional data file.S6 Figt-tests) is indicated by asterisks: *, p < 0.05; **, p < 0.01.Comparison of offspring per parasitized pupa , offspring per female wasp  among wild colonies or injected pupae in each batch. (-) means virus-free and (+) means virus-infected. Batch means the experimental pupae of the same day. Data represent means ± standard error. Statistical significance ((TIF)Click here for additional data file.S7 Fign = 3). SE bars annotated with the same letter are not significantly different (Tukey’s multiple comparison test).Viral load was determined in black pupae and adults at days 1 and 3 post-eclosion. Data represent means ± standard error ((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The mean death time (MDT) was statistically not different among the three age groups, ranging between 3.2 and 4.8 days. All broilers that became infected shed high levels of virus with transmission to contacts and demonstrated severe pathology. Mortality and virus shedding results indicated that age is not a determinant factor in susceptibility of broilers to H5N2 clade 2.3.4.4 HPAI virus. Previously, the Tk/MN/15 virus had a BLD50 of 3.6 log10 EID50 and MDT of 2 days in White Leghorn chickens and a BLD50 of 5.0 log10 EID50 and MDT of 5.9 days in turkeys, suggesting that the broiler breed is less susceptible to Midwestern H5N2 virus than the layer breed but similarly susceptible to turkeys. Therefore, genetic resistance of broilers to infection may have accounted only partially for the lack of affected broiler farms in the Midwestern outbreaks, with other contributing factors such as fewer outside to on farm exposure to contacts, type of production management system or enhanced biosecurity.In 2014–2015, the US experienced an unprecedented outbreak of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) virus. The H5N2 HPAI virus outbreak in the Midwest in 2015 affected commercial turkey and layer farms, but not broiler farms. To assess any potential genetic resistance of broilers and/or age-related effects, we investigated the pathogenesis and transmission of A/turkey/Minnesota/12582/2015 (H5N2) (Tk/MN/15) virus in commercial 5-week-old broilers, 8-week-old broilers, and >30-week-old broiler breeders. The mean bird lethal dose (BLD Anas acuta) [The Asian-origin H5N1 A/goose/Guangdong/1/1996 (Gs/GD) lineage of high pathogenicity avian influenza (HPAI) virus has become widespread across several continents, affecting wild birds, poultry, and humans. In late 2014, Eurasian H5N8 and reassortants H5N2 and H5N1 Gs/GD lineage clade 2.3.4.4 HPAI viruses were reported in North America [While the majority of initial H5 HPAI viruses were detected in wild waterfowl, wild and captive birds of prey, and backyard flocks along the Pacific flyway , most caCoturnix c. japonica) had stronger immune responses than pubescent and aged birds, suggesting an age-related difference in immune function [Albeit HPAI viruses produce high morbidity and mortality in gallinaceous domestic poultry regardless of age –15, age-function .The different genetic background between layer-type and broiler-type chickens may have an effect not only on performance but also on genetic expression and immunological responses –23. DiffDuring the outbreaks in the Midwest in 2015, turkey and layer-type chicken farms were affected but broiler farms were not . The absth 2015. The virus was propagated and titrated by allantoic sac inoculation of 9–10 day-old embryonating chicken eggs (ECE) by standard methods [The influenza A isolate A/turkey/Minnesota/12582/2015 (H5N2) (Tk/MN/15) was used as challenge virus. The virus was isolated from a turkey farm in Minnesota from a sample collected April 18 methods .7 generations. At least three independent chains were combined to ensure adequate sampling of the posterior distribution of trees. BEAST output was analyzed with TRACER v1.4 with 10% burn-in. The FigTree v.1.4.2 program was used to construct and visualize the maximum clade credibility tree.The Tk/MN/15 virus was selected because it is representative of the Midwest H5N2 cluster both phenotypically  and by pCrossed-line WPR × Cornish broiler birds from a commercial producer in GA, USA (an AI-free state during the outbreak) were utilized. The birds had been reared under field management conditions, including a typical US vaccination program which comprised a broad range of vaccines, but not exposed to or vaccinated against AI virus. One week prior to challenge, birds were moved from the field to the controlled environment in SEPRL ABSL-3 facilities to maintain biosafety and biosecurity barriers for H5N2 HPAI virus challenge. Birds were obtained at three different ages: 5-week-old (5w) broilers as early processing age, 8-week-old (8w) broilers as late processing age, and >30-week-old broiler breeders (>30w). Broiler breeders were in lay; all eggs produced during the study were discarded. Prior to inoculation, 30% of chickens of each age group were ensured to be serologically negative for AI virus infection as determined by hemagglutinin inhibition (HI) test. Also, oral swabs were collected before challenge to confirm absence of virus shedding as determined by quantitative real-time RT-PCR (qRRT-PCR). Each experimental group was housed separately in negative pressure isolators with HEPA-filtered ventilation within the animal biosafety level 3 enhanced facilities at Southeast Poultry Research Laboratory. The birds had ad libitum access to feed and water.50) and lethal (BLD50) doses at different ages, each age group was divided into three groups (n = 5/group), each inoculated intranasally with 2 (low dose), 4 (medium dose), or 6 (high dose) log10 mean egg infectious dose (EID50)/0.1 mL of Tk/MN/15 virus . Clinical signs were monitored daily. Oral swabs were collected from all birds on 2 and 4 dpc, placed in 1.5 mL of BHI with penicillin , gentamicin  and amphotericin B , and stored at −80 °C until use. Severely sick birds were euthanized and counted as dead for the next day in MDT calculations. At 14 dpc, surviving birds were bled to evaluate antibody titers and euthanized by intravenous administration of sodium pentobarbital (100 mg/kg body weight).To evaluate the mean bird infectious (BID10 EID50/0.1 mL) of Tk/MN/15 virus to harvest tissue. Upon death, the birds were necropsied and portions of nasal cavity, brain, thymus, trachea, lung, proventriculum, duodenum, pancreas, jejunum–ileum, spleen, kidney, adrenal and gonad, liver, skeletal muscle, comb, and heart were collected in 10% buffered formalin  for histopathologic evaluation and for detection of AI virus in tissues by immunohistochemistry. Brain, spleen, heart, and lung were also collected in BHI with antibiotics to a 1% (wt/vol) concentration for viral RNA quantification by qRRT-PCR.For each age group, two additional birds were challenged with the high dose  following the manufacturer’s instructions. In tissue homogenates, and in order to standardize the amount of nonspecific RNA from the tissue, the resulting viral RNA extracts were quantified by NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific) following the manufacturer’s instructions and accordingly diluted with phosphate buffered saline to obtain 50 ng/μL. The resulting viral RNA extracts, diluted (tissue homogenates) or undiluted (swabs), were quantified by one-step qRRT-PCR which targets the influenza matrix gene [10 EID50/mL (3.0 log10 EID50/g for tissue homogenates); for statistical purposes, qRRT-PCR negative samples were given a numeric value of 1.9 log10 EID50/mL (2.9 log10 EID50/g).Swabs and tissues in BHI were processed for qRRT-PCR to determine viral RNA titers. Viral RNA was extracted using MagMAX™-96 AI/ND Viral RNA Isolation Kitrix gene  using 75p value of <0.05 was considered to be significant.The D’Agostino and Pearson test was used to assess the normality of distribution of investigated parameters. All parameters in our study were not normally distributed. Significant difference for mean viral titers in tissues between groups was analyzed using Kruskal–Wallis test or Mann–Whitney test (GraphPad Prism™ Version 5 software). A Tissues in 10% formalin were processed for routine hematoxylin/eosin staining. Tissues were also processed for immunohistochemical staining using a mouse-derived monoclonal antibody  specific for type A influenza virus nucleoprotein, as previously described .3) were considered negative.Sera samples were tested by HI assays against the challenge antigen. The antigen was prepared as previously described  and the The HA gene of H5N1, H5N2, and H5N8 viruses were grouped by subtypes and geographical regions . The surviving inoculated birds did not show evidence of clinical disease and they were all serologically negative based on HI data, thus all survivors were considered uninfected. The BID50 and the BLD50 for Tk/MN/15 virus were the same in this study and resulted in 5.0 log10 BLD50 for all the age groups . At 4 dpc, mean virus titers of 4.0, 6.4, and 7.6 log10 EID50/mL were shed orally by 5w, 8w and >30w, respectively  at 1 dpe (2 dpc in the figure)  to 9.2 log10 EID50/g of heart (5 and >30w) .Brain, spleen, heart, and lung were collected for viral RNA quantification by qRRT-PCR from two necropsied birds per age group challenged with the high dose of Tk/MN/15 virus. Mean virus titers per tissue were calculated using the two birds from each age group necropsied at 3 dpc. High virus loads were detected in all the tissues and in all necropsied birds, ranging from 6.4 logThe H5N2 HPAI virus outbreaks in the Midwest during 2015 affected commercial turkey and layer chicken farms, but not broiler farms . To dete50 of 4.7 log10 EID50 was determined to be the upper cut-off for sufficient poultry adaptation for sustained transmission and spread of viruses between two or more premises in the field [10 EID50 became infected and died which resulted in an estimated BID50 and BLD50 for Tk/MN/15 virus of 5.0 log10 EID50 in all age groups. This concurs with a previous study with 3 and 6w commercial broilers that experienced 100% mortality following inoculation with 6.5 log10 EID50 of this same virus . In our study, the BID50 obtained is similar to the upper cut-off value, suggesting inadequate or sub-optimal adaption for sustained transmission in broilers. The MDT was statistically not different among the three age groups, being 4.8 dpc for 5-week-old broilers, and 3.2 dpc for 8-week-old broilers and the >30-week-old broiler breeders. Collectively, the BID50 and BLD50 results suggest that differences in age are not a determining factor in the susceptibility of broilers for Tk/MN/15 virus, and that this virus lacks optimal adaptation to broilers and/or broilers are mildly genetically resistant to infection by this virus.Previously, the experimental BIDhe field . In the 10 EID50 of Tk/MN/15 virus shed high levels of virus prior to death resulting in transmission to contact exposed birds. The lack of significantly different amounts of virus shed further confirmed the absence of age-related differences in susceptibility. Broilers of all ages inoculated with the low and medium doses of Tk/MN/15 virus did not become infected based on lack of mortality, virus shedding, and seroconversion. Collectively, our findings concur with DeJesus et al. [Virus shedding was in line with mortality results. Broilers of all ages inoculated with 6.0 logs et al.  that obss et al. , 37, 40.50 and BLD50 of 5.0 log10 EID50 as broilers, but a longer MDT of 5.9 dpc [50 and BLD50 of 3.6 log10 EID50 and MDT of 2.0 dpc in 4-week-old specific pathogen free (SPF) White Leghorn chickens. All the studies combined suggest that the broiler breed is less susceptible to Midwestern H5N2 poultry viruses than the layer breed, but similarly susceptible as turkeys. Interestingly, 80 and 90% of the layer and turkey farms, respectively, affected during the Midwestern outbreaks were premises with birds over 9 weeks old, and no premises with birds younger than 4 weeks old were affected [Recently, 4-week-old turkeys experimentally challenged with Tk/MN/15 virus had the same BID 5.9 dpc  using thaffected . These daffected , 43. Alsaffected .50, the Tk/MN/15 virus is slightly less adapted to broilers than to layers, which may partly explain why layer chickens and not broiler chickens were affected during the outbreak. However, the high pathogenicity of the virus and the readiness of replication and transmission in broilers exposed to high doses of Tk/MN/15 virus indicate that good biosecurity practices and disease control measures likely contributed to the prevention of HPAI virus being introduced and spread in broiler premises.In conclusion, the present study indicates that age is not a determining factor in susceptibility of broilers to H5N2 clade 2.3.4.4 HPAI virus. The epidemiology of infected premises of the H5N2 outbreak in the Midwest in 2015 also appears consistent with the data presented in this study. Based on BLD


In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3− as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3− as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.Alginate beads represent a simple and highly reproducible IMPORTANCEPseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation. Interestingly, P. aeruginosa growing without NO3− showed a higher growth rate during the initial 12 h of growth than with NO3− (P < 0.001). After 24 h, the apparent growth rate was highest for the NO3− group (P < 0.001), while there was no significant difference between the two groups after 48 h, and after 72 h, the apparent growth rate was again the highest for the NO3− group (P < 0.001). There was a negative correlation between time and apparent growth rate for beads without NO3− (P = 0.005) but not for beads with NO3−; this difference in effect was significant (P = 0.003). Peripheral colonies directly on the surface of the bead  were excluded from the statistical analysis as these colonies had an ample supply of O2.When grown in alginate beads,  CF lung . The agglung (3− . We foun< 0.001) . The appwth rate . We foun3− (P = 0.0059). The same pattern applied to the total biomass (P = 0.0012). To assess the suitability of the alginate bead model for mimicking aggregate size as observed in chronic infections, we used area measurements (rather than volume) as data on CF lung tissue and chronic wound samples are only available in two dimensions (2D). The average (± standard deviation [SD]) cross-sectional areas of P. aeruginosa aggregates in the bead model after 24 h of growth were 77 μm2 ± 59 μm2 and 175 μm2 ± 100 μm2 in the absence and presence of NO3−, respectively. To determine the spatial structures of aggregates in the beads, we looked at the aggregates closest to the bead surface (T [top]) and compared them with aggregates deeper in the beads (B [bottom]). Our results revealed that significantly more biomass was situated in the bead periphery at each time point without nitrate , but in the NO3−-supplemented beads, the differences disappeared after 24 h, resulting in an almost equal distribution of biomass after 72 h. Furthermore, we observed significantly larger aggregates in the bead periphery after 48 and 72 h of growth  in beads without NO3−   .2 measurements showed a linear decrease in O2 concentration over time . Respiration rates of P. aeruginosa grown planktonically at 100 and 180 rpm were significantly higher  than the volumetric respiration rate of alginate-encapsulated P. aeruginosa grown at 100 rpm , with 17 upregulated and 153 downregulated genes. Some of the most notable upregulated genes were ibpA and the Anr-regulated genes arcDABC, uspK, and uspN (see Table S2 for roles and descriptions). When comparing alginate-encapsulated P. aeruginosa to an alginate-encapsulated reference with NO3− supplementation, 141 genes were >3-fold differentially expressed, with 29 exhibiting upregulation and 112 showing downregulation. Besides the previously mentioned upregulated genes, we found oprG and the ccON2-encoded gene to be >3-fold induced (data not shown).In support of the fact that alginate-encapsulated bacteria display a significantly lower volumetric respiration rate than their planktonic counterpart and physiological zonations are present in the beads due to steep OP. aeruginosa supplemented with NO3− versus planktonic culture) we found similar genetic expressions between the two  were identical to the 170 genes from the first comparison . Accordi the two  and B, a the two   when comparing the NO3−-supplemented beads to the reference without NO3−, and likewise, when comparing to the planktonic reference, we found an ∼2.5-fold induction of narK1 and narI and an ∼2-fold induction of norB.None of the genes related to denitrification were induced >3-fold when comparing alginate-encapsulated rpm, rpl, and rps) (framed in Table S2). Furthermore, we found a broad repression of genes associated with iron regulation. The following genes, all regulated by the ferric uptake regulator (Fur), were repressed in alginate-encapsulated P. aeruginosa in comparison to the planktonic reference: sigma factor PvdS PA2426 (PvdS), ferri-siderophore receptor genes PO2398 (fpvA) and PA4221 (fptA), siderophore (pyochelin) biosynthesis genes PA4226 (pchE) and PA4228 to PA4231 (pchDCBA), and siderophore (pyoverdine) system-related genes PA2386 (pvdA), PA2394 (pvdN), PA2396 to PA2399 (pvdFEAD), and PA2401 (pvdJ) (see Table S2).The downregulated genes were involved in translation, posttranslation, and degradation, predominantly genes encoding ribosomal proteins in the 30S and 50S subunits of the 70S ribosome (P. aeruginosa cells were susceptible to tobramycin at 100× the MIC (100 μg ml−1) immediately after encapsulation, when the bacteria were still in a planktonic state (P. aeruginosa prevailed for 24 h in the presence of tobramycin at 100× the MIC (100 μg ml−1)  and viability staining with Syto9 and propidium iodide (PI) revealed that alginate-encapsulated ic state . Howeverμg ml−1)  and F.in vitro model system that recapitulates the physical aspects of nonattached aggregate growth observed in chronic infections and provides a versatile platform for studying bacterial aggregates. The model uses alginate-encapsulated , which is within the same range. In a review by Bjarnsholt et al. . Thus, alginate-encapsulated P. aeruginosa had an aggregate size , PA4352 (uspN), PA5170 to PA5173 (arcDABC), PA4067 (oprG), and PA1557 (ccON2-encoded gene) in alginate beads corresponds to results from previous studies showing that these genes were highly expressed in P. aeruginosa biofilms    and rhlR (fis) (sodM) and PA4470 (fumC) were also downregulated, which is concordant with the general repression of genes related to iron limitation, as sodM  –45 encod E. coli , but is  E. coli . In the fication . Thus, wnarGHJI)  when coma et al. , the elearginine  and pyruarginine . We founlR (fis) . The genlR (fis) . PA4468  as sodM  and fumCdM  constitutively expressed by plasmid pMRP9    of 0.1 in alginate. Droplets of the alginate with bacteria were dispensed via a 21-gauge needle placed 3 cm above the surface of a stirred 0.25 M CaCl2 solution, wherein the beads were hardened for 1 h. This procedure was previously reported to yield spherical and stable beads  , 35. An le beads . We prod2CO3 and citric acid  of the cut surface with an emphasis on visualizing the edge of the bead and as much of the bead interior as possible. Images were recorded as z-stacks in 1-μm increments with a 40×/1.3 numerical aperture (NA) oil immersion objective. Image analysis was performed using Imaris v8.3.1 . To calculate 2-dimensional cross-sectional areas of aggregates in the beads after 24 h of growth with and without NO3−, we excluded aggregates <10 μm2 and aggregates touching the edge of the image to avoid planktonic bacteria and incomplete aggregates. To elucidate whether the ability to grow anaerobically impacted the spatial structure and distribution of the bacterial aggregates in beads with and without NO3−, the images were split in half ∼106 μm from the surface of the bead across the x axis to separate the top (T) half from the bottom (B) half of the image, and T and B were compared statistically. Total biomass , average aggregate volume (object volume), and z positions were calculated with the ImarisVantage module. The total biomass was calculated by first subtracting background fluorescence from all 3-dimensional image stacks. The background fluorescence in the green channel was calculated by creating histograms of three different areas of the edge of the image, and the highest voxel value was determined. The three values were then averaged and a value of 92 voxels was determined as the background fluorescence. Isosurfaces were created of the remaining voxels and the sum of all individual objects was used to calculate the total biomass. The average aggregate volume was also calculated.For image analysis of the spatial organization and growth of bacterial aggregates, alginate-encapsulated Imaging of the alginate bead sections for quantitative PNA-FISH was performed with the same settings used for acquiring all the pictures. Fluorescence images were recorded as 1-μm z-stacks at a resolution of 4,096 by 4,096 pixels, with an averaging of 2 at 16-bit color depth, using a 63×/1.4 NA oil immersion objective and 594 nm laser excitation. Microcolony fluorescence was quantified using ImageJ  using a previously described procedure . Colony P. aeruginosa was grown with and without NO3−-supplemented alginate and medium, was sampled chronologically after 12, 24, 48, and 72 h, and was stored at 4°C in 4% formalin  with 0.25 M CaCl2 for stabilization. The beads were embedded in paraffin, cut in 4-μm sections with a standard microtome, fixed on glass slides, and kept in the dark at 4°C until further treatment. The sections were deparaffinized and stained with a Texas Red-conjugated 16S rRNA probe  specific for P. aeruginosa as previously described . The optodes were calibrated (in units of μmol O2 liter−1) by a two-point calibration procedure before each experiment using measurements in air-saturated and O2-free R2A at the experimental temperature (37°C) and pH (7.6).Molecular oxygen concentrations were measured with Oor spots , 62 moun2 depletion in the cuvette over time, and the total respiration rate of the beads was calculated from linear parts of the declining O2 concentration versus time curve (in units of μmol O2 liter−1 h−1). Respiration rates of planktonic bacteria were measured in a similar way using planktonic bacteria grown at two different flow speeds, namely, 100 rpm (similar to the beads) and 180 rpm (standard for planktonic cultures). If active cells do not exhibit a homogenous distribution in the beads, the respiration rate (R) will be underestimated. We compensated for the heterogeneous distribution of bacterial cells due to the clustering of bacterial aggregates in the periphery of the beads by using the calculated values of r  at the different time intervals to recalculate R as the volumetric respiration rate . The O2 penetration depth in the alginate beads, r, was calculated from the measured cell density and concentration of O2 at the surface of the beads, 0C .For respiration rate measurements on alginate-encapsulated bacteria, beads were drawn with a transfer pipette from the culture flask, rinsed 3 times with prewarmed 0.9% NaCl, and then transferred to a cuvette filled with prewarmed (37°C) sterile R2A and a glass coated magnet. The cuvette was closed air tight, mounted on a magnetic stirrer, and fitted with the fiber-optic readout cable. Each measurement followed the OcellR, and bead volume-specific, R, respiration rates by using simple diffusion-reaction relations for a spherical geometry as outlined in the supplemental material.Respiration measurements were performed hourly during the first 8 h and the experiments lasted ∼30 h. The respiration rate experiments were conducted on 4 biological replicates, and bacterial cell counts within each experiment were performed in duplicates. Total respiration rates were combined with quantifications of bacterial numbers and growth zonations to estimate cell-specific, 2 microsensor  was mounted on a motorized micromanipulator (MU1) and connected to an O2 meter (FireStingO2); all components were obtained from Pyro-Science GmbH, Germany. Calibration of the microsensor was performed as specified by the manufacturer via measurements in air-saturated and O2-free medium. The position where the sensor touched the bead  was determined visually with the help of a USB microscope . Microsensor positioning and data acquisition were performed with dedicated profiling software . Data were analyzed in Origin Pro 9.0.A single bead was submerged in a petri dish filled with R2A after 24 h of growth in a culture flask. The petri dish was placed on a heated plate (set to 37°C) and gently aerated by a fine air stream directed toward the surface via a Pasteur pipette connected to an air pump. A fiber-optic OdP. aeruginosa after 24 h of growth with and without NO3−. For alginate bead cultures, the beads were harvested and rinsed three times in sterile, prewarmed 0.9% NaCl to remove planktonic bacteria before mixing with two volumes of RNAlater . The samples were stored ON at 4°C before freezing at −80°C until further use. To dissolve the alginate beads before RNA isolation, the frozen beads were thawed at 4°C and ultrasound  was administered at the lowest intensity until the alginate beads appeared completely dissolved. Cells were harvested by centrifugation at 7,000 × g for 15 min at 4°C. The supernatant was removed, and the cell pellet lysed with 100 μl 1 mg ml−1 lysozyme  at room temperature for 13 min. RNA isolation was performed with an RNeasy mini purification kit , and contaminating chromosomal DNA was removed by RQ1 RNase-free DNase treatment . RNA quality and quantity were detected with a NanoDrop spectrophotometer . cDNA synthesis and hybridization were performed by the Microarray Center at the Copenhagen University Hospital (Denmark), and the arrays were scanned in the Affymetrix GeneArray 3000 7G scanner. Cell intensity files (CEL files) were generated in the GeneChip Command Console software (AGCC) . Gene expressions were analyzed using the software Arraystar .RNA was isolated from stationary-phase planktonic and alginate-encapsulateP. aeruginosa was investigated by challenging the beads with 100× the MIC of tobramycin for P. aeruginosa (100 μg ml−1 tobramycin)  , 10 in tt test. Aggregate volumes and biomass were analyzed by multiple t tests. A P value of <0.05 was considered significant.Data were analyzed for statistical significance with SPSS 22 software  and illustrated in GraphPad Prism 6 software  and Origin Pro 9.0 . Respiration rate data were analyzed by linear mixed models. Quantitative PNA-FISH data were compared by a linear regression analysis, a Mann-Whitney U test, and an independent