Severe acute respiratory syndrome coronavirus 2 viral load in the upper respiratory tract peaks around symptom onset and infectious virus persists for 10 days in mild-to-moderate coronavirus disease . RT-PCR cycle threshold (Ct) values correlate strongly with cultivable virus. Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset; it is similar in asymptomatic and symptomatic persons. Asymptomatic persons represent a source of transmissible virus. Since the emergence of coronavirus disease (COVID-19) at the end of 2019, rapid tracing and isolation of confirmed cases and close contacts with restrictions on social movement have played an important role in controlling onward spread of the virus. Understanding the duration of infectiousness in persons who test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to developing evidence-based public health policies on isolation, contact tracing and return to work. Virus detection by reverse transcription-PCR (RT-PCR) from respiratory samples is widely used to diagnose and monitor SARS-CoV-2 infection and, increasingly, to infer infectivity of an individual. However, RT-PCR does not distinguish between infectious and non-infectious virus. Propagating virus from clinical samples confirms the presence of infectious virus but is not widely available, requires biosafety level 3 facilities, and the results are not timely to inform public health actions. The aim of this work was to understand how RT-PCR detection relates to cultivable virus, which can be used as a proxy for infectiousness and can inform and support decisions on infection control.Upper respiratory tract (URT) samples from persons with suspected COVID-19 were tested at the national respiratory virus reference laboratory at Public Health England to support routine clinical care and surveillance activities during the COVID-19 pandemic. Samples included nose, throat, combined nose-and-throat and nasopharyngeal swabs, or nasopharyngeal aspirates; the majority were taken by clinical staff but some were self-sampled nose swabs.In the first 3 months of the COVID-19 pandemic in the United Kingdom (UK) (late January to early April 2020), we received 754 URT samples from 425 symptomatic cases that tested positive for SARS-CoV-2 by RT-PCR targeting the RNA-dependent RNA polymerase (RdRp) gene [2. Cells were inspected for cytopathic effect daily up to 14 days. Presence of SARS-CoV-2 was confirmed by SARS-CoV-2 nucleoprotein staining by enzyme immunoassay on infected cells. Cultivable virus was isolated from 133 (41%) samples (from 111 cases).Virus culture was attempted from 324 URT samples (from 253 cases) that tested positive for SARS-CoV-2 by RT-PCR. Samples were obtained from a range of clinical scenarios including community and healthcare worker surveillance, symptomatic persons tested as part of the early epidemic response and samples acquired in outbreak investigations. Selection of asymptomatic cases was through swabbing of contacts or facility/family/household testing in the context of outbreak investigations; we cannot be certain of their date of exposure or start of infection. Vero E6 cells were inoculated with clinical specimens and incubated at 37 °C, 5% COMedian Ct of all 324 samples was 31.15 (interquartile range (IQR): 27.50–33.86; range: 17.47–41.78). Some 233 cases (92%) were classified as non-severe (asymptomatic or mild-to-moderate) and 20 (8%) had severe illness . There was no difference in Ct values between those with asymptomatic , mild-to-moderate  or severe  illness (p = 0.79). A stratified comparison of the severe cases over time showed a similar result as described above: Ct values were lower  in week 1 than week 2. There were 62 samples from 61 asymptomatic cases and no difference in culture positivity rate was observed: 21 of 62 samples from asymptomatic individuals vs 112 of 262 samples from symptomatic individuals (estimated odds ratio (OR) = 0.66; 95% CI: 0.34–1.31; mixed effects logistic regression model, p = 0.23).We observed a strong relationship between Ct value and ability to recover infectious virus. The estimated OR of recovering infectious virus decreased by 0.67 for each unit increase in Ct value (95% CI: 0.58–0.77) . Virus pThere were 246 samples from 176 symptomatic cases where the date of symptom onset was known, of which 103 (42%) samples from 81 cases were culture-positive. Detection of cultivable virus peaked around the time of symptom onset . Median More than half the samples  tested (from 91 cases) were received more than 7 days after symptom onset and 21% of those (27 samples from 18 cases) were culture-positive; none of the 91 patients had severe illness or were immunosuppressed. Most of these late culture-positive samples (25/27) were taken between 8 and 10 days after symptom onset.There was no significant difference in Ct values (p = 0.12) or culture positivity (p = 0.63) from URT samples received across the different age groups, although this dataset included few children younger than 16 years. The proportion of asymptomatic cases was similar across age groups, except for 81–100 year-olds who were more likely to be asymptomatic than the other age groups  . There wReadouts from semiquantitative RT-PCR using Ct values provide a valuable proxy for infectious virus detection and may help to inform decision-making on infection control. This study adds to the evidence base on duration of infectiousness following mild-to-moderate COVID-19, demonstrating that infectious virus can persist for a week or more after symptom onset, declining over time. At 10 days after symptom onset, in line with current guidance from the World Health Organization  and the This study identified that Ct values and the presence of infectious virus were similar in samples from asymptomatic and presymptomatic persons, compared with those who were symptomatic, and is one of the first reports of virus isolation from cases who remain completely asymptomatic. The findings suggest that asymptomatic and presymptomatic persons do represent a source of potentially transmissible virus. Extensive data on cultivable virus from asymptomatic or presymptomatic individuals are lacking, with one outbreak investigation in a care home reporting detection of cultivable virus in one asymptomatic and 17 presymptomatic cases . AlthougOf note, recall bias may affect the interpretation of timing of virus detection in relation to symptom onset, particularly in elderly patients and those presenting with atypical symptoms. Duration and cessation of symptoms is also not well recorded. For asymptomatic cases, the time when infection was acquired is not known. A further limitation is that this dataset comprises real-world data and subjects were not sampled systematically; therefore, there may be bias in the timing of sampling related to the clinical scenario. Finally, the sensitivity of virus propagation from clinical samples is dependent on laboratory expertise, cell lines and protocols used, and may be affected by sample quality, storage and transport conditions, meaning it is difficult to directly compare data between laboratories described in other literature.Based on the real-world data described here, we recommend that infection control measures for persons with mild-to-moderate COVID-19 be particularly focussed immediately after onset of symptoms and retained for 10 days. Asymptomatic and presymptomatic persons are likely to be a source of infectious virus. Detection of cultivable SARS-CoV-2 from URT samples is valuable as a proxy for infectiousness; however, as the human infectious dose remains unknown, the significance of low titres of infectious virus for human-to-human transmission remains uncertain. Correlation with observational epidemiological data analysing known infector–infectee pairs is required to fully understand the dynamics of infectiousness and viral transmissibility.


Background: Clinical complications of long-term anticoagulation in patients with atrial fibrillation cause significant morbidity and have a substantial economic impact on the healthcare system.Objective: To assess the cost-benefit by implementing patient self-testing (PST) in German patients anticoagulated with vitamin K antagonists (VKA) compared to treatment with the new oral anticoagulant drugs (NOAC) apixaban, dabigatran, edoxaban, and rivaroxaban.Methods: A deterministic decision-analytic model was developed simulating the number of major bleedings, ischemic strokes, and hemorrhagic strokes and their associated costs by utilizing PST compared to those of treatment with NOAC. Data on the rates of these adverse events in both groups during the 1st year of treatment was taken from the NOAC approval studies. Direct costs were evaluated from the perspective of the Statutory Health Insurance (SHI) considering the use of resources directly related to PST testing and costs incurred by hospital treatment of the adverse events. Univariate sensitivity analysis was performed to examine the extent to which our calculations were affected by varying the parameters considered in our model within plausible extremes. To capture the interactions between multiple inputs, we also provided a probabilistic sensitivity analysis (PSA).Results: When achieving an average time in therapeutic range (TTR) of 78%, implementing PST in VKA patients reduces cost per patient compared to NOAC treatment between €603.38 [USD 681.52] (edoxaban) and €762.64 [USD 861.40] (rivaroxaban) during the 1-year observation period. In line with the TTR increase, the initially higher number of adverse events per VKA patient compared to NOAC-treated patients in the approval studies becomes largely aligned; the difference in associated hospital costs per patient in the NOAC groups is then only €1.03 [USD 1.16] (in favor of dabigatran), €23.41 [USD 26.44] (in favor of apixaban), €0.53 [USD 0.60] (in favor of edoxaban) and €52.62 [USD 59.43] (in favor of VKA anticoagulation in the rivaroxaban group).In PSA, implementation of self-management results on average in a cost saving between €619.20 [USD 699.39] and €785.24 [USD 886.93] per VKA patient in favor of the SHI. Under all reasonable assumptions, PST remains constantly less expensive irrespective of which NOAC is administered.Conclusion: Implementing PST in German VKA patients may significantly reduce SHI expenditures compared to utilizing NOAC. Atrial fibrillation (AF) increases the risk of stroke by a factor of 4–5 and accounts for almost 15% of all ischemic strokes.Currently, the non-vitamin K oral anticoagulants (NOAC) dabigatran, rivaroxaban, apixaban, and edoxaban are approved as potential alternatives of VKA treatment.However, one major point of criticism as stated by the Drug Commission of the German Medical AssociationIn a cohort study of German AF patients using claim data of the most representative German SHI, the “Allgemeine Ortskrankenkassen”  NOAC exposure was associated with significantly higher incidence rate ratios for death or non-specified strokes, myocardial infarction and severe bleeding suggesting that NOAC therapy doesn’t seem to be more effective and safer than a VKA therapy in “real life.”Roseet al.Indeed, there is evidenceRose’s approach we defined mathematical formulas that allowed us to simulate the effects on the number of adverse events in the warfarin control groups of the original NOAC approval studies. We calculated the number of events that occurred when the original TTR in the studies would now uniformly increase to a mean TTR of 78%Derived from In a second step we assessed the costs of NOAC and VKA treatment of AF patients when PSTOur aim was to examine the possible economic advantages of implementing PST in VKA patients compared to administering NOAC, when supervised by a properly trained general practitioner (GP). Since in Germany the short-acting warfarin is only rarely prescribed replaced it in our model of long-acting phenprocoumon, which is consumed by 98% of the German VKA patientsEthical approval was not necessary as only publicly available secondary data were used.Our model was parametrized by data on the rates per patient and year of three main adverse events in the study populations of the four NOAC approval publications.® system, test strips and lancets, seeWe assume that a relevant increase in the TTR is possible by PST under the supervision of a GP. Only costs to be carried by the SHI that arise on the legal basis of the German social security statute book (SGB) V are included. These are the costs of medication (NOAC and phenprocoumon), aids (CoaguChek®] 2019), respectively. Any discounts granted by the manufacturers of NOAC to some insurances are not taken into account for reasons of transparency.The outpatient costs refer to the National Association of Statutory Health Insurance Physicians’ Uniform Assessment Standard (EBM),A deterministic, patient-based decision-analytic model was developed, simulating the incremental costs of using one of the NOAC, compared to conventional VKA treatment, with and without PST. The perspective taken is that of the SHI itself . In this figure, the decision tree simulates the direct costs of the two alternative anticoagulation strategies per patient until the end of the first year for one year. We used TreeAge Software  for model building and analysis and examined our inputs over a wide range in sensitivity analyses to identify influential factors that would alter the base-case findings.Univariate sensitivity analysis was performed using all variables to examine the extent to which our calculations are affected by varying selected assumptions. Variation was done using either a) the lower and upper bounds of the parameter if present or b) the lower and upper bounds of a parameter’s 95% confidence interval. Where this is not applicable, we vary parameter values by ± 20% of the base-case value according to international practice, unless stated otherwise.Furthermore, in order to capture the interactions between multiple inputs we provide a probabilistic sensitivity analyses (PSA) by assigning an appropriate statistical (probability) distribution to all input parameters, from which values are randomly drawn in a 2nd order Monte-Carlo simulation . All costs are reported in 2019 Euros (€). Values were calculated in EUR and the average exchange  at https://www.ecb.europa.eu/stats/policy_and_exchange_rates/euro_reference_exchange_rates/html/eurofxref-graph-usd.en.html) was used to convert the EUR amounts to USD.Input parameters are shown together with their probabilistic distributions in Table 1.Rose’s publication15 was utilized to calibrate a model that captures the influence of a change  in TTR (∆TTR) on the number of ischemic strokes (IS) and major hemorrhages (MH) in relationship to those TTR-changes. As MH and hemorrhagic strokes (HS) were not assessed separately in Rose’s publication we assumed that the effect of TTR increase on MH and HS would be the same.The information from Table 5 of Rose et al.We standardized the data from Rose et al., i.e., we utilize the following relationship to estimate mean relative savings:The estimated coefficients were used to generalize the findings of Relative Savings in IS = 0.0134 + 0.6754 * ∆TTR andRelative Savings in MH = 0.0101 + 1.4282 * ∆TTRMultiplying the relative savings in IS or relative savings in MH, resulting from a particular ∆TTR, with the baseline IS or MH defines the savings in IS and MH related to a particular ∆TTR.To incorporate and compare results from different studies we standardize effects to a patient at risk in the respective study population using the following relationship:Saving IS: Baseline of study (number per study patient at risk in % per year) * (0.0134 + 0.6754 * ∆TTR)Saving MH: Baseline of study (number per study patient at risk in % per year) * (0.0101 + 1.4282 * ∆TTR).The risk of having an ischemic stroke (IS), hemorrhagic stroke (HS) or major bleedings (MB) in the NOAC approval studies are separately shown for the respective NOAC and the warfarin control group in Table 3, together with the mean time in therapeutic range (TTR) of the warfarin group members.3.1 Reimbursement in favor of hospitals (DRG case flat rates) of the three adverse events  was calculated using the webgrouper of the University of Münster.3.2. Drug costs3.2.1 Costs of the vitamin K antagonist phenprocoumon® and the generics available in Germany contain 3 mg of phenprocoumon per tablet (Tbl.). The defined daily dose (DDD) of phenprocoumon is 3mg (one Tbl.) According to the German Rote Liste® 2019 the price of 100 Tbl. is €17.89 [USD 20.21]; therefore, the costs per day amount to €0.1798 [USD 0.2031].The target range for treatment with VKA is in the INR range 2 to 3. In order to reach the target area, a saturation phase is required. MarcumarThe scheme for phenprocoumon saturation in a normal weight adult with no hepatic dysfunction is as follows:1st day: Three Tbl.: €0.54 [USD 0.61]2nd day: Two Tbl.: €0.36 [USD 0.41]3rd day: Two Tbl.: €0.36 [USD 0.41]4th day: One Tbl.: €0.18 [USD 0.20]5th day: Measuring the INR value and dosage according to the result.If the INR value is above the therapeutic range on day five, there is an overdose and a medication break is taken for at least one day. Thereafter, treatment should be continued with half a tablet. After four days, the INR is measured again and the dose adjusted if necessary.This means as a minimum in the first 9 days €1.8 [USD 2.03] (€1.44 [USD 1.63] plus €0.09 € [USD 0.10] x 4).If the INR value is below the therapeutic range on day five, the saturation phase is extended by one day by adding two tablets. This is followed by the administration of 1.5 tablets for three days. After four days, the INR is measured again and the dose adjusted if necessary. This means a maximum of €2.61[USD 2.95] in the first 8 days (€1.44 [USD 1.63] plus €0.36 [USD 0.41] plus €0.27 [USD 0.30] x 3).Since the usual maintenance dose for phenprocoumon is between 1.5 mg  and 4.5 mg (1.5 Tbl.), an average value of 1 Tbl. is assumed.3.2.2 Costs of NOAC (German “Red List” 2019)3.2.2.1 Apixaban® ) is 2 x 5 mg. 200 Tbl. of 5 mg (or even in the lower dose 2.5 mg) amount to (reimbursement amount by the GKV) €343.49 [USD 387.97]. The daily cost are €3.43 [USD 3.87]. The annual costs are €3.4349 [USD 3.88] x 365= €1,253.74 .The recommended daily dose of apixaban (Eliquis3.2.2.2 Rivaroxaban® is 20mg. 98 Tbl. of 20 mg each (or 15 mg) cost €320.74 [USD 362.28]. The daily dose thus amounts to €3.27 [USD 3.69] (rounded). The annual costs are 365 x €3.27 [USD 3.69] = €1,194.59 .The recommended daily dose of Xarelto3.2.2.3 Edoxaban® is 60 mg once a day. 98 Tbl. of each 60 mg (or 30 mg) cost €292.25 [USD 330.10]. The daily dose costs €2.98 [USD 3.37] (rounded). The annual costs are 365 x €2.55 [USD 2.88] = €1,099.48 .The recommended daily dose of Lixiana3.2.2.4 Dabigatran®) 150 mg twice a day is the recommended daily dose. 180 hard capsules of 150 mg (or 110 mg) cost €295.45 [USD 333.71]; the daily dose (€295.34 [USD 333.59]: 90) thus costs €3.2815 [USD 3.7065]. The annual costs are 365 x €3.28 [USD 3.70] = €1,197.77 . Of note, also the reduced doses, e.g. for patients suffering from impaired kidney function, are not less expensive.Dabigatran . An autonomous self-adaptation of the phenprocoumon dose requires training, a sufficient stock of test strips and the presence of a (maintenance-free) test device.According to §31 (3) SGB V, there is no copayment obligation of the insured persons for blood test strips. Since lancets, test strips and the measuring device according to § 31 (1) SGB V constitute an accepted aid, there is a claim for full reimbursement by the SHI. The costs of the necessary training are on average €106.64 [USD 120.45], varying between €84 [USD 94.88] and €150 [USD 169.43] in the German federal states (own investigation). Training costs for physicians and/or the entire practice team are not considered, since these are not carried by the SHI.® Softclix lancets is currently €9 [USD 10.17] for 50 pieces; therefore, the costs for 57 tests amount to €10.26 [USD 11.59] (57 x €0.18 [USD 0.20]).For the INR tests until the end of the first month  9 strips  are needed; then 1 strip per week. A test strip costs €2.86 [USD 3.23] (2 x 24 test strips each €137.31 [USD 155.09] - as of 18.3.2019), of which a total of 57 (for 9 + 48 days) are required per year and thus a total of €163.06 [USD 184.18] (rounded). The annual costs of CoaguChek® INRange System (PZN11296110) are €671.35 [USD 758.29] (current date 1.6.2019). The CoaguChek®-INR device is depreciated over 60 months, as is customary for medical measuring instruments .Current best prices for the CoaguChek3.4. Laboratory costs in the GP’s practice® INR system is in accordance with the laboratory value as a reference, it is generally assumed that the blood will be drawn at the GP’s practice and subsequently transferred to the collaborative laboratory. The laboratory community will receive €0.6 [USD 0.68] per examination in accordance with fee schedule position (GOP) 32113. Thus, costs of €3.60 [USD 4.07] (€0.6 [USD 0.68] [GOP 32113] x 6) incur at the expense of the SHI.According to the statements in the Federal Gazette, a total of 6 additional medical check-ups are accepted in medical practice  differ from those of phenprocoumon patients. Therefore, the same costs, as for VKA patients, of €113.40 [USD 128.09] are assumed as payment for the GP per year. The costs for determining those routine parameters are not calculated separately for reasons of insignificance  and are not considered in our model.Achieving a mean TTR of 78% by implementing patient’s self- management (PST) is on average between €603.38 [USD 681.52] and €762.63 [USD 861.39] less costly in the first year per VKA patient, compared to utilizing NOAC (see Table 4 a-b). Assuming that an increase of the original mean TTR as reported in the respective approval studies by approximately 16 percentage points for apixaban, 14 points for Dabigatran, 13 points for edoxaban, and 23 points for rivaroxaban to the target of 78% can be achieved, the number of severe adverse events in VKA patients decreases, and accordingly the associated hospital costs become largely aligned. Thus, after TTR adjustment, the cost difference for those severe events per VKA and NOAC patient in the various groups is then only €1.03 [USD 1.16] (in favor of Dabigatran), €23.41 [USD 26.44] (in favor of apixaban), €0.53 [USD 0.60] (in favor of edoxaban) and €52.62 [USD 59.43] (in favor of VKA anticoagulation in the rivaroxaban group).Univariate sensitivity analysis, in which all variables in the decision trees receive assigned values within their respective ranges, reveals that the drug costs of the single NOAC per patient and year have the highest impact on the absolute amount of cost savings resulting in a linear increase or decrease of 20% - the chosen lower upper and lower bounds of sensitivity analysis (Table 5 a-d). The break-even point when the costs of the NOAC strategy would fall below those of VKA treatment would only be achieved when the annual drug costs of the NOAC would be reduced between 55.43%  and 63.84% .®) between only 3 years and 10 years results in a difference of €156.65 [USD 176.94] in favor of the shorter period. Potential cost savings also depend on the amount of training costs that is mandatory for independently performing INR measurement and varies between €84 [USD 94.88] and €150 [USD 169.43]in the German federal states. Also, the relative small variation of the costs of one test strip by €0.57 [USD 0.64] results in additional or lower costs per VKA patient and year of €66.00 [USD 74.55]. In contrast, varying the G-DRG costs of hospitalization for treating the ischemic or hemorrhagic stroke or major bleeding as potential adverse events by ± 20% resulted in minor changes of total cost. This is because costs incurred for those adverse events would always change in parallel in both arms of the decision tree.Three other parameters still have moderate impact on the cost difference between the NOAC and VKA strategy: Varying the depreciation period for the expensive INR measurement device  and €785.24 [USD 886.93] . Here treatment with phenprocoumon remains constantly less expensive than treatment with one of the four NOAC.The present study is a differentiated cost-benefit analysis of the implementation of PST in VKA (phenprocoumon) patients. The cost savings in favor of VKA treatment under country-specific German conditions are predominantly attributable to the drastically higher drug costs of the 4 NOAC the annual average costs that are more than 16 (edoxaban) to 18 (apixaban) times higher compared to those of phenprocoumon with annual average costs of only €66.78 [USD 75.43].In contrast, the risk per patient of suffering from one of the three severe adverse events (ischemic or hemorrhagic stroke or major bleeding) under VKA or NOAC is in fact only in the single-digit percent level or lower.Increasing the TTR from the respective mean baseline level of the VKA population in the approval studies  to 78%, the realistic target of our model, nearly outweighs the initially lower number of strokes and bleedings in NOAC patients. Accordingly, the remaining differences in the associated costs compared to those incurring for severe advents in the respective NOAC groups are low. Therefore, although the costs of implementing PST, especially the costs of the INR measurement device and the patient mandatory training, in VKA patients are not negligible, NOAC drug costs remain the high cost component in our cost-benefit analysis.Probabilistic sensitivity analysis (PSA) that considers realistic assumptions of uncertainty, demonstrates that performing PST is consistently less expensive than a NOAC. Thus, even when comprehensive discount agreements on the pharmacy retails between NOAC manufacturers and individual health insurance organizations are taken into consideration, self-managed VKA treatment can be considered the strategy of choice as far as economic aspects in favor of the German SHI are concerned.Our study has some limitations that must be considered when interpreting our results: First, the effects of TTR increases on the number of adverse events and their associated costs are based on a mathematical approach that has been derived from a previous U.S. study without a direct relationship to the NOAC approval studies.Second, in all NOAC approval studies warfarin was used as VKA, however, is rarely prescribed in Germany in favor of phenprocoumon. Thus, in our analysis we set warfarin and phenprocoumon, that have identical costs per tablet in Germany, to the same level of adverse effects reported in the NOAC approval studies. We should note our model may overestimate the warfarin-induced adverse effects as reported in the respective approval studies. This is because warfarin has a shorter half-life than phenprocoumon, that patients using phenprocoumon have more often INR values in therapeutic range than warfarin and thus phenprocoumon seems preferable for use in long-term therapeutic anticoagulation.Third, our calculations refer only to the treatment of strokes and major bleedings immediately after diagnosis in a German hospital. Excluded from the model are costs for aftercare services provided by special rehabilitation centers, or physiotherapeutical services offered in ambulatory outpatient settings.Fourth, the results of the model can only be generalized if all VKA patients are mentally able to participate in PST and are willing to perform the ongoing INR checks, and that all NOAC patients regularly take their drug in the suggested doses. Indeed, a 100% implementation of PST for all patients who actually are anticoagulated with vitamin K antagonist may be a quite unrealistic option. However, in a large prospective cohort study in Switzerland those patients who decided to participate in PST had a high adherence of about 90% during a median follow-up of 4.3 years.On the other hand, there are concerns whether the results of the double-blinded approval studies of the four NOAC under consideration can also be assumed to be valid in real life: For example, in US-Veterans the adherence of AF patients to Dabigatran was reported to be only 72.2%.Thus, to validate our estimates, more cost studies, preferably with a multicenter and prospective study design, are required.The utilization of PST in anticoagulated German VKA patients with atrial fibrillation is likely to reduce overall costs. As such, routine implementation of PST may have also direct and positive impact on the control of clinical complications, especially stroke and major bleeding rates. Prospective clinical studies should be undertaken to prove our model and to further evaluate its economic advantages in the immediate future.


Prolonged treatment of immunocompromised influenza patients with viral neuraminidase (NA) inhibitors is required, because the immune system of such patients fails to eradicate the viruses. Here, we attempted to eradicate influenza virus from the respiratory organs of nude mice, which is a model of immunocompromised hosts, by using combination therapy of the viral polymerase inhibitor favipiravir and monoclonal antibodies (mAbs) against the receptor-binding site (RBS) and stem of viral hemagglutinin (HA). Although monotherapy or combination therapy of two antivirals (two mAbs or favipiravir plus a mAb) suppressed virus replication, they failed to eradicate viruses from nude mice. In contrast, the triple combination therapy of favipiravir plus anti-Stem and anti-RBS mAbs completely stopped virus replication in nude mice, resulting in virus clearance. Triple combination approaches should be considered for the treatment of human immunocompromised patients with severe influenza. Another polymerase inhibitor, favipiravir (FAV), which is also known as T705, was approved in Japan in 2014 with an indication limited to treatment of novel or re-emerging influenza virus infections unresponsive or insufficiently responsive to available antivirals2. Mutant viruses resistant to FAV are rarely isolated in vitro and in vivo with one exceptional case3. Although detection of viruses that exhibit reduced susceptibility to NA inhibitors or baloxavir marboxil in immunocompetent patients has frequently been reported9, such viruses usually do not dominate susceptible viruses with the exception of the worldwide spread of oseltamivir-resistant H1N1 virus in the 2007–2008 season. Recently, broadly protective human monoclonal antibodies (mAbs) against conserved regions of HA, including the receptor-binding site (RBS) and stem region, have been evaluated16 and studies for their clinical application are being conducted20. However, no human mAbs are currently available for clinical use.Treatment of seasonal influenza has relied on neuraminidase (NA) inhibitors and a polymerase inhibitor, which target the viral sialidase and polymerase activities of the virus, respectively. The time to influenza symptom alleviation is similar between one of the polymerase inhibitors, baloxavir marboxil, and the NA inhibitors; however, treatment with baloxavir marboxil efficiently reduces the viral load compared with treatment with an NA inhibitor within 1 day of initiation of treatment23. Therefore, prolonged treatment with NA inhibitors is required for immunocompromised patients, which leads to frequent emergence of viruses that are resistant to these drugs23. The resistant viruses, in turn, compromise the effectiveness of the NA inhibitors, resulting in substantial mortality among immunocompromised patients24. In contrast, polymerase inhibitors have not been well studied as a treatment for immunocompromised patients. A reliable approach to achieve rapid eradication of the influenza virus from immunocompromised patients is required.Virus clearance is usually delayed in immunocompromised patients, because the immune responses of such patients are insufficient to suppress virus replication. To inhibit virus replication in these patients, NA inhibitors are widely used but they fail to eradicate the virus from the respiratory organs25. In nude mice infected with influenza virus, virus clearance is delayed and the survival rate is reduced compared with wild-type mice26. In our previous study, monotherapy with an NA inhibitor (oseltamivir or laninamivir) for 28 days did not improve the survival time of infected nude mice, whereas monotherapy with a virus polymerase inhibitor  or combination therapy of FAV plus an NA inhibitor (oseltamivir or laninamivir) for 28 days increased it27. Although these treatments were effective in terms of survival, they fail to eradicate the virus from the respiratory organs of the infected nude mice, resulting in relapse and death due to the remaining infectious viruses after termination of treatment. The development of a protocol to eradicate influenza virus from nude mice could contribute to improved treatment outcomes in human immunocompromised patients. Here we attempted to eradicate influenza virus from nude mice by using combination therapy of FAV and mAbs against the RBS and stem of HA.As an immunocompromised patient model for influenza infection, the nude mouse, which is immunologically deficient, because it lacks a thymus, has been used since 19813 plaque-forming units (PFUs) of MA-CA04. The infected mice were treated with FAV at 100 mg/kg once a day for 28 days, a human mAb against the HA stem (clone CR9114) at 5 mg/kg once per 3 days for 14 days, or a human mAb against the RBS of HA (clone F3A19) at 1 mg/kg once per 3 days for 14 days, alone or in combination . Mice treated with the combination of FAV plus anti-Stem mAb, FAV plus anti-RBS mAb, or anti-Stem mAb plus anti-RBS mAb survived for significantly longer than the control groups, but died between 39 and 155 days post infection . The hazard ratio was decreased by all treatments tested  and did not harbor detectable viruses in the lung at day 188. These results show that the triple combination therapy of FAV, anti-Stem mAb, and anti-RBS mAb can eradicate influenza virus from immunocompromised hosts.To assess virus clearance from an immunocompromised host, we intranasally infected nude mice with 103 PFU of MA-CA04 were treated as described in Table 10 PFU/g, respectively, at day 7  and/or virus polymerase inhibitors  increased survival time but failed to achieve virus clearance, resulting in death after termination of treatmentFor resistant viruses to emerge, they must replicate under the suppressive pressure of antivirals. Therefore, it is important to sufficiently suppress or ultimately block virus replication to prevent the emergence of resistant viruses. Our data show that the triple combination therapy of FAV, anti-Stem mAb, and anti-RBS mAb completely stopped virus replication in infected nude mice, leading to virus clearance. Our triple combination therapy is, therefore, less likely than current monotherapies to lead to the emergence of resistant virus in immunocompromised hosts.Even though the combination therapy of FAV plus the anti-RBS mAb reduced virus titers in the lung to below the detection limit during drug treatment, infectious viruses have likely remained at the termination of treatment, leading to the deaths of nude mice likely due to virus replication. This finding indicates that termination of treatment when the virus titer in the tracheal swab sample of an immunocompromised patient is below the detection limit carries a risk of relapse due to remaining infectious viruses. Therefore, we contend that immunocompromised influenza patients should be closely monitored after the termination of treatment.All animal experiments were conducted in accordance with the University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo. The committee acknowledged and accepted both the legal and ethical responsibility for the animals, as specified in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.nu/nu; Japan SLC, Inc.) per group were anesthetized with isoflurane and intranasally infected with 103 PFUs of mouse-adapted A/California/04/2009 30. The infected mice were treated with FAV (100 mg/kg), the anti-Stem mAb clone CR911411 (5 mg/kg), and the anti-RBS mAb clone F3A1931 (1 mg/kg) alone or in combination  cells. Viral RNA was extracted from lung samples collected at 28 days post infection and RT-PCR was performed using primers against the NS segment.N-tosyl-l-phenylalanine chloromethyl ketone)-treated trypsin, and various concentrations (0.01–100 nM) of FAV. The plates were the incubated at 37 °C for 2–3 days and plaques were counted to determine IC50 values.Using the lungs of mice that received treatments that included FAV, we attempted to isolate viruses using MDCK cells in 24-well plates. The isolated viruses were titrated and ∼50 PFU of isolated viruses were then inoculated into confluent MDCK cells in 6-well plates. After infection, the cells were overlaid with MEM containing 0.3% bovine serum albumin, 1% agarose, 1 μg/ml TPCK . The cDNA was synthesized by using Superscript III reverse transcriptase (Invitrogen) and a U12 primer (5′-AGCAAAAGCAGG-3′). The cDNA products were amplified by PCR using primers specific for the HA segment. The PCR products were sequenced with the BigDye terminator 3.1 kit on an ABI 3130xl (Applied biosystems). The sequence data were submitted to Genbank (accession numbers LC537233–LC537240).50 of the isolated virus and then incubated at 37 °C for 30 min. The mixtures were inoculated into MDCK cells in quadruplicate and incubated for 1 h at 37 °C. BSA-MEM containing TPCK-treated trypsin was added to each well and the cells were incubated for 3 days at 37 °C. The cytopathic effect was examined and antibody titers required to reduce virus replication by 50% (IC50) were determined by using the Spearman–Karber formula.Two-fold serially diluted purified anti-Stem or anti-RBS mAb was mixed with 100 TCIDAmino acid positions were plotted on the crystal structure of CA04 HA  by using the PyMOL molecular graphics system to visualize the trimer.One-way analysis of variance followed by Dunnett’s test and the log-rank (Mantel–Cox) test followed by Benjamini–Hochberg correction were performed by using GraphPad Prism 7.04. Each sample size is defined in the main text, footnotes, or legends. No samples were excluded from the analysis.Further information on research design is available in the Reporting summaryPeer review file


Myodes glareolus) are host to many zoonotic viruses. As bank voles inhabiting areas contaminated by radionuclides show signs of immunosuppression, resistance to apoptosis, and elevated DNA repair activity, we predicted an association between virome composition and exposure to radionuclides. To test this hypothesis, we studied the bank vole virome in samples of plasma derived from animals inhabiting areas of Ukraine  that differed in level of environmental radiation contamination. We discovered four strains of hepacivirus and four new virus sequences: two adeno-associated viruses, an arterivirus, and a mosavirus. However, viral prevalence and viral load, and the ability to cause a systemic infection, was not dependent on the level of environmental radiation.Bank voles ( Myodes glareolus), a small rodent that is common in woodland habitats throughout much of Europe and northwest Asia, is an zoonotic hyper-reservoir [Metagenomic analyses of rodent fecal and pharyngeal viromes indicate that rodents host a diverse community of DNA and RNA viruses, with viral diversity apparently depending on host taxonomy more than geographical location, although rodent viruses can often shift between hosts ,2. The beservoir . Bank voeservoir , flavivieservoir , orthonaeservoir , cowpox eservoir , and piceservoir ; moreoveeservoir ,10, alpheservoir , polyomaeservoir , herpesveservoir ,14, aneleservoir , and hepeservoir . As bankeservoir ,18.The effects of environmental radiation on host–viral dynamics in natural systems are unknown. While high doses  of ionizing radiation inactivate viruses ,20, expoTo test our prediction, we isolated and characterized the virome from plasma samples from bank voles inhabiting (1) the Chernobyl exclusion zone (CEZ) and (2) from areas close to Kyiv, Ukraine, where the habitat is not contaminated by radionuclides. The CEZ is an area surrounding the former Chernobyl nuclear power plant that presents a mosaic of areas contaminated by radionuclides . We ideng for 10 min, after which the supernatant was pipetted into sterile 1.5 mL tubes and frozen at −20 °C until further processing. Environmental radiation levels at each trapping location were measured at 1 cm above the ground using a Geiger–Mueller dosimeter . In addition, every individual was subjected to γ-spectrometry  to estimate whole-body radionuclide (137Cs) burden and internal radiation exposure  or CEZ area (west: N = 10)  at room temperature for 10 min. Samples were then filtered (0.45 µm) and centrifuged with a 38% sucrose cushion at 175,000 g for 3 h. Virus DNA and RNA was isolated from the pellet using a GeneJET Viral DNA/RNA Purification Kit (ThermoFisher). Double-stranded cDNA was synthesized with Maxima H Minus Double-Stranded cDNA Synthesis Kit (ThermoFisher); the samples were concentrated with PureLinkTM PCR Micro PCR kit, and sent for sequencing (100 bp paired-end reads on an Illumina HiSeq4000) to BGI Tech Solutions .Virus particles were isolated from pooled plasma samples. A total 100 µL of plasma was pooled from bank voles collected from the Kyiv control area (east:  N = 10) . Sampleswww.csc.fi). Adaptor sequences and low quality reads were removed using Trimmomatic [www.ncbi.nlm.nih.gov/orffinder), Blastp (nr), and Hhpred [www.cbs.dtu.dk/services/TMHMM) was used to predict transmembrane domains. Remapping and mapping of reads from the transcriptome data to virus sequences was done with BWA [Raw reads were deposited on the National Center for Biotechnolgy Information’s (NCBI) Sequence Read Archive (SRA) (bioproject ID: PRJNA557363). Servers and software for bioinformatic processing and analysis were provided by The Finnish IT center for Science  with Muscle  using neNfemales = 9, Nmales = 10; Kyiv west: Nfemales = 10; CEZ east: Nfemales = 3, Nmales = 9; and CEZ west: Nfemales = 17, Nmales = 10; Virus DNA and RNA was isolated with GeneJET Viral DNA/RNA Purification Kit (ThermoFisher) from 100 µl of plasma (Kyiv east: www.r-project.org).All statistical analyses were done in R v3.5.0 , where there is no elevated level of radiation, and from a site within the Chernobyl exclusion zone (CEZ Nwest = 10), where soil was contaminated with radionuclides  and replication protein (REP). Parvoviruses are non-enveloped viruses with two terminal hairpin structures, which prime replication [Parvovirinae) and invertebrate (Densovirinae) hosts [Parvovirinae is divided into eight genera based on the proteins they transcribe: Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Dependoparvovirus, Erythroparvovirus, Copiparvovirus, Protoparvovirus, and Tetraparvovirus [Dependoparvovirus genera; Myodes glareolus adeno-associated virus 1 (MgAAV1) and 2 (MgAAV2) had 4927 nt and 4517 nt long genomes, respectively (lication . Parvovie) hosts . The subrvovirus . Dependoectively a. MgAAV1ectively b. MgAAV1Arteriviruses are enveloped, positive-strand RNA viruses in the order of Nidovirales. The Arteriviridae infect mammals, including Grey red-backed voles [Arteriviruses infect macrophages and the infection can be asymptotic, persistent, or disease-associated [Arterivirus genome is polycistronic, poly-adenylated, and codes for 11 proteins. We found nine ORFs from MgAV1, with the largest proteins (2491 and 1410 aa ORFs) similar to replicase polyprotein 1a and 1b of the Grey red-backed vole arterivirus 1  (Arteriviridae family. The remaining ORFs of MgAV1 were (in order from the 5′ end): envelope protein E , envelope glycoprotein 2 , envelope glycoprotein 3 , envelope glycoprotein 4 , envelope glycoprotein 5 , envelope protein 5a , membrane protein , and nucleocapsid protein . Surprisingly, our draft genome MgAV1 sequence did not contain the nonstructural protein 2 transframe fusion product (nsp2TF), which most of the identified arteriviruses had. The MgAV1 sequence had a 19 nt long poly-A tail. ICTV species demarcation criteria states that each arterivirus species is host-specific.ed voles . Arterivsociated . Arterivctively) c. MgAV1 ctively) d and is Flaviviridae family. Hepaciviruses infect a range of mammals, including bank voles [e-value = 3.1 × 10−25) and p7 . According to ICTV guidelines, hepaciviruses with less than 0.25 amino acid p-distances  in a conserved region of NS3 are conspecific [Hepaciviruses are positive-strand RNA viruses that belong to the nk voles . From thnk voles . We founnk voles ). In phynk voles . Using Bspecific . Since tMosavirus (mouse stool associated picornavirus) is a novel genus in the Picornaviridae family. There are currently only four identified mosavirus genomes, and thus the genus is not yet well defined. The first described mosavirus genome was isolated from a stool sample of North American canyon mouse (Peromyscus crinitus) [Coracias garrulus garrulus) [Marmota himalayana) [Apodemus syhaticus) [e-value = 6.4 × 10−14) and VP4  of foot-and-mouth disease virus and protease 2A of human poliovirus 1 . Foot-and-mouth disease virus and poliovirus are both picornaviruses. The Ukrainian bank vole mosavirus clustered with the type A mosaviruses (rinitus) , the secarrulus) , the thialayana) , and thehaticus) . As Eurohaticus) . The Ukraviruses h, and thNfemales = 9, Nmales = 10, and Kyiv west: Nfemales = 10) and two sites in the Chernobyl exclusion zone  . In females, MgAAV1 amounts in plasma were significantly lower in CEZ west animals than in animals collected from CEZ east (p = 0.044) or from control east (p = 0.033). In addition, MgAAV2 plasma amounts in females were lower in the CEZ west than in control east area (p = 0.033).We collected plasma samples from bank voles inhabiting two control areas near Kyiv (Kyiv east: es = 10) . Virus pes = 10) a,b. We des = 10) , nor did amounts . Howeverp = 0.043). Median values for virus plasma amounts for all animals (irrespective of host collections site or sex) were MgAAV1 = 241, MgAAV2 = 1695, MgAV1 = 4182, MgHV3 = 166, MgHV4 = 205.5, MgHV5 = 282.5, and MgMV1 = 375.6 , the plasma virus amounts were similar between the sexes at each collection site. The only statistically significant difference in plasma viral load between females and males was that females had more MgAV1 in plasma than males in control east  and MgAAV1 . Males had MgMV1 more frequently than females and females had MgAAV1 more frequently than males. Virus prevalences in plasma samples were analyzed separately for each sex and across populations a,b. The p-value < 0.0001), and also between MgAV1 and MgHV5  and MgMV1 . The correlation analyses were done also separately for males and females  or the other control location near Kyiv (N = 10). We used spleen and liver RNA-sequencing data for bank voles from Ukraine  NCBI SR, all froN = 40, Mann–Whitney: U = 61, p = 0.298).To estimate whether viruses had caused long-term adverse effects in these five animals, we compared their body condition index to the animals without virus reads in spleen or liver ,62. The To investigate the presence of the newly classified virus strains in other bank vole populations, we also searched for the virus sequences from all available wild bank vole liver and spleen transcriptomes . We identified all of the eight virus sequences from five Swedish adult bank vole spleens . There wMyodes rufocanus) and the Northern red-backed vole (M. rutilus). Similar to Wu et al. [We identified four new bank vole virus genomes from three families, increasing the known diversity of viruses in bank voles to nearly 20 species ,14,15,16The two adeno-associated parvoviruses, MgAAV1 and MgAAV2, are the first known parvoviruses from bank voles. A positive correlation in MgAAV1 and MgAAV2 plasma viral load within individuals implies that their replication is regulated by a common factor, such as through the action of a (presently unknown) helper virus or some inherent feature of the host that allows both viruses to become co-abundant. Adeno-associated viruses are not known to be pathogenic, which is in line with our results, as the prevalence and plasma particle amounts of MgAAV2 were the highest among the identified viruses, yet the read amounts in the RNAseq data from liver and spleen were among the lowest, indicating that the viruses did not infect the tissues. Viral pathogenesis generally requires the virus to be able to spread in the body and multiply to large enough numbers.The arterivirus MgAV1 had very high prevalence (90–100%) and particle amounts in plasma samples in comparison to other viruses; however, unlike the adeno-associated viruses, MgAV1 also had the highest read amounts in spleen and liver transcriptomes, indicating that it may have caused infection. Spleen and liver are lined with macrophages that ingest pathogens and activate immune defenses. Several viruses, including arteriviruses , infect The fact that fewer animals (12.5%) had detectable amounts of virus in their liver and spleens  than in plasma  indicates that these apparently common (at least in the Ukrainian bank voles) viruses rarely become systemic. Interestingly, the animals that had viral genetic material in their liver or spleen were apparently infected by most of the viruses, which may indicate generally poor health or a compromised immune system, which aided virus replication and infection. However, the five Ukrainian bank voles from the transcriptome analysis that had viruses in spleen and/or liver did not have lower body condition index compared to the animals without viruses in these tissues, indicating that these animals had not suffered from poor body condition for extended period of time. Thus, the pathogenic potential of the identified viruses remains unresolved. Counter to our expectations, we found no significant differences in virus characteristics between bank voles from high environmental radiation and control areas. Significant differences in plasma viral load and prevalence among collection sites could indicate that viral dynamics are more dependent on the local ecology and demography of the host population rather than radionuclide contamination.


L. de Swart et al.  and “Long-term measles-induced immunomodulation increases overall childhood infectious disease mortality” by M.Rory de Vries works in the field of viral pathogenesis and focuses on interactions between respiratory viruses (or corresponding vaccines) and the host immune system. In this mSphere of Influence article, he reflects on how the articles “Predominant infection of CD150 + lymphocytes and dendritic cells during measles virus infection of macaques” by R. L. de Swart et al.  and “Long-term measles-induced immunomodulation increases overall childhood infectious disease mortality” by M. J. Mina et al.  made an impact on him. These articles studied interactions between measles virus and the host and influenced him by making two important points. (i) It is crucial to use nonadapted (recombinant) viruses in disease-relevant model systems when studying virus-host interactions. (ii) Studying viral pathogenesis requires a combination of in vitro, ex vivo, and in vivo studies, and a group of researchers with multiple expertises. He learned that only when all these aspects are combined, can one truly answer the question: “How does a virus cause disease?”Rory de Vries works in the field of viral pathogenesis and focuses on interactions between respiratory viruses (or corresponding vaccines) and the host immune system. In this mSphere of Influence article, he reflects on how the articles “Predominant infection of CD150 I have implemented these lessons in our current studies on virus-host interactions of human respiratory syncytial virus (HRSV), where we make use of recombinant viruses directly based on clinical isolates and study their behavior in respiratory organoids or differentiated primary airway epithelial cells cultured at the air-liquid interphase , and scientists should always remain vigilant and “critical.” If one has a hypothesis that deviates from the current dogma, you should be ambitious and perform the experiments to either prove or disprove that hypothesis with perseverance and indomitable spirit.Both articles described here were game-changers in the field of measles research; combined with follow-up studies, they led to revisions of the textbooks. Shaping me as a researcher, this is one of the “life lessons” I have taken from these articles: what you read in textbooks or articles is not right


To provide baseline data for the anatomy of the external female genitalia and to investigate the correlation between those measurements and sexual function and genital perception.This prospective cohort study consisted of 208 healthy premenopausal women. The Female Sexual Function index (FSFI) and the Female Genital Self-image scale (FGSIS) questionnaires were administered. Participants were divided into two groups according to their female sexual dysfunction (FSD) status. External genital measurements and anterior and posterior vaginal length were measured.The external female genital measurements were : clitoral prepuce length 2.05±0.48; clitoral glans length 0.87±0.21; clitoral glans width 0.60±0.15; clitoris to urethra 2.24±0.55; anterior fornix depth 7.75±0.92; posterior fornix depth 9.25±0.75; labia minora width, right 2.12±0.86, left 2.20±0.96. A weak negative correlation was found between total FGSIS scores and clitoral prepuce length , whereas a weak positive correlation was seen between total FGSIS scores and anterior-posterior vaginal lengths . No statistically significant difference was found between the genital measurements of participants with FSD  and those without FSD , and the total FSFI scores and orgasm subdomain scores.The female genital measurements were found to be distributed over a wide range. Although the relationship between genital measurements and genital perception varied, no significant relationship was found between genital measurements and sexual functions or orgasm. These findings suggest that a more cautious approach should be taken towards genital surgeries for cosmetic purposes. PRECIS: The female genital measurements were found to be distributed over a wide range and no significant relationship was found between genital measurements and sexual functions or orgasm.,2,3,4,5. There are few reports in the literature regarding overall ‘normal’ female genital appearance or ‘normal’ dimensions, and exact positioning of the vagina, clitoris, and labia minora and majora,5. There has been a significant increase in female genital cosmetic surgery rates over the years,2,3,4,6. For the degree of increased female genital cosmetic surgery, the main motivator was found as improvement in genital appearance integrated with their aesthetic and sexual demands.External female genital measurements vary widely,9. Negative body image has been found to reduce sexual desire and arousability and was associated with fewer orgasms and less sexual satisfaction. Among sexually active women, discomfort with the appearance of their genitals leads to anxiety and inhibitions during sexual activity,12. A positive correlation has been determined between genital perception and sexual function. However, it is not possible to evaluate sexual satisfaction and genital perception only with anatomic features due to the complexity and multifactorial structure of female sexuality. It is important for the physician to recognize whether this discomfort is due to an anatomic reason or the perception of a defect. The media can negatively influence the genital perception of whom external genitalia are even in normal ranges. In patients demanding an esthetic procedure in order to increase sexual satisfaction, an attempt should be made to correct genital perception before performing the surgery.It is known that body image and genital perception are related to sexual satisfactionDue to limited data on the distribution of genital measurements, this study aimed to provide baseline data for healthy, reproductive-age female external genital anatomic measurements and to determine the relationship between these measurements and genital perception and sexual function.Our prospective cohort study consisted of 208 healthy female participants. Our study included premenopausal patients aged over 18 years who were sexually active, and were seen in a medical faculty hospital polyclinic for routine gynecologic examinations between October 2017 and February 2018. The measurements and questionnaires were administered to healthy female participants who reported no known illnesses. and the Female Genital Self-image scale (FGSIS) were administered under the supervision of a physician.Exclusion criteria included postmenopausal and pregnant patients; those with previous vaginal and/or perineal, gynecological, or aesthetic surgical interventions; patients with stage >2 pelvic organ prolapse; urinary incontinence; menstrual irregularities; gynecological cancer; Polycystic Ovary syndrome (PCOS); patients using oral contraceptives or antidepressants; and those with intrauterine devices. Participants were taken to a quiet room, demographic data was recorded, and validated Turkish versions of the Female Sexual Function index (FSFI). Female sexual dysfunction (FSD) was defined as a total score of 26 or less from a maximum possible score of 36. The participants were divided into two groups according to their FSD status. The FGSIS is a seven-question survey that reveals female genital perception. Genital perception is considered to be higher as the total score increases, with a maximum possible score of 28. The Beck Depression inventory was administered to patients who met the inclusion criteria and patients with a score of 17 and above were excluded from the study.The FSFI is a brief instrument consisting of 19 questions for the assessment of sexual function. Questions are scored for the domains of libido, arousal, lubrication, orgasm, satisfaction, and painSubsequently, the participants were taken to the examination room and genital measurements were taken while in the lithotomy position. External genital measurements were made using a digital stainless-steel Vernier caliper, which can measure to 1/10 mm, and vaginal measurements were made using a hysterometer. The calipers were sterilized using ethylene oxide or were used by passing them through disposable bag gloves. The clitoral glans was measured by pulling back the prepuce. Labia minora and majora length and width were measured bilaterally. All measurements were made by two gynecologists who each had at least 10 years’ experience.The template in The primary outcome was the determination of the measurements of the external female genitalia. The FSFI and FGSIS were administered to the participants in order to understand the degree to which the results were clinically relevant. The secondary outcome was the determination of the relationship of the measurements to sexual function and genital perception and the relationship between genital measurements and age, body mass index (BMI), and parity.The institutional Ethics Committee approved the study , and written informed consent was obtained from all individual participants included in this study, which was conducted in accordance with the Helsinki Declaration.2) test, depending on the type of variables. Statistical significance was defined as p<0.05.Statistical analyses were performed using the Statistical Package for the Social Sciences for Windows 22.0 software . Descriptive statistics were calculated for subject demographics and dependent variables and were given as the mean, standard deviation, frequency and percentiles of 5%, 50% and 95%. Student’s t-test was used for the comparison of quantitative variables between groups. Correlations between continuous data were analyzed using the bivariate Pearson correlation coefficient. Relations between categorical data and genital parameters were evaluated using Pearson’s chi-square  were nulliparous and 82.7% (n=172) were multiparous. It was determined that 58.4% (n=101) of the multiparas had normal deliveries, 32% (n=55) delivered via caesarean section, and 9.3% (n=16) delivered both via caesarean and normally. Episiotomy was performed in 65.6% (n=66) of the 101 patients who delivered normally. Of the patients, 41.3% were smokers. When the genital measurements of the nulliparous and multiparous patients were compared, the anterior vaginal length was longer in multiparous patients (p=0.009) and the clitoris-urethra distance was shorter in multiparous patients (p=0.03). When comparing the types of delivery, the anterior and posterior vaginal lengths of the patients who had delivered normally were longer compared with those of patients who had undergone cesarean section . In patients who had undergone episiotomy, the anterior vaginal length was longer (p=0.008); there was no difference in posterior vaginal length (p=0.12).The mean age of the participants in the study was 35.2±9.1  years, and the mean BMI was 25.1±4.6  kg/mThe external female genital measurements were distributed over a wide range. The external female genital measurements detected were as follows: clitoral prepuce length 2.05±0.48; clitoral glans length 0.87±0.21; clitoral glans width 0.60±0.15; clitoris to urethra 2.24±0.55; anterior fornix depth 7.75±0.92; posterior fornix depth 9.25±0.75; labia minora width, right 2.12±0.86, left 2.20±0.96; labia minora length, right 3.60±1.17, left 3.79±1.26; labia majora width, right 3.02±0.59, left 2.98±0.63; labia majora length right 7.43±0.95, left 7.40±0.79 . The distribution of the genital measurements and the mean ± SD, range, and the 5, 50, and 95 percentile values are shown in According to the FSFI scores, a comparison of the patient group with FSD  and the group without FSD  is shown in Correlation analysis was performed between the genital measurements and the variable parameters of age, parity, and BMI . There w=0.32).A statistically significant weak negative correlation was detected between the total FGSIS scores and clitoral prepuce length, and there was a statistically significant weak positive correlation between the total FGSIS scores and anterior-posterior vaginal lengths . As parity increased, the total FGSIS score decreased (p=0.001). There was a positive correlation between the total FSFI score and the total FGSIS score  and found different orgasm ratios below and above the clitoris-urethral meatus distance (CUMD) of 2.5 cm. A difference of 0.7 mm was found between the means of the CUMD (2.2 vs 2.9) in these two older studies. This difference was explained by the fact that in one study the measurement started from the middle of the glans, whereas the other started with the prepuce. We took our measurement from the middle of the glans, and our results were consistent with the literature. In our study, unlike in that study, no significant difference was found between the FSFI groups and the CUMD. Upon examination of the relationship between the FSFI orgasm subdomain and the genital perception scores, no significant difference was detected.Wallen and LloydThe FSFI orgasm subdomain was analyzed separately from the total score because it was thought that there could be a clinical relationship between the measurements and orgasm. However, no significant relationship was found between genital measurements, especially clitoral prepuce and labium minora length, and orgasm or sexual function. The very weak inverse relationship between genital perception scores and prepuce length can be explained by the influence of parity. With the increase in parity, an increase in the clitoral prepuce size and a decrease in genital perception were detected. This suggests that after clitoral hood reduction surgeries performed for the purpose of increasing sexual satisfaction, sexual outcomes should be compared with the status before the surgery.. The study found that, in the long term, these surgeries improved genital perception and sexual function. However, it was observed that the body-image perceptions, genital perceptions, and sexual functions of those who wanted surgery were lower than those of the control group.Female genital esthetic operations were evaluated and sexual functions and their relationship with body image were investigated over a two-year period, in a prospective study. It is also known that genital and body perceptions can be changed by exposure to visuals such as video and photographic images. There was a positive correlation between FSFI and FGSIS, associating high genital perception with high sexual function,13. Our study results are consistent with other studies that found a positive correlation between FSFI and FGSIS. For this reason, it is necessary to evaluate the pre-operation genital and body perceptions of women who desire esthetic surgery for reasons other than function.Many factors may affect genital perceptionThe examination of female genital measurements and sexual functions along with genital perception created a powerful avenue of the study. The limitation of the study is that the study group reflected a part of society thought to be healthy. Multiparous patients were in the vast majority, and the lack of a categorical evaluation according to age and weight were other limitations. Another limitation was that the Body Dysmorphic Disorder scale was not administered to the patients. Future investigations dealing with the effects of surgical outcomes on sexual function and genital perception are recommended.Considering the breadth of the distribution of the measurements, the fact that ‘normal’ female genital measurements can vary should be discussed with patients. For patients without functional impairment who want genital aesthetic surgery because of a desire to increase sexual satisfaction, an attempt should be made to first correct their genital perception. More research is needed on the subject of esthetic surgical procedures that change genital measurements and the contributions of this surgery to sexual function and genital perception.The wide range of genital measurements observed makes it difficult to draw the boundaries of ‘normal’ regarding female external genitalia. Genital cosmetic surgeries should be considered more cautiously because negative genital perceptions are open to verbal and visual influence and because there is no significant relationship between genital measurements and sexual functions.


Musk deer can produce musk which has high medicinal value and is closely related to human health. Viruses in forest musk deer both threaten the health of forest musk deer and human beings.Using viral metagenomics we investigated the virome in 85 faeces samples collected from forest musk deer.Smacoviridae, pCPa-like virus and pPAPh2-like virus. UJSL001 (MN621482), UJSL003 (MN621469) and UJSL017 (MN621476) fall into the branch of unclassified CRESS-DNA virus (CRESSV1–2), UJSL002 (MN621468), UJSL004 (MN621481) and UJSL007 (MN621470) belong to the cluster of Smacoviridae, UJSL005 (MN604398) showing close relationship with pCPa-like (pCRESS4–8) clusters and UJSL006 (MN621480) clustered into the branch of pPAPh2-like (pCRESS9) virus, respectively.In this article, eight novel CRESS-DNA viruses were characterized, whole genomes were 2148 nt–3852 nt in length. Phylogenetic analysis indicated that some viral genomes were part of four different groups of CRESS-DNA virus belonging in the unclassified CRESS-DNA virus, The virome in faeces samples of forest musk deer from Chengdu, Sichuan province, China was revealed, which further characterized the diversity of viruses in forest musk deer intestinal tract. Forest musk deer is a national protected animal, mainly distributed in Sichuan province, Guangxi province and other places, China , 2. The Circoviridae [Genomoviridae.Viruses with small circular rep-encoding ssDNA (CRESS-DNA) genomes encode a replication associated protein (Rep), mainly includes oviridae , GenomovSmacoviridae [Geminiviridae, Nanoviridae and Bacilladnaviridae [, Smacovioviridae , Geminivaviridae . These aaviridae , 13, plaaviridae –17, dragaviridae –21, mosqaviridae , rats [2aviridae , bats [2aviridae , duck [2aviridae , cattle aviridae , pigs [2aviridae , 28, dogaviridae , human [aviridae –33, and aviridae . CRESS-Daviridae .In this study, the virus community in the intestinal tract of forest musk deer was analyzed by virus metagenomics. The results of this study put forward for the first time on CRESS-DNA viruses propagating among forest musk deer.In 2016, 85 forest musk deer faeces samples were collected from Chengdu, Sichuan province, China. Samples were collected by disposable materials and transported to the laboratory on dry-ice and store in the − 80 °C refrigerator. Samples were put into 1.5 ml tubes containing phosphate buffered saline (PBS). The supernatants of fecal samples were collected after vigorous eddy current for 5 min and centrifugation for 10 min  , 37.500 μl of supernatant was filtered through a 0.45 μm filter (Millipore) to remove eukaryotic and bacterial cell sized particles. The viral particle enrichment filtrate was then treated with uncleases to digest nonparticle protected nucleic acid at 37 °C for 90 min . RemainiPutative open reading frames (ORFs) in the circular genomes were predicted by Geneious software version 2019.0.3 , and theThe Rep protein sequences of these novel virus were homology alignment with the reference sequences in GenBank using the ClustalW program in MEGA7.0. Phylogenetic analyses were constructed using full-length rep protein of novel virus and other genetically close relatives , 46. SavSmacoviridae and 473 reads sequence similar to the virus of Circoviridae. Table The 85 faeces samples of the eight libraries generated a total of 6, 153, 736 unique sequence reads using illumine Miseq sequencing runs with 250 base pair terminals. The Ensemble program was used to read the de novo assembly sequence  and BLASFour complete CRESS-DNA genomes showing the highest sequence identity to CRESS-DNA virus. Genomes were 3518 nt , 3212 nt , 2148 nt  and 3025 nt  in length. As shown in Fig. Three complete CRESS-DNA genomes showing the highest identity to Smacovirus. Genomes were 2665 nt , 2866 nt  were obtained through inverse PCR, and 2526 nt  in length, respectively. Figure Circoviridae. Genome was 3852 nt  in length. UJSL005 genome was acquired through inverse PCR based on a large contigs from library 6 and Sanger sequencing. Figure Circoviridae (NC_026635.1) (Table A complete CRESS-DNA genome showing the highest sequence identity to P. pulchra plasmids) from GenBank, a phylogenetic tree was constructed [Based on the alignment of the Rep amino acid sequences herein detected with the best matches of BLASTp search in GenBank and those of representative CRESS-DNA genomes including 6 groups of unclassified CRESS-DNA virus (CRESSV1–6), two GasCSV-like viruses, Bacterial plasmids (pCRESS1–9) and a small group of Eukaryotic plasmids , UJSL001 and UJSL003 belong to the cluster of CRESSV2, UJSL001 showing close relationship with CRESS_AUM21936, UJSL003 showing close relationship with CRESS_AXH77830  and pPAPh2-like (pCRESS9). For the first time, CRESS-DNA virus in the faeces of forest musk deer was mentioned, which was beneficial to further understanding of the genetic and evolutionary diversity of these viruses.Our report describes viral nucleic acids enriched in forest musk deer faeces, shows that CRESS-DNA virus sequences are present in all libraries and have the most reads compared to other viruses. This suggests that these viruses are likely to replicate in forest musk deer host cells, but there is no evidence for this. Based on phylogenetic analysis, four different groups of CRESS-DNA genomes in forest musk deer faeces were detected, which belonged to CRESS-DNA virus, Circoviridae [Geminiviridae [Nanoviridae [Circoviridae range in size from 1.7 to 2.1 kb and contain two major ORFs, which encode Rep and Cap proteins. According to the International Committee on Taxonomy of Viruses (ICTV), the ssDNA has genomes between 1.7-6 kb. Eight CRESS-DNA virus extracted in this study, the genomes range in size from 2.1 kb to 3.5 kb. Previous research on the stem-loop structure of diverse circovirus and cycloviruses, a highly conserved stem-loop structure is also found [CRESS-DNA viruses with small, circular replication-associated protein (Rep)-encoding single stranded (CRESS) DNA genomes, are largely identified based on conserved rolling circle replication proteins . It consoviridae , which civiridae  and Nanooviridae  which caso found , 52, 55,In the recent years, a large number of CRESS-DNA genomes have been determined in human and any other mammals, birds, insects, plants, fungi, and environment samples which bringing to light a high level of genetic diversity among these virus , 52, 56.In conclusion, this study is the first to discover a variety of new CRESS-DNA viruses in the intestinal tract of forest musk deer and analyze their genomic characteristics, which is of great significance for the study of forest musk deer virus and the genetic and evolutionary diversity of CRESS-DNA virus. At the same time, the host adaptability and pathogenicity of the new CRESS-DNA virus need further study.The virome in faeces samples of forest musk deer from Chengdu included the viruses showing sequence similarity to CRESS-DNA viruses, where eight divergent genomes of CRESS-DNA viruses were identified in detail. The contents include genome protein structure, stem-loop structure and rep protein phylogenetic analysis. Although CRESS-DNA virus is prevalent in forest musk deer, its pathogenicity has not been known. This study increased the knowledge of the diversity of viruses in forest musk deer faeces.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.


Mumps cases continue to occur, also in countries with a relatively high vaccination rate. The last major outbreaks of mumps in the Netherlands were in 2009–2012 and thereafter, only small clusters and single cases were reported. Molecular epidemiology can provide insights in the circulation of mumps viruses. The aims of the present study were to analyze the molecular epidemiology of mumps viruses in the Netherlands in 2017–2019 and to compare the phylogenetic trees built from sequence data of near complete mumps virus genomes or from the SH gene and non-coding regions (SH+NCRs). To this end, Sanger sequence data from SH+NCRs were analyzed from 82 mumps genotype G viruses. In addition, near complete genomes were obtained from 10 mumps virus isolates using next-generation sequencing. Analysis of SH+NCRs sequences of mumps genotype G viruses revealed the presence of two major genetic lineages in the Netherlands, which was confirmed by analysis of near complete genomes. Comparison of phylogenetic trees built with SH+NCRs or near complete genomes indicated that the topology was similar, while somewhat longer branches were present in the phylogenetic tree with near complete genomes. These results confirm that analysis of SH + NCRs sequence data is a useful approach for molecular surveillance. Furthermore, data from recent mumps genotype G viruses might indicate (intermittent) circulation of mumps genotype G viruses in the Netherlands in 2017–2019. Orthorubulavirus of the family of Paramyxoviridae. Infection of humans with mumps virus results in acute illness which is usually characterized by a temporary unilateral or bilateral parotitis. Occasionally, mumps virus infections can result in serious complications [Mumps viruses are single stranded negative-sense RNA viruses belonging to the genus ications , 2, but ications , 3.In 1987, vaccination against mumps virus was implemented in the National Immunization Program in the Netherlands. Although this resulted in a rapid decline of mumps cases, in recent years multiple outbreaks occurred mainly among vaccinated young adults , 5. The Molecular epidemiology can provide insights in the circulation of mumps viruses. Sequencing of the SH gene and adjacent non-coding regions provides information about the genotype and some information of circulating strains , 8. IncrThe aims of the present study were to analyze the molecular epidemiology of mumps viruses in the Netherlands in 2017–2019 and to compare phylogenetic trees built using near complete genomes with SH+NCRs. To this end, we investigated all typable specimens generated from isolated viruses of molecular confirmed mumps cases in the Netherlands from 2017 to 2019.Clinical samples  from suspected mumps cases  were submitted by municipal health services or clinical microbiological laboratories in the Netherlands via regular mail at room temperature to the National Institute for Public Health and the Environment (RIVM) for molecular diagnostics. Upon arrival at RIVM, samples were stored at +4°C until further processing. RNA was extracted by automated extraction using the MagNA Pure 96 extraction (Roche Diagnostics) with DNA and Viral NA Small Volume kit and molecular detection of mumps virus RNA was performed by real time quantitative PCR (RT-qPCR) using primers as described previously . In addi2. Subsequently, 1 ml of Dulbecco’s Modified Eagle Medium  supplemented with 100U/ml Pen-Strep (Lonza), 2 mM L-Glutamine (Lonza), 12.5 μg/ml Amphotericin B (Biowest) and 24 units/ml Nystatin suspension (Sigma) was added and plates were incubated at 37°C/5% CO2. Wells were checked daily for the presence of cytopathic effect (CPE). If no CPE was present after five days, cells were trypsinized (0.25% Trypsin-EDTA(1X) Gibco) and subsequently about 1/3 of trypsinized cells were transferred to another well and fresh medium was added. Again, cells were checked daily for the presence of CPE. If no CPE was present for 12 days after the start of the first incubation, culture was considered negative. If CPE was detected, medium was harvested and subsequently passaged one or two times over confluent Vero cells to produce a virus stock in a T25 cm2 flask or T75 cm2 flask (Corning) resulting in 8 or 25 ml cell culture supernatant medium containing mumps virus.In total 73 clinical materials  were used for virus isolation. Confluent Vero cells (ECACC 84113001) in 24 wells plates (Greiner Bio-One) were inoculated with clinical material  and cells were incubated for one hour at 37°C/5% COet al, manuscript in prep; https://github.com/DennisSchmitz/Jovian).Mumps virus isolates were processed for full genome sequencing using a viral metagenomics approach. To this end, 200μl of each cell culture supernatant was pretreated and processed for next-generation sequencing using the Illumina NextSeq 550 platform essentially as described elsewhere  –25. For Between January 2017 and October 2019, 246 mumps cases were notified in the Netherlands based on clinical symptoms and laboratory confirmation, or an epidemiological link with a laboratory confirmed case. A mumps virus genotype could be determined for 123 cases (50%), of which 108 cases (88%) belonged to genotype G. The other 15 cases belonged to either genotype C (five cases), D (one case), H (seven cases), J (one case) or K (one case). From in total 82 mumps genotype G cases could NCRs sequence data be obtained in addition to the SH sequences used for genotyping (GenBank accession numbers MT238691-MT238955).Inoculation of nine throat swabs and one oral fluid specimen (14%) resulted in a mumps virus isolate as determined by the presence of cytopathic effect. Eight mumps virus isolates belonged to genotype G, while the other two mumps virus isolates belonged to genotype C or K . Near coComparison of nucleotide sequence data of SH+NCRs obtained from mumps viruses detected in clinical materials by Sanger sequencing with consensus sequence data from isolates obtained by NGS revealed in total three differences. At position 144 of the SH gene of MuVi/Deventer.NLD/12.19 a C was present (corresponding to position 6413 of the complete genome of Mumps virus -strain Jeryl-Lynn- live major vaccine component), while a T was detected at that position in the original material of MuVs/Deventer.NLD/12.19. Sanger sequencing confirmed the presence of a C at this position in the isolate. Additional Sanger sequencing revealed that the nucleotide shift from T to C was already present after the first passage.At position 443 of the N-P NCR (corresponding to position 2141 of the complete genome of Mumps virus -strain Jeryl-Lynn- live major vaccine component), a C was detected in the sequence data from MuVi/Amsterdam.NLD/29.17/1, while an A was detected in the sequence data obtained from the original material (MuVs/Amsterdam.NLD/29.17/1). Since this nucleotide position was in the phosphoprotein gene, this nucleotide change resulted also in an amino acid change (lysine to glutamine). The presence of the C in the isolate was confirmed by Sanger sequencing. Additional Sanger sequencing analyses of the various passages indicated that the C was already detected at this position as a minor variant after the first passage, while two peaks of equal size were detected after the second passage. In the P-M NCR of MuVs/Utrecht.NLD/35.19, two peaks of similar size (A/G) were detected in the Sanger sequence data (both forward and reverse sequence) at position 76 (corresponding to position 2924 of the complete genome of Mumps virus -strain Jeryl-Lynn- live major vaccine component), while the consensus sequence of MuVi/Utrecht.NLD/35.19 was a G at that position. This nucleotide position was also in the phosphoprotein, but this nucleotide difference did not result in an amino acid change. Additional analysis of the first passage and second passage of this isolate by Sanger sequencing indicated that in all these passages both nucleotide variants were present with two peaks of equal size. No other nucleotide differences were detected between the sequence data obtained from the original materials and the isolates.Phylogenetic analysis of concatenated SH+NCRs mumps genotype G virus sequences indicated that two main lineages of genotype G virus strains were detected in the Netherlands in 2017–2019 with respectively 49 and 17 mumps viruses , which dWithin these three lineages, multiple sequence variants were detected in the Netherlands. In addition, various other virus variants were detected in the Netherlands in 2017, 2018 and 2019, but these variants were only detected in a limited amount of cases. Based on epidemiological data, 69 mumps viruses included in this phylogenetic analysis were detected in cases that were infected in the Netherlands. From 9 cases, it was known that they were infected in another country, while the country of infection was unclear in 3 mumps cases. Mumps viruses detected in 6 from these 9 cases that were not infected in the Netherlands belonged to lineage I, while the other three mumps viruses belonged to either lineage II (one mumps virus), lineage III (one mumps virus), or one of the viruses that were not part of a lineage (one mumps virus) .Comparison of the genetic differences provided by concatenated SH+NCRs sequences and near complete genomes revealed an increase in the number of nucleotide differences if near complete genomes were analyzed. For example, SH+NCRs sequences of MuVs/Utrecht.NLD/35.19 and MuVs/Eindhoven.NLD/42.18/1 were identical, while 11 nucleotide differences were detected by analysis of near complete genomes. The pairwise identities for these two viruses were 100% for the SH+NCRs and 99.97% for the near complete genomes. In addition, one nucleotide difference was present between the SH+NCRs sequences of MuVs/Amsterdam.NLD/29.17/1 and MuVs/Beugen.NLD/16.18, while 16 nucleotide differences were present between the isolated near complete genomes of these two viruses, although one of these differences was confirmed to be related to isolation of the virus. Pairwise identities between the isolates of these viruses were 99.91% for the SH+NCRs and 99.87% for the near complete genomes. Furthermore, MuVs/Deventer.NLD/12.19 and MuVs/Utrecht.NLD/15.19 were identical for SH+NCRs sequences, while four nucleotide differences were present between the near complete genomes of these isolated viruses (including one related to isolation of the virus), resulting in pairwise identities of 99.96% for the SH+NCRs and 99.97% for the near complete genomes . The aveComparison of the phylogenetic topology between SH+NCRs and near complete genomes indicated that a similar branching pattern was observed in both phylogenetic trees, except for the branching pattern of MuVi/Utrecht.NLD/35.19 and MuVi/Gelderland.NLD/26.19/2. The two nucleotide changes in SH+NCRs of MuVi/Deventer.NLD/12.19 and MuVi/Amsterdam.NLD/29.17/1 compared to clinical samples resulted in increased branch lengths, but did not change the phylogenetic topology .Alignment and subsequent phylogenetic analysis of the near complete genomes (excluding 3’ and 5’ termini) obtained from mumps virus isolates detected in the Netherlands with several representative and closely related mumps viruses indicated that these viruses also belonged to two separate groups of viruses . PairwisMumps cases continue to occur, also in countries with a relatively high vaccination rate. Molecular epidemiology could contribute to the understanding of mumps virus circulation. In the present study, the molecular epidemiology of mumps virus was evaluated using SH+NCRs mumps virus sequences obtained from mumps cases in the Netherlands in 2017–2019. Analysis of these regions with the relatively highest variation of the mumps virus genome revealed the presence of two major lineages in the Netherlands. The presence of these two lineages was confirmed by analysis of near complete genomes of a selection of mumps viruses.The presence of two lineages with multiple closely related mumps viruses in recent years, might indicate that there was (intermittent) circulation of mumps viruses in the Netherlands in recent years. This is supported by the fact that near complete genomes of both lineages are distinct from mumps viruses detected recently in mainly the United States of America and Canada , 29. Of Of interest, comparison of SH+NCRs sequence data from original materials with that of isolates indicated that passaging of mumps viruses over cells resulted in three nucleotide differences. Since the sequence of 2270 nucleotides of in 10 total viruses were compared, it was calculated that 0.013% of all nucleotide positions changed due to passaging (15384*0.00013 = ~2 nucleotide changes for a full mumps virus genome). Therefore, sequence data from isolates should be interpreted carefully, especially if used to understand exact transmission trees. To study transmission trees, preferably complete genomes obtained from original materials or eventually viruses that are passaged only a single time over cells are used , 29.Most mumps viruses from which a near complete genome was obtained in this study were collected from mumps cases that occurred several months after each other, and therefore it cannot be concluded whether these viruses are part of the same transmission chain. Of interest, mumps viruses MuVi/Noord-Holland.NLD/9.19, MuVi/Deventer.NLD/12.19 and MuVi/Utrecht.NLD/15.19 were collected within six weeks. While the sequence of MuVi/Noord-Holland.NLD/9.19 was relatively distinct from the other two viruses, between MuVi/Utrecht.NLD/15.19 and MuVi/Deventer.NLD/12.19 were only four nucleotide differences present. Since both mumps cases also lived in the same city, they were most likely part of the same mumps cluster, but there was no direct epidemiological link between these two cases.Comparison of phylogenetic trees prepared by analysis of SH+NCRs and near complete genomes indicated that the topologies of both trees were similar, while branches lengths were different. Furthermore, the mean pairwise identity of the SH+NCRs regions was slightly lower than the mean pairwise identity of the near complete genomes (99.60% vs. 99.66%), which confirms that the SH+NCRs regions are relatively variable regions of the mumps virus genome . TherefoIn conclusion, while in recent years no major outbreaks of mumps occurred in the Netherlands, the presence of two main lineages of multiple mumps genotype G viruses with identical or very similar SH+NCRs sequences might indicate (intermittent) circulation of mumps viruses in the Netherlands in 2017–2019.


If a woman becomes infected with the Zika virus while pregnant, the virus can cross the placenta and infect the baby, causing abnormalities of the eyes and the rest of the body. A detailed eye examination is essential.Flavivirus), first isolated in 1947 from a monkey in the Zika forest of Uganda. The virus is endemic in areas of Africa and Asia.Zika virus (ZIKV) is a flavivirus (genus Aedes mosquitoes (which are active in the daytime) or via sexual contact, infected blood and trans-placental transmission in utero.The virus can be spread by “Reports of Zika virus have increased recently, with cases being reported in new countries outside of Africa.”People carrying the virus can introduce Zika virus into new countries; however, Aedes mosquitoes are required to continue local transmission.Aedes aegypti mosquitos.Reports of Zika virus have increased recently, with cases being reported in new countries outside of Africa. In early 2015, Zika infection was confirmed in Brazil, causing a large outbreak due to the lack of immunity in the population and the abundance of Most people infected with Zika virus are asymptomatic. Those who have symptoms may complain of mild fever, rash, painful joints and muscles, headache and conjunctivitis. Rarely, patients may develop acute uveitis and/or a maculopathy which is characterised by macular retinal pigment epithelium (RPE) changes with a grey annulus around the fovea on posterior segment examination and disruption of outer retinal and RPE integrity, as evidenced by an optical coherence tomography OCT scan.The Zika virus may be found in tears, therefore good hand hygiene to prevent person-to-person contamination is important.If a woman becomes infected with the Zika virus while pregnant, then the virus can cross the placenta and infect the unborn foetus. This results in congenital Zika syndrome (CZS), which consists of a spectrum of clinical manifestations observed in babies who have been exposed to Zika virus while in utero.The main features are severe microcephaly with partially collapsed skull  and braiThe eye abnormalities seen in CZS include retinal pigment mottling, chorioretinal atrophy, optic atrophy/hypoplasia and coloboma  and 2b. Exposure to the virus during the first three months of pregnancy appears to be associated with more severe manifestations, although CZS may occur after maternal exposure to the virus at any point during her pregnancy.All babies born to women who may have been exposed to Zika virus during pregnancy should undergo a full clinical evaluation by a paediatrician and examination of the eye (including dilated fundoscopy) by an ophthalmologist to determine if there is any evidence of CZS which will require management and follow-up.When the infection is acquired, most cases are asymptomatic and the symptoms, if present, are non-specific. If the patient is pregnant and concerned that she may have contracted Zika, then laboratory tests are required to confirm the infection.Real-time polymerase chain reaction (RT-PCR) can identify the virus in blood samples 4 to 7 days after clinical onset. It is also possible to identify viral ribonucleic acid (RNA) in the urine up to 15 days after symptoms, even if the virus is no longer present in the bloodstream. Immunoglobulin (IgM) is increased between the 2nd and 12th week after clinical presentation; however, there may be cross-reactivity with other flaviviruses.Zika virus infection can be prevented by avoiding mosquito bites , particularly between sunrise and sunset, when Aedes mosquitos are most active.The risk of sexual transmission of Zika virus is reduced by using condoms when staying in an endemic area and for 8 weeks after returning from this area. If symptoms of Zika infection have been noted, then condom use is recommended for 6 months after the infection.Travellers returning from Zika-endemic areas should wait 28 days from their date of return before they can donate blood.Currently, there is no specific antiviral treatment and no effective vaccine to prevent the infection.