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Despite a rapid expansion of Porcine reproductive and respiratory syndrome virus (PRRSV) sublineage 8.7 over recent years, very little is known about the patterns of virus evolution, dispersal, and the factors influencing this dispersal. Relying on a national PRRSV surveillance project established over 20 years ago, we expand the available genomic data of sublineage 8.7 from China. We perform independent interlineage and intralineage recombination analyses for the entire study period, which showed a heterogeneous recombination pattern. A series of Bayesian phylogeographic analyses uncover the role of Guangdong as an important infection hub within Asia. The spatial spread of PRRSV is highly linked with a composite of human activities and the heterogeneous provincial distribution of the swine industry, largely propelled by the smaller-scale Chinese rural farming systems in the past years. We sequence all four available modified live vaccines (MLVs) and perform genomic analyses with publicly available data, of which our results suggest a key "leaky" period spanning 2011-2017 with two concurrent amino acid mutations in ORF1a 957 and ORF2 250. Overall, our study provides an in-depth overview of the evolution, transmission dynamics, and potential leaky status of HP-PRRS MLVs, providing critical insights into new MLV development., (© 2024. The Author(s).)

Different factors influence the spread of SARS-CoV-2, from the inherent transmission capabilities of the different variants to the control measurements put in place. Here we studied the introduction of the Alpha, Delta, and Omicron-BA.1 variants of concern (VOCs) into Spain. For this, we collected genomic data from the GISAID database and combined it with connectivity data from different countries with Spain to perform a phylodynamic Bayesian analysis of the introductions. Our findings reveal that the introductions of these VOCs predominantly originated from France, especially in the case of Alpha. As travel restrictions were eased during the Delta and Omicron-BA.1 waves, the number of introductions from distinct countries increased, with the United Kingdom and Germany becoming significant sources of the virus. The largest number of introductions detected corresponded to the Delta wave, which was associated with fewer restrictions and the summer period, when Spain receives a considerable number of tourists. This research underscores the importance of monitoring international travel patterns and implementing targeted public health measures to manage the spread of SARS-CoV-2., Competing Interests: Declaration of interest There are no conflicting interests.

The dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron-BA.1 variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the United States became increasingly significant. The number of detected introductions varied from 96 and 101 for Alpha and Delta to 39 for Omicron-BA.1. Most of these introductions left a low number of descendants (<10), suggesting a limited impact on the evolution of the pandemic in Galicia. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Phylogeographic analyses are able to exploit the location data associated with sampled molecular sequences to reconstruct the spatio-temporal dispersal history of a pathogen. Visualisation software is commonly used to facilitate the interpretation of the accompanying estimation results, as these are not always easily interpretable. spread.gl is a powerful, open-source and feature-rich browser application that enables smooth, intuitive and user-friendly visualisation of both discrete and continuous phylogeographic inference results, enabling the animation of pathogen geographic dispersal through time. spread.gl can render and combine the visualisation of several data layers, including a geographic layer (e.g., a world map), multiple layers that contain information extracted from the input phylogeny, and different types of layers that represent environmental data. As such, users can explore which environmental data may have shaped pathogen dispersal patterns, that can subsequently be formally tested through more principled statistical analyses. We showcase the visualisation features of spread.gl on several representative pathogen dispersal examples, including the smooth animation of a phylogeny encompassing over 17,000 genomic sequences resulting from a large-scale SARS-CoV-2 analysis., Competing Interests: Competing interests The authors declare no competing interests.

BackgroundThe earliest recognised infections by the SARS-CoV-2 Omicron variant (Pango lineage B.1.1.529) in Belgium and Switzerland suggested a connection to an international water polo tournament, held 12-14 November 2021 in Brno, Czechia.AimTo study the arrival and subsequent spread of the Omicron variant in Belgium and Switzerland, and understand the overall importance of this international sporting event on the number of infections in the two countries.MethodsWe performed intensive forward and backward contact tracing in both countries, supplemented by phylogenetic investigations using virus sequences of the suspected infection chain archived in public databases.ResultsThrough contact tracing, we identified two and one infected athletes of the Belgian and Swiss water polo teams, respectively, and subsequently also three athletes from Germany. In Belgium and Switzerland, four and three secondary infections, and three and one confirmed tertiary infections were identified. Phylogenetic investigation demonstrated that this sporting event played a role as the source of infection, but without a direct link with infections from South Africa and not as a superspreading event; the virus was found to already be circulating at that time in the countries involved.ConclusionThe SARS-CoV-2 Omicron variant started to circulate in Europe several weeks before its identification in South Africa on 24 November 2021. Accordingly, it can be assumed that travel restrictions are usually implemented too late to prevent the spread of newly detected SARS-CoV-2 variants to other regions. Phylogenetic analysis may modify the perception of an apparently clear result of intensive contact tracing.

In spring 2021, an increasing number of infections was observed caused by the hitherto rarely described SARS-CoV-2 variant A.27 in south-west Germany. From December 2020 to June 2021 this lineage has been detected in 31 countries. Phylogeographic analyses of A.27 sequences obtained from national and international databases reveal a global spread of this lineage through multiple introductions from its inferred origin in Western Africa. Variant A.27 is characterized by a mutational pattern in the spike gene that includes the L18F, L452R and N501Y spike amino acid substitutions found in various variants of concern but lacks the globally dominant D614G. Neutralization assays demonstrate an escape of A.27 from convalescent and vaccine-elicited antibody-mediated immunity. Moreover, the therapeutic monoclonal antibody Bamlanivimab and partially the REGN-COV2 cocktail fail to block infection by A.27. Our data emphasize the need for continued global monitoring of novel lineages because of the independent evolution of new escape mutations., (© 2022. The Author(s).)

The ongoing SARS (severe acute respiratory syndrome)-CoV (coronavirus)-2 pandemic has exposed major gaps in our knowledge on the origin, ecology, evolution, and spread of animal coronaviruses. Porcine epidemic diarrhea virus (PEDV) is a member of the genus Alphacoronavirus in the family Coronaviridae that may have originated from bats and leads to significant hazards and widespread epidemics in the swine population. The role of local and global trade of live swine and swine-related products in disseminating PEDV remains unclear, especially in developing countries with complex swine production systems. Here, we undertake an in-depth phylogeographic analysis of PEDV sequence data (including 247 newly sequenced samples) and employ an extension of this inference framework that enables formally testing the contribution of a range of predictor variables to the geographic spread of PEDV. Within China, the provinces of Guangdong and Henan were identified as primary hubs for the spread of PEDV, for which we estimate live swine trade to play a very important role. On a global scale, the United States and China maintain the highest number of PEDV lineages. We estimate that, after an initial introduction out of China, the United States acted as an important source of PEDV introductions into Japan, Korea, China, and Mexico. Live swine trade also explains the dispersal of PEDV on a global scale. Given the increasingly global trade of live swine, our findings have important implications for designing prevention and containment measures to combat a wide range of livestock coronaviruses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

At the end of 2020, several new variants of SARS-CoV-2-designated variants of concern-were detected and quickly suspected to be associated with a higher transmissibility and possible escape of vaccine-induced immunity. In Belgium, this discovery has motivated the initiation of a more ambitious genomic surveillance program, which is drastically increasing the number of SARS-CoV-2 genomes to analyse for monitoring the circulation of viral lineages and variants of concern. In order to efficiently analyse the massive collection of genomic data that are the result of such increased sequencing efforts, streamlined analytical strategies are crucial. In this study, we illustrate how to efficiently map the spatio-temporal dispersal of target mutations at a regional level. As a proof of concept, we focus on the Belgian province of Liège that has been consistently sampled throughout 2020, but was also one of the main epicenters of the second European epidemic wave. Specifically, we employ a recently developed phylogeographic workflow to infer the regional dispersal history of viral lineages associated with three specific mutations on the spike protein (S98F, A222V and S477N) and to quantify their relative importance through time. Our analytical pipeline enables analysing large data sets and has the potential to be quickly applied and updated to track target mutations in space and time throughout the course of an epidemic., (© 2021. The Author(s).)

Rabies is a neglected zoonotic disease which is caused by negative strand RNA-viruses belonging to the genus Lyssavirus . Within this genus, rabies viruses circulate in a diverse set of mammalian reservoir hosts, is present worldwide, and is almost always fatal in non-vaccinated humans. Approximately 59,000 people are still estimated to die from rabies each year, leading to a global initiative to work towards the goal of zero human deaths from dog-mediated rabies by 2030, requiring scientific efforts from different research fields. The past decade has seen a much increased use of phylogeographic and phylodynamic analyses to study the evolution and spread of rabies virus. We here review published studies in these research areas, making a distinction between the geographic resolution associated with the available sequence data. We pay special attention to environmental factors that these studies found to be relevant to the spread of rabies virus. Importantly, we highlight a knowledge gap in terms of applying these methods when all required data were available but not fully exploited. We conclude with an overview of recent methodological developments that have yet to be applied in phylogeographic and phylodynamic analyses of rabies virus.

Background: The present study was undertaken to examine the usefulness of both vitrification and assisted hatching (AH) on blastocysts that originate from embryos showing different qualities during their cleavage stage., Methods: A total of 281 blastocysts were vitrified (93 vitrification-warming cycles) in a mixture of ethylene glycol-dimethylsulphoxide-Ficoll and sucrose using the Hemi-Straw (HS) carrier system. After warming, AH using the partial dissection technique was performed in 36 cycles., Results: After warming and culture for 24 h, a total of 168 blastocysts (60%) was suitable for embryo transfers and a total of 25 ongoing pregnancies (27%) was obtained. Forty-nine transfers of 96 no-AH blastocysts and 36 transfers of 72 AH blastocysts resulted in an implantation rate of 13 and 22% respectively (P < 0.05). The percentage of transfers with at least one hatching blastocyst was significantly higher after application of AH (69 versus 33%) (P < 0.001). In all, 73 and 38% of blastocysts showing respectively optimal and non-optimal embryo development during the early stage were available for transfer (P < 0.001). Consequently, implantation rates of 19 and 6% were obtained after transfers of blastocysts showing respectively optimal and poor embryo development., Conclusions: Artificial opening of the zona pellucida after warming of vitrified blastocysts significantly improved the rate of transfers with hatched blastocysts and the implantation and pregnancy rates. The percentage of blastocysts that survived the HS vitrification procedure and were available for embryo transfer is related to their previous developmental quality.

Background: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity., Method: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted., Results: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01)., Conclusions: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.

Objectives: To evaluate pregnancy outcome after selective embryo reduction by transcervical aspiration or transvaginal puncture and intrathoracal injection with potassium chloride (KCl) in triplet pregnancies occurring after assisted procreation and to compare this outcome with that for triplets not undergoing embryo reduction., Design: Retrospective case series., Setting: In vitro fertilization program of the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University, Belgium, which is a tertiary referral institution., Patients: Seventy-two patients presenting a triplet pregnancy after assisted procreation., Intervention: Transcervical aspiration embryo reduction at 8 to 9 weeks of pregnancy or transvaginal puncture and intrathoracal injection of KCl at 9 to 10 weeks of pregnancy., Main Outcome Measures: Rate of spontaneous embryo reduction, complications relating to the procedure, pregnancy, and neonatal outcome., Results: The rate of spontaneous reduction was 18%. Among the 14 patients undergoing transcervical aspiration, 3 aborted and 4 lost an additional fetus. The transvaginal puncture technique had a lower complication rate (2/19). Neonatal outcome was improved in pregnancies after selective embryo reduction. After transvaginal puncture, the outcome was comparable with that for twin pregnancies after assisted procreation., Conclusions: Triplet pregnancies after assisted procreation had a poor neonatal outcome. The outcome was improved after spontaneous reduction. Transcervical aspiration should not be used because of its high rate of early and late complications. Transvaginal puncture had less early complications, but the technique might be associated with prematurity and third trimester fetal death. In triplet pregnancies, embryo reduction decreases the number of babies going home per patient, but the quality of life of the remaining babies is improved.

In a retrospective study (1985-1989) based on data from the Centre for Reproductive Medicine in Brussels, a total of 23 ectopic pregnancies (2.24%) occurred after 3800 embryo, zygote or gamete transfers. This number was low compared with the data published elsewhere. Tubal damage was a major risk factor towards developing an ectopic pregnancy after in-vitro fertilization and embryo transfer. The number of ectopic pregnancies after the association of clomiphene citrate and human menopausal gonadotrophin (HMG) was significantly higher in patients with tubal (7.8%) and non-tubal indications (2.1%) compared with those stimulated with gonadotrophin-releasing hormone (GnRH) and HMG (2.18% and 0.84%, respectively). The number of replaced embryos was not associated with the rate of ectopic pregnancy and neither did transfer technique (intra-uterine or intra-Fallopian transfer) influence the ectopic pregnancy rate.

Laparoscopic removal of benign mature teratoma is safe and effective in pre-menopausal patients. Fourteen patients underwent laparoscopic removal of such tumours. There were no operative complications. Post-operative hospitalization was short (average: 2 days). All patients undergoing a second-look laparoscopy had mild adhesions around the operated ovary. Two spontaneous and one in-vitro fertilization pregnancy occurred. If a teratoma is diagnosed during laparoscopy or oocyte retrieval, it should be promptly removed as malignant transformation is not excluded. In this case, the embryos should be frozen for later transfer.

Sixty-three normo-ovulatory infertile women were randomly divided into two groups. All women were first desensitized with the gonadotrophin-releasing hormone agonist (GnRHa), buserelin. Thereafter, ovarian stimulation with human menopausal gonadotrophins (HMG) was started in both groups but in group A the GnRHa was stopped on the same day. In group B, the GnRHa was continued during HMG treatment until the ovulatory human chorionic gonadotrophin stimulus was given. Premature luteinization was not observed in either group, although the preovulatory basal luteinizing hormone (LH) secretion was significantly higher in group A. An equal number of embryos of comparable quality was transferred in both groups and the pregnancy outcome was similar. However, the supernumerary embryos of group A were of a lower morphological quality and survived the cryopreservation process less well. We concluded that the continuous administration of a GnRH agonist during HMG treatment resulted in better quality of supernumerary embryos.

Objective: To compare zygote intrafallopian transfer (ZIFT) and in vitro fertilization and embryo transfer (IVF-ET) as treatments of male-factor infertility., Design: Patients were prospectively randomized to ZIFT or IVF-ET., Setting: In vitro fertilization program of the Centre for Reproductive Medicine of the Dutch-speaking Brussels Free University, Belgium, which is a tertiary referral institution., Patients: One hundred fifty-seven couples were enrolled in the study. Inclusion criteria allowed only first trials of couples with long-standing infertility caused by a male factor. Female factors were excluded., Interventions: In ZIFT, up to three fertilized oocytes were transferred into one single patient fallopian tube by means of laparoscopy 18 hours after insemination. In IVF-ET, cleaving embryos were replaced into the uterine cavity about 48 hours after insemination., Main Outcome Measures: Fertilization and transfer rates, implantation and pregnancy rates, pregnancy outcome, and cost per procedure were evaluated., Results: Implantation rates of 12.3% and 10% per replaced conceptus were achieved for ZIFT and IVF-ET, respectively., Conclusions: This study demonstrates no therapeutic advantage of ZIFT over IVF-ET in male-factor infertility in terms of reproductive outcome or economic benefit.

Objective: To study the relationship between the quality of the transferred embryos and the occurrence of multiple pregnancies. Embryo quality was defined by the cleavage rate and by morphological parameters such as blastomere size and the presence or absence of anucleate fragments., Design: A retrospective analysis of 1,915 consecutive transfers of fresh embryos between January 1986 and December 1989., Setting: All the embryo transfers (ETs) were performed in patients from the in vitro fertilization program of the Center for Reproductive Medicine, Vrije Universiteit Brussel, Brussels, Belgium., Results: We confirmed the relationship between the number of embryos transferred and the pregnancy rate (PR): 11.9% of the single, 19.0% of the double, and 34.1% of the triple ETs were successful. Thirty-one percent of these triple embryo replacements resulted in a multiple gestation. At the time of transfer (44 to 48 hours after insemination), we observed that embryos that had undergone at least two mitotic divisions implanted better than two-cell embryos of comparable morphological appearance (implantation rate per transferred embryo: 21.3% versus 12.3%, P less than 0.001) and that heavily fragmented embryos did not implant as well as embryos without or with fewer anucleate fragments (1.5% versus 14.1%, P less than 0.001). The PR, implantation rate, and the incidence of multiple pregnancies increased significantly with the number of good quality embryos that were transferred., Conclusions: Our study indicated that embryo quality based on morphological observations could predict the occurrence of multiple pregnancies.

The in-vitro fertilization and embryo transfer (IVF-ET) procedure was compared in 175 couples with male infertility and 480 couples with tubal infertility. In cases of male infertility, more oocytes were recovered but fewer oocytes were fertilized. Although the cleavage rate was decreased, no difference in embryo quality was found. In male infertility, fewer transfers were performed and the average number of embryos per transfer was lower. The total pregnancy rate was also lower per cycle (12.8 versus 22.9%), but not per transfer (25.4 versus 25.7%). The further obstetrical outcome was similar in both groups. We conclude that male infertility can be treated by IVF-ET but results are still disappointing when compared to a control group with normal spermatozoa.

Buserelin was inadvertently administered during 13 early pregnancies in 12 women with long-standing infertility, who had started the GnRH-agonist for gonadotrophin desensitization prior to ovarian stimulation for IVF. Six women delivered a healthy child and one pregnancy continues uneventfully. Three patients aborted before the sixth week and three women with tubal disease had an ectopic pregnancy. Corpus luteum function was normal in 11 of the 13 pregnancies. Although no evidence of embryotoxic effects of buserelin was observed, barrier contraceptive methods should be advised during the first days of its administration.

This retrospective study concerns the incidence of multiple pregnancy after the replacement of three conceptus using different techniques of assisted reproduction. During a 2-year period, 713 in vitro fertilization-embryo transfers (IVF-ETs) with three embryos, 190 gamete intrafallopian transfers (GIFT) with three oocytes, and 161 zygote intrafallopian transfers (ZIFT) with three zygotes were performed. Although we observed significant differences in implantation and pregnancy rates (PRs), the three techniques resulted in high multiple PR. At 20 weeks, 16% of GIFT pregnancies, 27% of ZIFT pregnancies, and 32% of IVF-ET pregnancies were multiple. Therefore we recommend to limit the number of conceptus transferred to a maximum of three in all cases.

The banked sperm of 5 of 13 Hodgkin patients was thawed for assisted reproduction. The post-thaw sperm characteristics were extremely impaired. Nevertheless, in 80% of all trials fertilization in vitro was obtained and conceptus could be transferred. All spouses became pregnant and seven healthy infants were born. One could recommend every Hodgkin patient to bank his sperm before chemotherapy. In vitro fertilization techniques with this frozen-thawed sperm reveal promising results in comparison with artificial insemination to maintain the reproductive capacity of male Hodgkin patients.

Ten endocrinologically normal women were injected subcutaneously with 500 micrograms D-Ser(TBU)6-EA10-LHRH (buserelin) on days 3,4 and 5 after the start of the menses. Two types of response were observed. Five women (group A) responded promptly and had a mean number of 13.4 oocytes retreived after 11.4 days of stimulation. In the second group (B), two to three times more HMG was needed to obtain a mean number of 7.3 oocytes after 17.2 days of stimulation. The response upon stimulation could be predicted by the serum gonadotrophin output on days 4 and 5 of the cycle. One woman from group B had a premature LH rise on day 16 and luteinization; her cycle was abandoned. In the four other patients of group B, serum and urinary LH concentrations showed that pituitary gonadotrophin secretion had recovered before the ovulatory stimulus, without signs of premature luteinization. Two women in each group became pregnant, one of whom aborted. This short-term GnRH agonist treatment could be an alternative method for ovarian stimulation, although it did not totally prevent the occurrence of an endogenous LH surge.

In many in-vitro fertilization (IVF) programmes, the transvaginal approach is now the most widely used method for ultrasound-guided oocyte retrieval. Sometimes, however, the position of the ovaries is such that it might not be possible to aspirate the follicle transvaginally without puncturing the uterine wall. Between May and November 1988, we performed 518 transvaginal, ultrasound-guided oocyte retrievals for IVF or zygote intra-Fallopian transfer (ZIFT). During this period, 22 transuterine punctures (4.2%) were necessary to reach one of the ovaries. To investigate the effect of uterine laceration on the results of IVF/ZIFT treatment, the outcome of treatment was compared between patients who had a transuterine puncture and a control group of patients treated by IVF or ZIFT during the same period without transuterine puncture. Seven of 16 patients who had a transuterine puncture became pregnant after IVF, compared to 84 pregnancies out of 347 cases (24.2%) in the control group. After ZIFT, two pregnancies (2/6) were achieved in patients with uterine laceration, compared to 46 pregnancies out of 149 procedures (30.8%) in the control group. These differences were not statistically significant. Transuterine puncture during transvaginal oocyte retrieval does not seem to have an adverse effect on the results of human IVF and ZIFT treatment.