Due to widespread PSA testing incidence rates of localized prostate cancer increase but curative treatment is often not required. Overtreatment imposes a substantial economic burden on health care systems. We compared the direct medical costs of conservative management and radical therapy for the management of early-stage prostate cancer in routine care.An observational study design is chosen based on claims data of a German statutory health insurance fund for the years 2008–2011. Three hundred fifty-three age-matched men diagnosed with prostate cancer and treated with conservative management and radical prostatectomy, are included. Individuals with diagnoses of metastases or treatment of advanced prostate cancer are excluded.In an excess cost approach direct medical costs are considered from an insured community perspective for in- and outpatient care, pharmaceuticals, physiotherapy, and assistive technologies. Generalized linear models adjust for comorbidity by Charlson comorbidity score and recycled predictions method calculates per capita costs per treatment strategy.p < 0.0001). Complications increase costs of assistive technologies by 30% (p = 0.0182), but do not influence any other costs. Results are robust to cost outliers and incidence of prostate cancer diagnosis. The short time horizon does not allow assessing long-term consequences of conservative management.After follow-up of 2.5 years per capita costs of conservative management are €6611 lower than costs of prostatectomy  contains supplementary material, which is available to authorized users. Prostate cancer (PCa) is the second most common cancer in men worldwide and the most common in Germany , 2. AnnuThe conservative management (CM) strategies active surveillance (AS) and watchful waiting (WW) are proposed in this context for the treatment of localized PCa to reduce overtreatment and subsequent complications such as erectile dysfunction (ED) and urinary incontinence (IC). Under AS regular biopsies, PSA tests, and digital rectal examinations are performed to initiate curative treatment if tumor progression occurs. If life expectancy is less than 10 years or comorbidity does not allow any other form of PCa-treatment WW is recommended. This strategy has no standardized follow-up scheme; symptom-oriented, palliative therapy is initiated if disease progresses . A CM-stCurrent economic studies suggest that, while health outcome in terms of survival and quality of life is similar for CM and curative treatment, CM is a cost-saving strategy over the first 5 to 10 years of treatment –12. MostIn a German health care context, claims data of statutory health insurance (SHI) funds are a valuable evidence source as medical care, resource use, and costs are documented in detail and over a long period of time for a large cohort of patients. About 85% of the German population is insured within the social security system of SHI, which is characterized by pay-as-you-go financing and income-dependent insurance contributions. The remaining 15% are covered by private insurance. SHI covers inpatient and outpatient care, pharmaceuticals, physiotherapy, and assistive technologies. Co-payments of patients are compulsory, especially for pharmaceuticals and medical aids .It is the objective of this paper to analyze claims data of a German AOK SHI fund regarding costs of CM compared to RP in an age-matched and comorbidity-adjusted cohort of men with early-stage PCa.AOK Baden-Württemberg is the largest SHI fund in the south-western German federal state of Baden-Württemberg with about 3.8 million insured individuals. About 25% of the insured population is retired and over 65 years. Data on all claims incurred at AOK between 2008 and 2011 were provided on a patient level. Co-payments to medical services covered by SHI are included in the dataset, whereas patients’ out-of-pocket payments for other services are not. German data protection laws were considered during extraction and analysis of data and AOK approved of the intended use of the data. An ethics committee was consulted regarding this study; ethics approval is not necessary as identification of individuals is not possible in the dataset.A prospective, longitudinal study design is chosen, where a cohort of men diagnosed with early-stage PCa is followed from the point of treatment initiation.The 6-month pre-observation period (January 1st 2008 to June 30th 2008) is created as a basis for the calculation of the Charlson comorbidity score (CCS), in order to allow for comorbidity adjustment of costs.In the 12-month observation period from July 1st 2008 to June 30th 2009 PCa-cases are identified and categorized into the two treatment-groups CM and RP.Both treatment groups are followed-up for a period of exactly 2.5 years (follow-up period). In case of RP, follow-up time starts individually for each person after the date of the initial prostatectomy procedure and follows each man for 2.5 years on an individual basis. Men under CM, on the other hand, are followed for a fixed period from July 1st 2009 to December 31st 2011. The reason for this is, firstly, that the starting point of CM cannot be established in the cohort; an artificial starting point has to be created, which is the beginning of the observation period (July 1st 2008). Secondly, follow-up is not intended to start with the onset of CM. This is due to the cohort selection, where men under CM have to be surveyed for at least 12 months (the observation period) to be included in the cohort; men dying in this period are not considered for analysis. In the RP- group a single event in the observation period determines inclusion in the cohort, which in turn possibly includes men dying in this period. To account for this bias, follow-up of AS is offset by 12 months.The study period is January 1st 2008 to December 31st 2011 and is divided into three sections . After exclusion of 71 individuals not constantly insured at AOK Baden-Württemberg in the study period (defined by the coding of ‘transition to another insurance fund’ in the claims data) and three individuals with female gender coding, the baseline data set includes 25,293 individuals  are calculated at baseline before and after matching. For calculation of CCS the comorbidity-group ‘cancer’ is set 0, because diseases other than PCa are of interest for adjustment.We use an excess cost approach to display cost differences between treatment strategies for inpatient and outpatient care, pharmaceuticals, physiotherapy, and assistive technologies. All direct medical costs and co-payments are considered that are relevant for the perspective of the SHI scheme insured community (§ 35b (1) SGBV) . Comorbip-values less or equal than 0.05 are considered statistically significant [Ninety-five percent confidence intervals (CI) are calculated for costs via a non-parametric bootstrap approach based on 1000 replications. Difference in costs is tested via bootstrap hypothesis testing and nificant . The CCSnificant , 22. To In a sensitivity analysis we include only incident PCa cases, with no C61 code in the pre-observation period–assuming that CM is initiated with the first PCa-diagnosis–to estimate the influence of the cohort selection on outcomes.Statistical analyses are performed with the software package SAS, version 9.3.Mean age at baseline after matching is 69 years (STD 6.80) in the RP-group and 70 years STD 7.13) in the CM-group, with a mean CCS of 0.11 and 0.19, respectively. ED, IC and BPH are most prevalent in the RP-group at baseline  and outpatient costs  as well as costs for assistive technologies  than RP. Overall, total incremental costs of CM are significantly lower compared to RP  .Comparison of adjusted costs Table  displaysFor the analysis of inpatient, outpatient, pharmaceutical, physiotherapy, and assistive technology costs two-part models are used, as more than 10% of individuals have zero costs respectively. Total costs are analyzed in a one-part model, as no individuals have zero costs.p = 0.016) in the incident cohort.Analysis of costs is repeated including incident PCa-cases only, which comprised of an age-matched cohort of 320 men . Cost analysis reveals that mean comorbidity-adjusted total costs of CM increase by €1496 to €13,430 in total compared to base case. Outpatient costs of CM also increase compared to base case from €2332 to €2499. Overall, CM is still significantly less costly than RP  while costs of surveillance, especially AS, are more equally distributed over time –9. The sIn the base case analysis pharmaceutical costs are higher under CM compared to RP, not significantly though; this may be explained by pharmaceutical treatment of BPH in the CM-group which is not necessary in the RP-group.Published cost studies with a European health care context comparing CM and RP report similar cost differences to our study. Andersson et al.  report €Several US-based observational and modeling studies show that CM is the least costly strategy over the whole study duration, consistent with the results presented here , 8, 24. Our study has some limitations. Regarding cohort selection, there is no information on tumor stage or Gleason score included in claims data, which might allow clinical classification of PCa. To account for this limitation we define early-stage PCa as absence of both diagnoses of metastases and treatment associated with recurrence or advanced tumor progression. Inclusion in the CM-group cannot be based on clinical criteria or specific procedure codes and therefore a distinction between WW- and AS-patients is not possible. Seven Percent of men under CM receive at least one biopsy during follow-up which suggests that these are under AS, while the remaining men may be under WW. The cohort’s life expectancy, however, is with a mean age of 70 years at baseline greater than 10 years and men are–according to treatment guideline–not eligible to WW. Patients might actually be under AS, but are not surveyed by regular biopsies according to guideline. This might be due to current studies on adverse effects of serial biopsies on erectile function and infectious complications , 27. FurRegarding the study design, a 12-month observation period is chosen which excludes CM-patients who receive curative therapy in this period. The probability for curative treatment in the first year of CM is, however, low (<5%) . Also itRegarding cost analysis, calculation of costs for single procedures is not conducted in this study as case numbers are too small to be representative. CCS is not able to adjust for comorbidities with high outpatient and pharmaceutical costs as the score was developed to assess inpatient mortality. We do not account for dependency of data due to matching by using conditional regression analysis. Dependency of data is very low as we only match for age; also no longitudinal analysis of single individuals is intended.Generalizability of results to the general population is limited as claims data cannot report out-of-pocket-payments. Also, due to the excess cost approach absolute costs do not reflect PCa treatment specific costs and no conclusion can be inferred on the costs of RP and CM from this study.Overall, our analysis indicates that initial treatment costs as well as short-term follow-up costs of CM based on claims data of a German SHI fund are significantly lower than the costs of curative therapy with RP for early-stage PCa–predominantly due to the high initial costs of the surgery. Treatment of complications following initial therapy has a very small impact on costs. These results must be interpreted in light of the limitations regarding cohort selection in claims data.Further research is necessary to analyze costs of CM compared to curative treatment over a longer time horizon than reported in this study including costs of long-term complications and end-of-life care.


A woman with controlled HIV infection developed in late August 2016 a pruritic rash with fever and conjunctival hyperaemia after a trip to the French Caribbean islands. On day 3 after symptom onset, Zika virus RNA was detected in plasma, urine and vaginal samples with respective viral loads of 3.8, 6.1 and 5.3 log copies/mL. Notably, we demonstrated the presence of infectious Zika virus particles in the vaginal samples by isolation in cell culture. A female patient returned to France from the Caribbean islands with acute Zika virus infection. After obtaining informed consent, we collected vaginal swabs for both RNA detection and virus culture in order to investigate the infectivity of vaginal secretions.A French woman in her 40s, with a controlled HIV-1 infection, travelled to French Caribbean islands (Martinique for seven days then Guadeloupe for three weeks) in summer 2016. On the day of return to France, she felt asthenic with myalgia. Two days later, a pruritic rash appeared on her face, chest, back and arms, with abdominal pain and diarrhoea. She consulted her general practitioner, who referred her to our department on the same day.+ T-cell count over 500/mm3 under antiretroviral therapy (emtricitabine/tenofovir and nevirapine). Physical examination showed a widespread itching maculopapular exanthema, conjunctival hyperaemia and mild fever (38.1 °C). Intense asthenia and diffuse myalgia were still ongoing.Her medical history was limited to a virologically suppressed HIV-1 infection with CD4As the symptoms were consistent with acute Zika virus infection, the patient consented to have a vaginal swab, after being informed that there would be no direct benefit from knowing the test result. In the absence of a standardised collection protocol, vaginal secretions were collected by direct swab  and after instilling 5 mL of saline solution between the cervix and the posterior vaginal wall . At her request, her scheduled cervical Pap smear was performed at the same time.3  and C-reactive protein 8 mg/L (norm: < 5 mg/L). RT-PCR assays were negative for dengue and chikungunya viruses in plasma  and a test for NS1 antigen for dengue virus was also negative . Detection of Zika virus RNA by RT-PCR  was positive in plasma, urine and vaginal secretions, three days after onset of symptoms. The viral RNA load was 3.8 log copies/mL in plasma, 6.1 in urine, 5.3 in vaginal sample 1 and 3.9 in vaginal sample 2. The use of a saline solution instillation for the second sampling possibly led to a dilution of the sample. The two vaginal samples were inoculated on Vero and C6/36 cells, and Zika virus was isolated from both, demonstrating the presence of infective Zika virus. All methods were performed as detailed in the supplementary data of a previous publication [Standard laboratory tests showed moderate lymphocytopenia with 1,120 cells/mmThe patient’s fever subsided on day 3, and the rash on day 6 after symptom onset. A transient bilateral knee pain occurred on day 4.The patient consented to a second genital swab 10 days after the onset of symptoms: no viral RNA was detected in vaginal secretions and in cervical mucus. No serum collection was performed during the second visit. At that time, she had recovered from all symptoms except a mild persistent asthenia. Her husband did not present symptoms evocative of Zika virus infection during or after the journey. The patient stated that their sexual intercourses were always protected by condom use. Initially, Zika virus was thought to be exclusively transmitted through mosquitoes, but sexual transmission was subsequently reported. Transmission from men to their female or male sexual partners has been well and often demonstrated and has been observed up to 44 days after symptom onset -5, whereThis seldom reported and transient presence of Zika virus in the female genital tract contrasts with an extensive literature about its persistence in semen, where RNA has been detected up to six months after return from endemic areas ,10-14. MThe short time period during which we could detect Zika virus in our patient’s genital tract is consistent with our prior failure to detect the virus by RT-PCR from vaginal swabs in two other patients, seven and eight days after the onset of symptoms . AlthougRecently, a mouse model of Zika virus infection by vaginal exposure demonstrated that Zika virus replicated within the genital mucosa and could lead to a fetal infection , but no Recent evidence of extended presence of the virus in semen and of possible transmission from women to men has led to an update of the United States Centers for Disease Control and Prevention (CDC)’s guidance on the prevention of sexual transmission of Zika virus . Current


Brugia malayi MF in the blood of a dually-infected host and that the virus is carried into the mosquito hemocoel by the MF during their penetration of the mosquito midgut. The mechanism of MF enhancement may be more complex than simple leakage of viremic blood into the hemocoel during MF penetration. The affinity of arboviruses to adhere to or otherwise associate with MF may depend on the specific combination of the virus and MF involved in a dual host infection. This in turn may determine the relative importance that MF enhancement has within an arbovirus transmission system.Concurrent ingestion of microfilariae (MF) and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Within hours of being ingested, MF penetrate the mosquito midgut and introduce virus into mosquito hemocoel, creating a disseminated viral infection much sooner than normal. How virus is actually introduced is not known. In this report, we present experimental evidence that suggests that certain alphaviruses may adhere or otherwise associate with sheathed  The basic mechanism of arboviral infection in mosquitoes has been known for many years . Brieflyetc.). Laboratory studies have shown that MF in the blood can enhance the infectivity of arboviruses to mosquitoes by enabling virus to bypass the midgut barriers [microfilarial enhancement of arboviral transmission [Aedes aegypti, would produce not only a higher-than-normal incidence of dengue during the initial year of its introduction, but would also generate more frequent  epidemic waves over a 14-year simulation period.In nature, vertebrate hosts of arboviruses are frequently infected with filarial nematodes. As part of their life cycle, microfilariae (MF) penetrate the mosquito midgut after being ingested and move to their preferred site of development within the hemocoel , moderate in another (Ae. aegypti) and absent altogether in a third (Ae. triseriatus). These differences were attributed to differences in the quantity of blood that leaked into the hemocoel during MF penetration. However when a different virus—Venezuelan equine encephalitis virus (VEEV)–was tested in the same Aedes / B. malayi system but in gerbils that had both lower viremias (2- to 10-fold) and lower microfilaremias (ca. 2-fold) [less susceptible to VEEV  than to EEEV  , the mag to EEEV . ClearlyAe. albopictus collected in Polk County, Florida USA [Aedes mosquitoes were used; Ae. aegypti Rockefeller strain and Ae. taeniorhynchus Vero Beach strain. This study used seven male mongolian gerbils (Meriones unguiculatus) that were 7–12 weeks old. Microfilaremic gerbils, interperitoneally infected with Brugia malayi filarial parasites, were provided by the NIH/NIAID Filariasis Research Reagent Resource Center for distribution by BEI Resources, NIAID, NIH (NR-49238). Nonmicrofilaremic gerbils were obtained from Tumblebrook Farm, Brookfield, MA USA. Gerbils were maintained in standardized rat cages fitted with filter tops and supplied wood-chip bedding and enrichment toys . Gerbils were maintained on standard rat chow, room temperature and light cycle and were monitored twice daily by a trained animal care technician throughout the experiment. An institutionally approved protocol was in place that defined the early/humane endpoint and in the event that animals became visibly ill  gerbils were to be euthanized via CO2 inhalation. None of the animals became ill or died prior to the experimental endpoint. Gerbils were anesthetized with an approved anesthetic  prior to having mosquitoes feed on them. After the experiment, gerbils were euthanized via CO2 inhalation.Two viruses were used; VEEV, V3000 infectious clone of the epizootic subtype 1A Trinidad donkey strain  and EEEVrida USA . The VEErida USA , except To compare relative susceptibilities to hemocoelomically introduced virus, 4- to 10-day-old nulliparous female mosquitoes were inoculated  with VEEB. malayi yielded higher rates of viral dissemination that did studies with EEEV and B. malayi, despite the fact that host viremias were higher with EEEV. To investigate further these unanticipated findings, calculations were made to estimate the proportional amount of viremic blood ingested by a dually-infected mosquito that would have to have been introduced into the hemocoel by each penetrating MF in order to yield the level of enhancement observed in our trials. We call this estimate ‘parts per blood meal’ (ppBM) per MF. For each trial, the host viremia and numbers of penetrating MF had been recorded and thus we used these data, in combination with the dose-response equations generated from inoculation studies described above, to calculate ppBM estimates for each VEEV and EEEV trial. For example in a trial wherein Ae. taeniorhynchus were fed on a dually-infected gerbil, there was a 54% net increase in the EEEV dissemination rate over that in the control group. Substituting 0.54 for the Y-value in the dose-response equation for relative susceptibility of Ae. taeniorhynchus to inoculated EEEV and solving for the X-value, it was calculated that a hemocoelomically delivered dose of 0.096 PFU of EEEV virus per mosquito was required to infect 54% of the mosquitoes. The average viremic content of the actual blood meals was measured at 630 PFU per mosquito and the geometric mean number of penetrating MF per mosquito was 27.3. Therefore, the total proportion of viremic blood meal that must have been introduced into the hemocoel per MF to produce a 54% enhanced dissemination rate was: (0.096 PFU / 630 PFU) ÷ 27.3 MF or 0.0000056 parts of the blood meal per MF (ppBM). In this way we calculated ppBM values for each trial in which there occurred a significant enhancement of EEEV or VEEV dissemination due to concurrent ingestion of B. malayi MF by mosquitoes.As mentioned previously, our studies with VEEV and The kinetics of viral growth within the hemocoels of infected mosquitoes were compared between mosquitoes fed on dually-infected gerbils  versus mBrugia malayi MF were incubated for one hour at 35°C in 5 ml of cell culture medium to which was added either VEEV (starting titer = 105.6 PFU/ml) or EEEV (starting titer = 103.5 PFU/ml). Tubes were gently agitated every 5–10 minutes to ensure mixing of MF and virus. Afterwards, tubes were centrifuged for 5 minutes at 1,000 rpm to pellet the MF and 4.5 ml of the supernatant was removed and stored at -70°C for later viral quantification. The MF pellets were re-suspended by adding 4.5 ml of fresh medium. Tubes were vortexed for several minutes to ensure mixing and then centrifuged again at 1,000 rpm to pellet the MF. This procedure of washing the MF with serial 10-fold dilutions was repeated eight times in order to dilute virus beyond the theoretical limits of detection. After the final centrifugation and supernatant removal, the residual 0.5 ml of diluent containing the MF pellet was vortexed and the MF ‘slurry’ was assayed for virus by plaque assay. A small amount (ca. 100 μl) was reserved to inoculate into the thoraces of Ae. aegypti mosquitoes (3 μl per mosquito) as described above. Inoculated mosquitoes were maintained for 7 days and then homogenized individually and assayed for virus by plaque assay.Several thousand 10 as the independent variable and infection rates transformed to probits as the dependent variable. The software package, Statistix  was used with the 0.05 level of significance throughout.Rates of viral infection and dissemination were compared among groups by chi square analyses or Fisher’s exact test, depending on sample size. To construct dose response equations, linear regression analyses were performed with viral dose transformed to logGuide for the Care and Use of Laboratory Animals, National Research Council, 2011. The USAMRIID IACUC specifically approved this study.Research at U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) was conducted under an Institutional Animal Care and Use Committee (IACUC) approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other federal statutes and regulations relating to animals and experiments involving animals. This facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to the principles stated in the Ae. taeniorhynchus, the mean ppBM for VEEV introduced into the hemocoel per MF  was over 23 times greater than that estimated for EEEV   was 19 times greater than that estimated for EEEV   . Similar; n = 1) . ReplicaAe. taeniorhynchus mosquitoes were compared between mosquitoes whereby virus was introduced by MF (= virus + MF) versus direct inoculation into the thorax. Mosquitoes tolerated serial amputations well, with over 95% survival at the end of the 5-day trial  versus mosquitoes infected by intrathoracic inoculation , then the growth curves for the two sets of mosquitoes (inoculated versus per os) should have been identical. However, if the virus was not immediately available, then there would have been a delay in the growth curve. This is what was observed  of ay trial . It tookculation . Importaculation . Furtherobserved .3.5 PFU/ml and titers of the supernatants decreased logarithmically with each successive wash until the fourth wash, at which point there was no detectable virus left in successive supernatants (5.6 PFU/ml). During the dilution series, virus titers in the supernatants leveled off at 102.3 PFU/ml after the fifth wash and did not diminish with successive washings. After the final wash, MF pellets for both viruses were assayed by plaque assay and by mosquito inoculation. No EEEV was detected in the MF pellet by plaque assay. Examination of the plaque assay wells with an inverted microscope revealed an abundance of pelleted MF trapped within the solidified agar overlay, but no virus plaques were present. However, nearly half of the 24 Ae. aegypti mosquitoes inoculated with 0.3 μL of the pelleted MF suspension developed viral infections 1 week after inoculation. This indicated that residual EEEV had remained in or on the MF even though it was undetectable by direct plaque assay. Mosquito inoculations were more sensitive than the plaque assays perhaps because once inoculated, infective virions had an additional week in which to replicate within the mosquito hemocoel before being processed for plaque assay. In the VEEV trial, virus was detected at high levels both by plaque assay and mosquito inoculation. Pelleted MF within the agar of the plaque assays were surrounded by large coalescing plaques and all 24 inoculated mosquitoes became infected. These results suggest that both EEEV and VEEV became associated with B. malayi MF during the hour-long incubation and remained in or on the MF despite repeated washings. The higher viral concentration of VEEV in the MF pellet, as determined by both plaque assay and mosquito inoculation, suggests that viral affinity to B. malayi MF was higher with VEEV than with EEEV.The affinity of the MF/virus association was examined by incubating MF with virus, then subjecting the MF to repeated cycles of washing and then assaying the washed MF pellet for virus. In the EEEV trial, the starting virus titer was 10rnatants . For theB. malayi MF during their exodus from the midgut. This is different than the simple leakage of viremic blood into the hemocoel from holes or lacerations caused by penetrating MF. When mosquitoes feed on a host that is concurrently viremic and microfilaremic, both pathogens are ingested and infectious virions may enter the mosquito hemocoel and establish a disseminated viral infection sooner than normal [B. malayi MF  than for EEEV (44 x 10−7 ppBM)  . These cBrugia spp. MF passage through the mosquito midgut indicate that MF penetration occurs rapidly and passage is largely complete within 3 to 4 hours after an infective meal [Second, there was a delay of ca. 24 hours in the growth kinetics of virus within mosquito hemocoels when virus was delivered by MF versus by direct inoculation . Studiesive meal –21. TherThird, MF were incubated with virus and then subjected to a series of repeated washings so extensive that any residual virus should have been diluted beyond the theoretical limits of detection. Yet the washed MF still contained infectious virus . This inBrugia MF and VEEV and EEEV. Studies whereby Brugia pahangi MF were incubated in a suspension of cationic colloidal iron demonstrated that the microfilarial sheaths were strongly electronegative. However, once the MF had penetrated the mosquito midgut and shed their sheaths, the surfaces of MF became electro-neutral [Brugia MF retain their sheaths following ingestion by Aedes spp. mosquitoes and that sheaths are only shed after MF pass through the midgut [Ae. aegypti, exsheathment of ingested MF may occur prior to midgut penetration [B. malayi MF and EEEV/VEEV were mediated by charge interactions between the MF sheaths (= negative) and virus (= positive), and the sheaths were retained until after MF crossed the mosquito midgut, this would explain the 24-hour delay in viral growth kinetics between MF-mediated versus direct inoculation modes of viral entry into the mosquito hemocoel. Perhaps during the host viremia, virus adheres to the sheath of MF within the blood of a dually-infected host but disassociates only after MF pass through the midgut and the virus-coated sheaths are shed within the mosquito hemocoel.The precise nature of this association remains unknown but one possibility may be related to differences in the electrostatic charges between -neutral . Most ste midgut , 23–25. etration , 26. Theetration –29. If tAe. taeniorhynchus, we did not find evidence of virus leakage into the hemocoel. However, at higher viremias it is entirely possible that infectious virus might leak out from the exit sites created by MF in sufficient quantities to produce enhanced dissemination.In areas where arboviral and filarial infections co-occur and prevalence of filariasis is high, MF enhancement may act to intensify natural arboviral transmission cycles. However, MF enhancement can only occur if the following 5 conditions are met. First, a vertebrate host must be concurrently infected with both virus and microfilariae. Second, mosquitoes feeding on a dually infected host must ingest both virus and microfilariae. Third, microfilariae must penetrate the mosquito midgut. Fourth, upon microfilarial penetration, sufficient virus must pass into the mosquito body cavity to establish infection. Fifth, there must be no salivary gland barriers and the mosquito must be able to transmit the virus by bite. In this report, we have identified a complexity to the fourth condition. That is, potential introduction of virus into the hemocoel is dependent not only on the host microfilaremia (i.e. number of punctures caused by MF), viremia (i.e. dose of inoculum), and the amount of tissue damage produced during MF penetration (i.e. size of punctures) but also—and perhaps more importantly—on the affinity of virus to adhere to or otherwise associate with the MF. If the virus-MF affinity is weak or nil, then virus introduction into the hemocoel could only result from leakage from punctures. Relatively high levels of microfilaremia, viremia, and/or expansive midgut fissures created during MF penetration would be required to produce enhanced viral dissemination in the vector. The degree to which MF typically create midgut fissures remains unclear. Some histological studies on MF penetration have described lesions disrupting the full depth of the midgut wall –24 whereBrugia), then MF enhancement may occur at lower host microfilaremia and/or viremia, and require fewer, less destructive MF penetrations through the midgut. In this regard, it is important to note that some MF species are sheathed whereas others are not. The presence or absence of a sheath, as well as the physio-chemical composition of the sheath, may affect virus-MF affinity and thus influence the probability of MF enhancement occurring.If on the other hand, the affinity between virus and MF is strong Click here for additional data file.


This review discusses the pathophysiology, risk factors, and the advances in the prevention or treatment of graft-vs-host disease (GvHD) by exploiting adjunct virotherapy. In addition, nonviral adjunct therapeutic options for the prevention of GvHD in the context of allogeneic hematopoietic stem cell transplantation  are discussed. The role of oncolytic viruses to treat different HSCT-eligible hematological cancers is also considered and correlated with the issue of GvHD in the context of allo-HSCT.Poxviridae family, called myxoma virus (MYXV), not only exhibits oncolytic activity against various hematologic malignancies like multiple myeloma (MM) or acute myeloid leukemia (AML) but also, in addition, ex vivo MYXV treatment of human allogeneic-bone marrow transplants , or allo-peripheral blood mononuclear cell  transplants can abrogate GvHD in xenografted mice without impairing graft-vs-tumor (GvT) effects against residual cancer. To date, this is the first and the only oncolytic virus with a dual potential of mediating oncolysis against a residual cancer target and also inhibiting or preventing GvHD following allo-HSCT.Emerging therapies focused on the prevention or treatment of GvHD include the use of regulatory T cells (Tregs), mesenchymal stem cells (MSCs), microbiome manipulation, B cell inhibitors, among others. Our lab and others have reported that an oncolytic DNA virus from the This review discusses how oncolytic virotherapy can be applied as a potential adjunct therapy for the potential treatment of GvHD. In addition, we highlight major emerging nonviral therapies currently studied for the treatment or prevention of GvHD. We also review the emerging oncolytic virotherapies against different hematological cancers currently eligible for allo-HSCT and highlight the potential role of the oncolytic virus MYXV to decrease GvHD while maintaining or enhancing the positive benefits of GvT. For many malignant and nonmalignant hematological or immunological diseases, the only cure can be acquired in combination with allogenic hematopoietic stem cell transplantation  . The mosIn an effort to prevent induction of GvHD, or ameliorate its pathologic consequences, non-myeloablative and reduced conditioning regimens along with prophylactic drugs have been used with partial beneficial outcomes. Despite the significant progress achieved during the last decade to prevent, or overcome the severity of, GvHD after allo-HSCT, it remains the major cause of non-relapsing cancer mortality, being fatal for up to 15–20% of recipients undergoing allo-HSCT . TherefoGvHD occurs when donor T cells activate and respond to HLA mismatches on recipient’s tissue. Three stages contribute to the onset of aGvHD . First, The pathogenesis of GvHD is ascribed to the tissue-destructive activities of the donor leukocytes, in particular the activated T lymphocytes. In the setting of an allogeneic HSCT, in which the donor transplant sample is usually either derived from bone marrow or mobilized PBMCs, the T cells in the donor graft are activated upon interaction between donor T cell receptors (TCRs) and major (or minor) histocompatibility complex (MHC)-bound host allo-antigens, resulting in vigorous T proliferation, differentiation, and migration into multiple host target organs and tissues  Fig. 1)1). FurthCurrent prophylaxis and treatment for GvHD are partially effective with high risk of disease relapse, development of infections, and long-term adverse effects. Because the centerpiece of prophylaxis and treatment of GvHD involve T cell manipulation, different approaches have been used in clinical trials to prevent or treat GvHD. Table Besides targeting, or manipulating allo-reactive T cells, other approaches to prevent GvHD are focused on induction of regulatory T cells, targeting B cells, mesenchymal stem cells (MSCs), and the use of chemokine and cytokine antagonists such as maraviroc, TNF inhibitor, IL-2 receptor antagonist, and IL-6 inhibitor. In this section, we briefly discuss some of the more relevant emerging approaches used to abrogate GvHD.+CD25+FoxP3+ cells that promote immunotolerance and play a key role in inhibiting excessive immune responses, and preventing, or delaying rejection of the allografts [+ Tregs is maintained by IL-2, administration of low doses of IL-2 increased the proliferation of Tregs and increased the generation of thymic Tregs [Regulatory T cells (Tregs) are CD4lografts . Tregs slografts . Althougic Tregs , 40. ThuIncreasing evidence links B lymphocytes with the pathogenesis of aGvHD and cGvHD . There aMesenchymal stem cells (MSCs) are non-hematopoietic pluripotent progenitor cells that are found in adult BM as well as in adipose and perinatal tissues . MSCs suBecause MSCs support stem cell engraftment, inhibit lymphocyte responses, and are safe post-infusion, MSCs have emerged as potential treatment of complications related to allogeneic HSCT . In the Recent studies have demonstrated both negative pathologic consequences and positive anti-cancer benefits that can accompany cytomegalovirus (CMV) reactivation in terms of relapse of myeloid malignancies following allo-HSCT . However+ cells, which require rigorous conditions to upscale these TK+ cells under good manufacturing practice (GMP) conditions.In a different approach, allogeneic transplantation of donor lymphocytes engineered with the suicide gene thymidine kinase of herpes-simplex virus (TK) showed therapeutic potential to control GvHD. However, this methodology has been restricted due to the difficulty to ex vivo manipulation of donor lymphocytes and the limitation in the generation of the TKPoxviridae family. In nature, MYXV exhibits a highly restricted host range and is only pathogenic to European rabbits. Importantly, it has been shown that MYXV can also infect a wide variety of human cancers, including pancreatic, ovarian, melanoma, glioblastoma, and various hematologic malignancies such as MM and AML. Preclinical studies have also demonstrated that MYXV is a safe OV candidate even in highly immunodeficient mice [Because a variety of neoplasms remain incurable with current standard therapies, novel and innovative anti-cancer therapies are required in order to sustain long-term cancer regression and prolong patients’ lives. Both immunotherapy and oncolytic virotherapy are new promising approaches to clinical cancer therapeutics. The success of oncolytic viruses (OV) in the clinical setting depends on the selective tumor cell oncolysis by the therapeutic virus, followed by activation of cellular immune responses against both viral and tumor antigens. One common characteristic of many candidate OVs is that they are safe for normal healthy cells and tissues, but selectively infect and replicate in a wide spectrum of human cancer cells . Myxoma ent mice . MYXV isThere are at least 2 doz viruses that are now in the path to be translated form the bench to the bedside, including measles virus, vesicular stomatitis virus, adenovirus, reovirus, herpes simplex virus, parvoviruses, and two poxviruses, vaccinia virus, and MYXV –62. VaccThe use of OVs has garnered considerable interest as cancer therapeutics and is currently under intense clinical investigation. Among different hematologic malignancies, multiple myeloma (MM) has begun to emerge as a prime candidate for oncolytic virotherapy. MM is a clonal plasma cell (PC) malignancy with an estimated of 30,770 new cases and 12,770 patient deaths in 2018 . DespiteOf the many viruses that are currently under investigation for MM are the RNA viruses including measles virus, vesicular stomatitis virus, reovirus, and coxsackievirus 21 and DNA viruses such as adenovirus, vaccinia virus, and MYXV. To date, MYXV is the only OV shown to be capable of both ameliorating GvHD following allo-HSCT, in addition to possessing anti-cancer activities. However, to put this seemingly unique feature of MYXV in perspective, we discuss this virus in context with other OVs being currently developed against various hematologic malignancies that are currently eligible for HSCT.Morbillivirus under the family Paramyxoviridae, causes infections in the respiratory tract. Virus entry to the cells occurs through the interaction of the viral hemagglutinin-glycoprotein (H-glycoprotein) with the CD46 receptor, which is overexpressed in cancer cells like MM [Measles virus (MV), a negative-strand RNA virus that belongs to the genus  like MM . Edmonst like MM . Earlier like MM . Other M like MM . The MV- like MM . The pre like MM . Because like MM . This maReovirus is a non-enveloped double-strand RNA virus with minimal pathogenicity in humans . ReoviruRhabdoviridae family. The virus has the ability to cause vesicular lesions in farm animals [Vesicular stomatitis virus (VSV) is a negative-strand-enveloped RNA virus that belongs to the  animals . However animals , and whe animals . Because animals . Preclin animals , 88. ThePicornaviridae family. In humans, this virus causes myositis and respiratory tract infections [+ plasma cells at up to 98.7% with minimal effects in progenitor cell function [Coxsackievirus 21 (CVA21) is a non-enveloped positive-strand RNA that belongs to the fections , 90. Howfections . CVA21 ifections , 92. Prefections . Incubatfunction . Becausefunction . Immunocfunction . DespiteAdenovirus (Ad) members are non-enveloped double-stranded DNA viruses, and wild-type Ad may cause mild clinical infections of the upper respiratory tract; however, they may also cause significant morbidity and mortality in immune-compromised patients . Attenuaαvβ5 or αvβ3 are required for the internalization of the virus [Preclinical studies showed the efficacy of Ad to deliver the thymidine kinase (TK) gene into MM cell lines like OCI-My5 and RPMI 8226, as well as primary patient samples . Becausehe virus ; it was he virus . In a sthe virus . In effehe virus . Despitehe virus . In the he virus , 102. Thhe virus  and morthe virus . In addiPoxviridae family and is a close relative of the smallpox virus [Vaccinia virus (VACV) belongs to the ox virus . VACV isox virus . Differeox virus , 107. Mcox virus . This atox virus . In addiox virus .The first clinical study using VACV dates back to 1987 when a 67-year-old patient with IgA MM received intravenous injection of VACV Ankara strain (AS) resulting in the decrease of the levels of IgA from 1309 mg/dL in the early stage of the treatment to 432 mg/dL on the day 96 of the regimen and with no adverse effects . In 2008Poxviridae family whose natural tropism is restrictive to rabbits. There are no reported anti-MYXV antibodies in any human populations. Like vaccinia, MYXV is a double-stranded DNA poxvirus with its replication cycle strictly performed in the cytoplasm. Notably, MYXV is nonpathogenic to humans or any non-rabbit vertebrate hosts, including mice or any domestic animals. Studies performed in our lab have shown that MYXV had oncolytic activity against a wide variety of human cancer cells in vitro and in vivo [Myxoma virus (MYXV) is a member of the  in vivo . In 2009 in vivo . Althoug in vivo . For exa in vivo . In anot in vivo . It is k in vivo . Likewis in vivo .+) were spared by the virus [−/− (NSG) host. Importantly, MYXV did not compromise the engraftment of HSPCs from the CD34+ compartment because of the inability of MYXV to bind or infect this cell population [+ HSPCs that allows the virus to be used as an adjunct ex vivo therapy for either auto- or allo-HSCT. In addition, intravenous (i.v.) systemic delivery of MYXV into BALB/c mice bearing the murine MOPC315 myeloma cell line eliminates these cancer cells via induction of rapid cellular apoptosis mediated by the systemic MYXV treatment [In 2012, studies performed by Bartee et al. revealed that infection of human MM cell lines with MYXV resulted in the efficient eradication of these MM cancer cells via induction of rapid cellular apoptosis, whereas normal human hematopoietic and progenitor cells  that had presumably persisted within disease niches at the time of transplant and/or the re-infusion of myeloma cells that can contaminate the autograft. In order to improve disease-free responses and overall survival, tandem approaches including autologous transplantation, non-myeloablative allogeneic transplants, post-transplant maintenance, and immunotherapy strategies have been tested  model demonstrated that human bone marrow samples pre-treated ex vivo with MYXV and then transplanted into NSG mice greatly reduced mortality compared with control engrafted mice  . Death ophocytes . Authorsphocytes . Thus, aphocytes . Thus, MMore recently, in vivo experiments using a classic mismatched mouse to mouse allogeneic HSCT model  confirmed the oncolytic potential of MYXV to decrease tumor burden and increase the survival rates in the recipient mice . AlthougAs mentioned before, the standard treatment for MM is with myeloablative therapy along with autologous HSCT. However, to a lesser extent, some MM patients have also been treated with reduced-intensity conditioning followed by allo-HSCT . Thus, t+ hematopoietic stem cells and is harmless for immune engraftment, (2) the virus can target, infect, and eliminate a spectrum of hematological cancers in patients who are eligible for HSCT, (3) the virus can load onto other primary leukocytes within the allo-transplant sample and migrate with these cells to sites of disseminated cancer in the transplant recipient, and finally, (4) the virus can infect and compromise the effector functions of CD3+ T lymphocytes that normally become activated and drive GvHD following allo-HSCT.Up to now, MYXV is the first and the only oncolytic virus that has been reported to eliminate residual cancer  and also prevent GvHD in the setting of allo-HSCT. It is possible that other select oncolytic viruses might also possess these dual anti-cancer/anti-GvHD properties, as long as it could be demonstrated that (1) the virus cannot infect primary CD34Undoubtedly, a better understanding of GvHD biology will pave the way to develop novel treatment strategies with improved clinical benefits for patients receiving allogeneic HSCT. However, despite the myriad of biological and pharmacologic therapies used in combination with allogeneic transplantation procedures, GvHD still represents the major cause of morbidity and mortality of non-relapsed malignancies. In addition, the treatment of some very aggressive cancers like MM is mainly restrictive conventional therapies or autologous HSCT. However, although these therapies often do provide years of additional life, they still do not induce long-term survival in the majority of patients with MM. Combination therapies, including tandem treatments , are attractive but their application is restrictive to those patients that have matched MHC-related or unrelated donors. In addition, more extensive research is required in order to improve the efficacy of these tandem therapies. A new innovative strategy is the use of oncolytic viruses like MYXV in the setting of allo-HSCT. Recent studies have shown that MYXV could have a dual role not only inhibiting the development of GvHD but also maintaining or enhancing the positive benefits of GvT effects derived from allo-HSCT. However, more preclinical studies are required in order to understand the molecular mechanism used by MYXV to control allo-reactive T cells and to improve the effector mechanisms of GvT.


Since July in 2015, an emerging infectious disease, Fowl adenovirus (FAdV) species C infection with Hepatitis-Hydropericardium syndrome was prevalent in chicken flocks in China. In our study, one FAdV strain was isolated from commercial broiler chickens and was designated as SDSX1.The phylogenetic information, genetic mutations and pathogenicity of SDSX1 were evaluated.7 to 1011 per 100 mg tissue and liver had the highest virus genome copies.The phylogenetic analysis indicated that SDSX1 is a strain of serotype 4, FAdV-C. The amino acid analysis of fiber-2 showed that there were more than 20 mutations compared with the non-virulent FAdV-C strains. The pathogenic evaluation of SDSX1 showed that the mortality of one-day-old chickens inoculated SDSX1 was 100%. The typical histopathological changes of SDSX1 were characterized by the presence of basophilic intranuclear inclusion bodies in hepatocytes. The virus copies in different tissues varied from10In conclusion, the isolate SDSX1, identified as FAdV-4, could cause one-day-old chicks’ typical inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) with 100% mortality. The virus genome loads were the highest in the liver. Molecular analysis indicated that substitutions in fiber-2 proteins may contribute to the pathogenicity of SDSX1. Aviadenovirusof the family Adenoviridae [Fowl adenoviruses (FAdVs) are non-enveloped, double-stranded DNA linear viruses, belonging to the genus oviridae , 2. The oviridae –5. The vFAdVs are grouped into 5 species (FAdV-A to FAdV-E) based on genomic restriction enzyme digest patterns with 12 serotypes identified so far: FAdV-A (serotype FAdV-1); FAdV-B (serotype FAdV-5);FAdV-C (serotypes FAdV-4 and -10); FAdV-D ; and FAdV-E  . FAdV-4 From June to November in 2015, a disease emerged in large-scale broiler and egg-laying chickens in farms in Shandong, Henan, Hebei, Liaoning, Anhui and Jilin provinces in China. The disease inflicted mostly the 20-to-30-day-old broilers with a mortality rate of 20–30%, and occasionally affected layers aged 20–70 days or 200–300 days but with a lower mortality rate. Usually, there were no obvious clinical signs with a sudden death. The death peak was 4–8 days after onset and the course of disease could last 8–15 days. The diseased animals had flabby hearts with straw-colored fluid in the pericardial sac and the swollen liver with foci of hemorrhage and/or necrosis. The aim of this study was to molecular characterization and pathogenicity assessment of FAdV strain responsible for disease outbreak in chickens.The virus was isolated from the SPF chicken’s embryos by 5 blind passages and it was designated as SDSX1. The whole genome nucleotide sequence and hexon gene sequence of SDSX1 are available in the GenBank  and the complete genome for SDSX1 was found to be 43,630 bp in length. The percent sequence identity for available whole genomes showed that SDSX1 has 100% sequence homology with three isolates from China , CH/SDDZ/2015, CH/AHBZ/2015 , and CH/JSXZ/2015 (Table p < 0.0001). There were no obvious clinical manifestations for the inoculated chickens and the chickens had normal intake before death. Typical gross pathological changes in chickens were the flabby heart with straw-colored fluid in the pericardial sac and the swollen liver with foci of hemorrhages .The data indicated that SDSX1 was a highly pathogenic isolate. Regarding the pathogenicity of the SDSX1 isolate, the survival curve showed that all of 10 one-day-old SPF chickens inoculated with the SDSX1 died within 14 days. The results were consistent with several previous studies in which parts of serotype 4 were highly pathogenic strains , 25, 26.+ T cell proliferative response of the chickens immunized with fiber-2 protein were significantly higher than that of the chickens immunized with inactivated vaccine, and fiber-2 could provide 100% protection [It is reported that fiber-2 played a significant role in virulence of FAdV . Severalotection . The neuotection . In our In conclusion, the isolate SDSX1, identified as FAdV-4, could cause in one-day-old chicks’ typical inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) with 100% mortality. The virus genome loads were highest in the liver. Molecular analysis indicated that substitutions in fiber-2 proteins may contribute to the pathogenicity of SDSX1.Twenty 10~20-day-olddead “817” broilers were collected from various farms in Liaocheng in Shandong province. Gross lesions were examined, and liver tissues were fixed in 10% formalin for 48 h at room temperature and then HE staining was performed following routine histological procedures. Animal work performed in this study was approved by Research Animal Use and Care Committee, the Agricultural University of Hebei, in compliance with the Guidance for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China.A TaqMan-based real-time PCR assay developed in-house for specific detection of FAdV-4 was used to determine the presence of FAdV-4 in collected liver samples as described previously . The priThe whole sequence of the isolated virus was sequenced by Next Generation Sequencing (NGS). Briefly, the DNA samples were ultrasonicated to generate fragments less than 500 bp. The DNA fragments were polished using T4 polynucleotide kinase. The adaptors were then ligated and loaded on the HiSeq 2000 for sequencing.W/V), filtered and centrifuged. The supernatant from each sample was then collected and used for inoculation of 9-day-old specific pathogen free (SPF) chicken embryos  via allantoic cavity. The dose of inoculation was 0.1 ml per embryo. The allantoic fluid from infected embryos was harvested and used for repeated inoculation of chicken embryos. 5 embryos were used for each inoculum. The inoculation dose of allantoic fluid was 0.1 ml per embryo for each time. After 5 passages, allantoic fluid was harvested in the biohazard safety equipment and virus nucleic acid was extracted for DNA sequencing analysis. A specific fragment of 724 bp from the hexon gene was amplified by PCR for detecting the virus and the following primers were used: sense, 5’GACCTTCGCGGACTACTTGG3’; and antisense, 5’GACGGTTTGGTTGGAACTGAT3’.Virus isolation was performed on the samples with positive PCR results. The liver tissue was homogenized in 0.01 M PBS containing penicillin (100 IU/μL) and streptomycin (100 μg/μL) with the ration of 1:5, which were prepared by serial 10-fold dilution. The embryos were incubated at 37 °C for 6 days and observed every day. Embryos that died within the first 24 h were discarded, those that died than 24 h were selected for the allantoic fluid collection. PCR was used for detecting virus in allantoic fluid. Results were recorded.Virus titer in chicken embryos for the allantoic fluid of five passage viruses was determined by chicken embryos infectivity assay. The result was recorded as EID6 EID50 per chicken. Virus used for inoculation was prepared in the biohazard safety equipment. Chickens in the control group were mock inoculated with the same dosage of PBS. The dosage and pathway of inoculation was selected based on the pre-experiment (data not shown). All the chickens were observed daily for clinical signs up to all chickens are dead. The date and time of the death of each animal were recorded. When the chickens died, the liver samples were collected for the histopathological examination and virus detection.Twenty one-day SPF chickens were randomly allotted to two groups (10 chickens per group). For the inoculated group, each chicken was intraperitoneally inoculated with 1 ml SDSX1 virus containing 10Quantification of the SDSX1 load in each of the analyzed organs was performed by Real-time PCR method reported in the previous study . BrieflyData were expressed as mean ± standard deviations. The survival curve of the inoculated and control chickens and the photograph of SDSX1 virus distribution in experimented SPF chickens were made using GraphPad Prism (version 5.0). One-way analysis of variance test was used.


The Third Annual Meeting of the European Virus Bioinformatics Center (EVBC) took place in Glasgow, United Kingdom, 28–29 March 2019. Virus bioinformatics has become central to virology research, and advances in bioinformatics have led to improved approaches to investigate viral infections and outbreaks, being successfully used to detect, control, and treat infections of humans and animals. This active field of research has attracted approximately 110 experts in virology and bioinformatics/computational biology from Europe and other parts of the world to attend the two-day meeting in Glasgow to increase scientific exchange between laboratory- and computer-based researchers. The meeting was held at the McIntyre Building of the University of Glasgow; a perfect location, as it was originally built to be a place for “rubbing your brains with those of other people”, as Rector Stanley Baldwin described it. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The meeting featured eight invited and twelve contributed talks, on the four main topics: (1) systems virology, (2) virus-host interactions and the virome, (3) virus classification and evolution and (4) epidemiology, surveillance and evolution. Further, the meeting featured 34 oral poster presentations, all of which focused on specific areas of virus bioinformatics. This report summarizes the main research findings and highlights presented at the meeting. The European Virus Bioinformatics Center (EVBC) was conceived of in 2017 to bring together experts in virology and virus bioinformatics in Europe . T. T61]. TFor a virus to have epidemic potential in human populations, an infected individual must be capable of transmitting the infection to other individuals. However, for the majority of human RNA virus species, human infections are acquired only from non-human reservoirs. The evolution of human transmissibility is poorly understood. Through parallel analyses of 1755 RNA viruses, we identified at least 90 nodes across 39 genus-level phylogenies associated with transitions involving the gain of human infectivity and/or transmissibility. Human-infective and human-transmissible viruses evolve independently, and at least 73% of human-transmissible RNA virus lineages emerged directly from non-human virus lineages in diverse mammal or bird taxa. Negative sense single-stranded RNA virus lineages generate a higher proportion of strictly zoonotic viruses. Our analysis demonstrates that RNA viruses from mammal/bird lineages not currently known to be infective to humans are a likely source of future epidemics in human populations, a public health threat recently designated “Disease X”.http://evbc.uni-jena.de/events/3rd-evbc-meeting).Another important facet of this year’s annual EVBC meeting was the poster session on Thursday evening. The standard of the research presented was extremely high and, combined with a networking event in the Glasgow University Union, provided plenty of opportunity to meet the presenters. The relaxed atmosphere was instrumental to promoting discussions and developing new interactions between attendees. The list of poster presenters and titles can be found online (The Third Annual Meeting of the European Virus Bioinformatics Center brought together scientists in the field with expertise in different disciplines for scientific exchange and provided the opportunity for discussing ongoing and new collaborations. The meeting attracted new researchers to virus bioinformatics, which was reflected by several first-time attendees. The presentations strongly underlined the interdisciplinary “virology meets bioinformatics” character of the meeting. We enjoyed lively discussions after the speakers’ presentations, in the breaks, during the poster session and at the social events.evbc@uni-jena.de.We hope that speakers summaries provided in this report will give an interesting insight into the field of virus bioinformatics and will encourage interested researchers to join us at the Fourth Annual Meeting of the EVBC to be held in Switzerland in 2020. For more information, do not hesitate to contact us via


Oribivirus genus in the family Reoviridae. Genetic recombination through reassortment has been described in AHSV, but not through homologous intragenic recombination. The influence of the latter on the evolution of AHSV was investigated by analyzing the complete genomes of more than 100 viruses to identify evidence of recombination. Segment-1, segment-6, segment-7, and segment-10 showed evidence of intragenic recombination, yet only one (Segment-10) of these events was manifested in subsequent lineages. The other three hybrid segments were as a result of recombination between field isolates and the vaccine derived live attenuated viruses (ALVs).Intragenic recombination has been described in various RNA viruses as a mechanism to increase genetic diversity, resulting in increased virulence, expanded host range, or adaptability to a changing environment. Orbiviruses are no exception to this, with intragenic recombination previously detected in the type species, bluetongue virus (BTV). African horse sickness virus (AHSV) is a double-stranded RNA virus belonging to the  Culicoides have been associated with AHSV transmission and replication [African horse sickness virus (AHSV) causes a non-contagious, but highly infectious arthropod-borne disease of equids known as African horse sickness (AHS). In susceptible horses, the disease causes severe morbidity and mortality, while mules, donkeys, and zebra are less affected and considered to be vertebrate reservoirs ,2. The vlication . The Worlication . African horse sickness virus particles are non-enveloped, icosahedral, and consist of a double capsid layer containing the genome composed of ten linear, double-stranded RNA (dsRNA) segments ,7. The gGenetic recombination has no restrictions and can take place in segmented and non-segmented viruses, including the orbiviruses such as blue tongue virus (BTV) ,17,18. TA study by Woods  reportedComplete genome sequences of more than 100 AHSV isolates were used to analyze genomic recombination. The majority of the viruses were collected between 1933 and 2014 in Sub-Saharan Africa at the OIE World Reference Centre for AHSV and BTV at the Agricultural Research Council–Onderstepoort Veterinary Institute, South Africa. The complete genomes of 87 isolates were determined for this study (KP939368–KP940236 and KP009621–KP009790), while the sequences of an additional 14 were extracted from GenBank . Sequencv/v) foetal bovine serum (FBS), L-glutamine (200 mM) and 25 µg/mL Penicillin-Streptomycin-Amphotericin B (PSA) (Gibco) were infected with freeze-dried individual AHSV isolates (Monolayers of baby hamster kidney (BHK21) cells cultured in Dulbecco’s Modified Essential Medium (DMEM)  supplemented with 5%  v4.39 package . The RDPIndividual alignments of each of the ten virus genome segments were used to generate separate phylogenetic trees. Significant incongruences were detected between the tree topologies representing the ten genome segments, suggesting that recombination influences AHSV evolution. In order to detect evidence of intragenic recombination, the individual segment alignments were analyzed using different methods described in the RDP package. Potential recombination events were predicted in segment-1, segment-6, segment-7, and segment-10. These included both single and double cross-over events. Each putative recombination event was subsequently analyzed and will be discussed individually.p-values of RDP = 6.47 × 10−20, GENECONV = 3.37 × 10−15, BootScan = 2.903 × 10−21, MaxChi = 1.45 ×10−8, SiScan = 1.83 × 10−8, and 3Seq = 5.64 × 10−11  and SiSscan predicted p = 2.30 × 10−9  used in vaccines by Onderstepoort Biological Products (OBP). The alignments were used to identify possible recombination events and four genome segments provided evidence of intragenic recombination. The first genomic recombination event was predicted in Segment-1. The latter consists of 3965 base pairs and is therefore the largest of the AHSV genome segments. It codes for virus protein 1 (VP1), one of the enzymatic minor core proteins responsible for the RNA-dependent-RNA-Polymerase activity . In BTV,The second genome segment to present evidence of intragenic recombination is Segment-6 encoding VP5, which forms the inner layer of the outer capsid. VP5 has been shown to play a role in membrane destabilization prior to virus entry into cells . The genSegment-7 is conserved, 1167bp in size and codes for the 349 amino acid major core protein VP7 . MaximumThe last recombination event appeared in of segment-10 of multiple isolates. Segment-10 is the smallest and second most variable genomic segment, coding for two in frame non-structural proteins involved in virus release NS3/NS3A . In BTV,This study provides the first evidence of intragenic recombination in African horse sickness virus, albeit less abundant than in bluetongue virus. In comparison, BTV has more antigenic diversity than AHSV with an ever-growing number of serotypes described, 27 and nine respectively . The disUnfortunately, the intragenic recombination described here cannot account for the significant incongruence observed between phylogenetic trees generated from different AHSV segments. This necessitates subsequent investigations into the role of reassortment on AHSV evolution.


The idea that positive-sense RNA (+RNA) viruses may be to some extent taxonomically unified arose when unexpected similarities between animal and plant virus genomes were discovered (reviewed in reference The question thus arises, whether the membrane association of the polymerase was a primordial characteristic of viruses in branches 2 and 3, which may have facilitated virus replication in early eukaryotic cells possessing internal membranes. The phylogeny of RdRps cannot answer this question, since the membrane-binding sites in viral replicases reside outside the conserved core RdRp domain . Further


An influenza epidemic is still a problem despite the development of vaccines and anti-influenza drugs. Preventive measures such as handwashing are fundamental and important for counteracting influenza virus infection. In this study, we clarified the anti-influenza virus effects of surfactants, which are the main components of hand soaps for hand washing: potassium oleate (C18:1), sodium laureth sulfate (LES) and sodium lauryl sulfate (SDS). For a human influenza virus strain (H3N2), C18:1 reduced the infectivity by 4 logs or more, whereas LES and SDS reduced the infectivity by 1 log or less. Similar results were obtained when an avian influenza virus strain (H5N3) was used. The interaction between the surfactant and virus was then investigated by isothermal titration calorimetry. The LES-virus system showed a positive value of enthalpy changes (ΔH), meaning an exothermic interaction that indicated a hydrophobic interaction. In contrast, both the C18:1-virus system and the SDS-virus system showed negative values of ΔH, meaning an endothermic interaction that indicated an electrical interaction. The ΔH value of the C18:1-virus system was much higher than that of the SDS-virus system. A mixture of C18:1 and HA proteins similarly showed negative values of ΔH. These results indicate that influenza virus inactivation by a hydrophobic interaction of a surfactant with the viral envelope is insufficient to prevent infection, whereas inactivation by an electrical interaction of a surfactant with HA proteins is sufficient to prevent influenza virus infection. Orthomyxoviridae, has a virus particle (virion) of ca. 100 nm in diameter. The virion is covered with an envelope composed of a lipid bilayer and inserted viral spike proteins, hemagglutinin (HA), neuraminidase (NA) and M2 ion channel proteins. One influenza virus particle is estimated to contain roughly 700 HA proteins, 200 NA proteins and 20 M2 proteins [Influenza virus, which belongs to the family Influenza virus causes an outbreak every year, disturbing social activities at schools and workplaces and increasing medical expenses . InfluenInfluenza virus infection can be prevented by vaccines and can be treated with anti-influenza drugs. However, these measures might be ineffective due to antigenic changes or drug resistance of influenza viruses. Preventive measures such as handwashing and wearing masks are fundamental for counteracting influenza virus infection.A hand soap used for handwashing contains a surfactant as a basic ingredient. Synthetic surfactants such as sodium laureth sulfate (LES) and sodium lauryl sulfate (SDS) are used for hand soaps . A surfaThe aim of the present study was to clarify the anti-influenza virus effects of representative surfactants, LES, SDS and potassium oleate (C18:1), that are generally used in hand soap and to elucidate the mechanism of action in order to provide insights for the development of a new hand soap.2 gas.MDCK(+) cells, canine renal epithelial cells, were purchased from ATCC as MDCK cells (NBL-2: ATCC CCL-34) through Dainippon Pharmaceutical Co.  as described by Noma et al. , and theTen-day-old chicken embryonated eggs were purchased from Hiroshima Experimental Animals Corp. . The eggs were not in a specific-pathogen-free grade but maintained in a bioclean condition.Human influenza virus A/Udorn/72 (H3N2) and avian influenza virus A/whistling swan/Shimane/499/83 (H5N3) were provided by Dr. R. A. Lamb  and Dr. K. Otsuki , respectively, and propagated in embryonated chicken eggs.Sodium dodecyl sulfate (SDS), sodium laureth sulfate (LES) and oleic acid were purchased from Wako Pure Chemical Industries , NOF Corporation  and Tokyo Chemical Industry , respectively. Potassium hydroxide was purchased from Wako Pure Chemical Industries. All reagents were used without further purification.An influenza HA vaccine for the 2013–2014 season (manufacturing number HA130A), containing the HA proteins of A/California/7/2009(H1N1) pdm09, A/Texas/50/2012(H3N2) and B/Massachusetts/2/2012, was purchased from Biken Corporation Ltd. . The vaccine was a split vaccine made from purified virus particles and contained more than 90 µg/ml of the HA proteins from the three viruses.SDS and LES were dissolved in deionized water at 0.35 mmol/l. To enhance homogeneous dispersion, the solutions were heated at 40°C for 1 h. Oleic acid was dissolved in deionized water at 0.35 mmol/l, and then an equimolar amount of potassium hydroxide was added and the solution was heated at 75°C for 1 h for conversion to potassium oleate (C18:1). Finally, the pH of the aqueous C18:1 solution was adjusted to 10.3 by adding potassium hydroxide.50) was calculated. The anti-virus effect was estimated by comparing surfactant-treated infectivity with mock-treated infectivity.An anti-virus assay for influenza virus was performed as described previously . BrieflyInfluenza virus particles were purified as described previously . Briefly−1 mmol/l) was maintained in the ITC cell (1.4 ml) at 25°C with stirring at 300 rpm. Aliquots of virus solution  were injected into the ITC cell using a microsyringe. The volume of each injection was 5 μl. The duration of each injection was 14 s, and there was an interval of 250 s to allow for equilibration correction. The heat of dilution was subtracted even though its contribution to total heat was negligibly small. The enthalpy changes (ΔH) due to interaction were determined from the titration data on the basis of the amount of surfactant molecules.ITC experiments were performed at 25°C using a VP-ITC MicroCal microcalorimeter . An aqueous surfactant solution , whereas SDS and LES reduced the infectivity by 1 log or less, although the reductions were statistically significant. In the case of 10-fold concentrated surfactants  pdm09, A/Texas/50/2012(H3N2) and B/Massachusetts/ 2/2012, was injected into C18:1 solution (17.5 mmol/l). The C18:1-HA system showed a negative value of ΔH, indicating an attractive interaction between C18:1 and HA.(TIF)Click here for additional data file.


Glycogen storage disease type I (GSD I) is a relatively rare metabolic disease with variable clinical intensity. It is caused by deficient activity of the glucose 6-phosphatase enzyme (GSD Ia) or a deficiency in the microsomal transport proteins for glucose 6-phosphate (GSD Ib). We searched the most recent English literature (1997-2017) regarding any article with the key word of “glycogen storage disease type I” in PubMed, Science Direct, Scopus, EMBASE, and Google Scholar. We will present all of the published articles about the molecular genetic characteristics and old-to-new diagnostic methods used to identify GSD I in regard of methodology, advantages and disadvantages. Diagnosis of GSD type I and its variants is challenging because it is a genetically heterogeneous disorder. Many molecular methods have been used to diagnose GSD I most of which have been based on mutation detection. Therefore, we discuss complete aspects of all of the molecular diagnostic tests, which have been used in GSD type I so far. With the advent of high throughput advanced molecular tests, molecular diagnosis is going to be an important platform for the diagnosis of storage and metabolic diseases such as GSD type I. Next-generation sequencing, in combination with the biochemical tests and clinical signs and symptoms create an accurate, reliable and feasible method. It can overcome the difficulties by the diagnosis of diseases with broad clinical and genetic heterogeneity. Glycogen storage diseases (GSDs) are a group of metabolic disorders determined by the accumulation of glycogen in different tissues. GSDs are caused by the enzyme deficiencies effect on glycogen synthesis, glycogen breakdown or glycolysis (glucose breakdown), typically within muscles and/or liver cells. The disorders were numbered as they were discovered which classified chronologically by GSD type I (von Gierke disease) to GSD type XI . MajoriGSD (Glycogen storage disease) type I or von Gierke disease is the second most common type of GSDs. GSD type I typically presents in early childhood. Von Gierke disease is an autosomal recessive disorder. There is no report regarding ethnic difference in the incidence of GSD type I, however there are different types of mutations in Caucasian, Hispanic, Asian and Jewish populations . The ovHistorically, in 1929, Edgar von Gierke was the first to describe a glycogen storage disease (GSD), which initially was named in his honor  regarding any article with the key word of “glycogen storage disease type I” in PubMed, Science Direct, Scopus, EMBASE, and Google Scholar.GSD I is characterized by accumulation of glycogen and fat in the liver and kidneys, leading to hepatomegaly and renomegaly. The clinical manifestations of von Gierke disease include growth retardation, hepatomegaly, hypoglycemia, lactic acidemia, hyperuricemia, osteoporosis and hyperlipidemia . The paThe clinical presentations of GSD Ib is quite similar to that of GSD Ia, however the symptoms of the patients with GSD Ia, such as hepatomegaly, a characteristic ''doll-like'' face, short stature, and chronic fatigue are more severe. Unlike patients with GSD-Ia, most patients with GSD-Ib also suffer from neutrophil dysfunction and neutropenia, lead to frequent bacterial infections. Clinical differential diagnosis of GSD-Ia from Ib is difficult because neutropenia is sometimes periodic or never develops in GSD-Ib  was codified the enzyme translocase and mutations within this gene in GSD Ib patients were described  to translocate glucose-6-phosphate (G6P) from the cytosol to the lumen of the endoplasmic reticulum (ER) and two other transport systems to transport the reaction products phosphate (Pi) and glucose (G6PT2 and G6PT3 respectively) to the cytosol , which al., 1997). G6PT1 al., 2002). It hasal., 2002). G6PC and most patients with GSD Ic and Id have mutations in the G6PT1 gene SLC37A4, therefore GSD I is now divided into two general subtypes GSD Ia and GSD I non-a, respectively  and GSD-Ib, a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) . But wial., 1995; Veiga-Dal., 1995). Howeveal., 1995; Kishnanal., 1995; Janeckeal., 1995). Among al., 2001).The final and conclusive diagnosis of GSD needed pathologic diagnosis in combination with biochemical and clinical findings; however, liver biopsy is an invasive procedure . PractiPrompt and accurate diagnosis is the most important point for the proper treatment of metabolic diseases. Diagnosis of GSD I is sometimes complicated because there are common features between GSD I and III, including hepatomegaly, hypoglycemia, and hyperlipidemia. Validated and clinically useful tools with a positive predictive value > 90 % are necessary for the diagnosis of GSD I. The diagnosis of GSD I currently relies on clinical features, pathologic diagnosis of liver biopsy, biochemical, and molecular genetic tests  and pathologic findings of the liver biopsy in favor of GSD .G6PC or G6PT1 gene unique to Caucasian, Hispanic, Chinese/Japanese/ Korean, and Jewish GSD I patients have been described, suggesting separate ethnic founder effects for some mutations . Transfal. (1999) has conal. (1999) analyzeal. (1999) showed al., 2000; Nakamural., 2000).Eventually, even a single base alteration can be detected by the altered mobility of the single-stranded DNA molecule in SSCP. In 2004, in one study the frequency of two prevalent mutations of GSD Ia patient in Caucasian (the Q347X and R83C mutation) was reported to screen the Ashkenazi Jewish population by SSCP method as an accurate and easy technique which leads to a predicted prevalence  five times higher than in the general Caucasian population .According to the above literature, disadvantages of SSCP method include the requirement of highly standardized electrophoretic conditions in order to get constant results. Furthermore, some mutations may remain undetected, and accordingly definite absence of mutation cannot be proven. In HD method single-base substitutions are less stable and excessively sensitive to environmental changes. This fact reduces the sensitivity of this method for this type of mutations, which is frequently found in GSD I , two deletions (540del5 and 158delC) and one nonsense mutation (Q347X). Three of them have not been described previously and the R83C was the most common mutation among the Czech and Slovak patients  has been applied for screening of unknown point mutations. Identification of mutations in the al., 2000). This tal., 2000).G6PC (GSD Ia) and SLC37A4 (GSD Ib) full genes can be used for confirming the diagnosis of these diseases precisely  have launched that are referred to as massively parallel or next-generation sequencing (NGS). Direct sequencing has proven extremely successful because of its accuracy and affordability, but it is inappropriate for large-scale screening projects because it has been developed to sequence only one amplified DNA molecule at one time  in a given sample on both strands . Moreoval., 2014; Janeckeal., 2014). Directal., 2014; Gerin eal., 2014). Directal., 2017). Since al., 2017; Lam et al., 2017; Ihara eal., 2017; Triocheal., 2017; Chiang al., 2017; Kozák eal., 2017; Reis etal., 2017; Yuen etal., 2017; Qiu et al., 2017; Ki et aal., 2017; Miltenbal., 2017; Qiu et al., 2017; Tamhankal., 2017; Liang eal., 2017; Gu et aal., 2017; Lu et aal., 2017; Mahmoudal., 2017, Choi etal., 2017), Koreanal., 2017), Brazilal., 2017), Chinesal., 2017; Chiang al., 2017; Qiu et al., 2017; Gu et aal., 2017; Lu et aal., 2017), Indianal., 2017; Karthi al., 2017), Japaneal., 2017), Czech al., 2017), Hungaral., 2017) and Freal., 2017) patiental., 2017), Chinesal., 2017; Qiu et al., 2017; Liang eal., 2017) Japanesal., 2017) populatiga, 2018). Converiga, 2018; Wang etiga, 2018). Briefliga, 2018). SuccesWang et al. (2013) have deSeveral approaches have been used for detection of known mutations. These methods usually started with the polymerase chain reaction (PCR) and additional assay steps are performed based on the type of mutation such as RFLP-PCR . Ihara non-A patients which is advantageous for a primary molecular genetic diagnostic approach. They concluded that the principal advantages of DHPLC are its semi-automated nature, with rapid results (a few minutes per sample), and the feasibility to collect eluted DNA for next analyses, but DHPLC alone cannot provide the details about the nature of mutations. Also, the sensitivity of the method is dependent on the temperature of analysis, the selection of which is dependent on operator experience  technique after PCR . Thoughal., 2000). Lam etal., 2000) have real., 2000).G6PC gene because it is a major cause of GSD-Ia among Japanese patients. The high reproducibility and sensitivity of this technique indicates that the method may be safely applied to clinical diagnosis. Afterward, Kojima et al.  combined with a TaqMan fluorogenic probe (TaqMan-ASA)  used a al. (2004) performal. .Lately, high resolution melting analysis (HRMA), a simple real-time PCR-based method for detecting sequence variations for GSD Ia was developed. The basis of this method is that changes in amplicons of HRM are dependent on their DNA melting curves in the presence of saturating DNA binding dyes . TherefIn the last decade, the use of DNA microarray technique for diagnosis of genetic diseases has been reported , the cost, speed and accuracy of detection increase. The authors declare that they have no conflicts of interest.


Culex mosquitoes collected in Haiti in 2016. This finding raises questions about the role of SPONV as a human pathogen in Haiti and other Caribbean countries.Spondweni virus (SPONV) and Zika virus cause similar diseases in humans. We detected SPONV outside of Africa from a pool of  Spondweni virus (SPONV) and Zika virus are closely related flaviviruses that were first described in Africa in 1952 and 1947, respectively  within a 10-mile radius in Gressier, a semirural setting. Trap locations were selected based on environmental considerations, low risk for traps being disturbed, and known human arbovirus-caused illnesses in the area , and sex. All pools were screened for chikungunya virus, dengue virus (DENV) serotypes 1–4, and Zika virus RNA by real-time reverse transcription PCR (rRT-PCR) (Aedes and Culex mosquito species by molecular methods (Cx. quinquefasciatus mosquitoes (non–blood-fed) collected on July 4, 2016, rRT-PCR results suggested the presence of Zika virus RNA , but this same pool was negative for chikungunya virus and DENV RNA by rRT-PCR. After unsuccessful attempts to amplify Zika virus–specific amplicons using previously described Zika virus sequencing primers, we used an unbiased sequencing approach after treatment of virions in mosquito homogenate with cyanase (We detected SPONV from a pool of 7 mixed-sex rRT-PCR)  Table 1,rRT-PCR)  Table 2. cyanase  Table 3, cyanase . ThereafThe SPONV genome from Haiti shared 10,174 (98.8%) of 10,290 nt identity with a SPONV isolate from mosquitoes in South Africa in 1954 (GenBank accession no. DQ859064) and 9,958 (96.8%) of 10,287 nt identity with the SPONV Chuku strain from blood of a febrile human patient in Nigeria in 1952 (accession no. KX227369) (Table). When compared with the Zika virus reference strain from Uganda (accession no. KY989511), a strain from Puerto Rico (accession no. KU501215), and a strain from Haiti in 2016 (accession no. MF384325), Zika virus and SPONV clearly continue to diverge because the nucleotide and amino acid identities of SPONV are less similar to more recent strains of Zika virus (Table). Few SPONV sequences have been deposited into GenBank, resulting in insufficient information to predict how and when SPONV was introduced in Haiti.Cx. quinquefasciatus mosquitoes raises questions about the role of this species as a vector for this virus and highlights the need for ongoing surveillance for SPONV infection among humans in the Caribbean, combined with studies of potential vector populations.In the Americas and the Caribbean, SPONV is a potential emergent arbovirus and public health threat that manifests clinically with symptoms and signs similar to those of Zika virus infection (Culex quinquefasciatus mosquitoes, Haiti, 2016.Additional methods for study of Spondweni virus in