Metagenomic sequencing, molecular characterization, and Bayesian phylogenetics of imported type 2 vaccine-derived poliovirus, Spain, 2021

Introduction: In 2021, a type 2 vaccine-derived poliovirus (VDPV2) was isolated from the stool of a patient with acute flaccid paralysis (AFP) admitted to Spain from Senegal. A virological investigation was conducted to characterize and trace the origin of VDPV2. Methods: We used an unbiased metagenomic approach for the whole-genome sequencing of VDPV2 from the stool (pre-treated with chloroform) and from the poliovirus-positive supernatant. Phylogenetic analyses and molecular epidemiological analyses relying on the Bayesian Markov Chain Monte Carlo methodology were used to determine the geographical origin and estimate the date of the initiating dose of the oral poliovirus vaccine for the imported VDPV2. Results: We obtained a high percentage of viral reads per total reads mapped to the poliovirus genome (69.5% for pre-treated stool and 75.8% for isolate) with a great depth of sequencing coverage (5,931 and 11,581, respectively) and complete genome coverage (100%). The two key attenuating mutations in the Sabin 2 strain had reverted (A481G in the 5'UTR and Ile143Thr in VP1). In addition, the genome had a recombinant structure between type-2 poliovirus and an unidentified non-polio enterovirus-C (NPEV-C) strain with a crossover point in the protease-2A genomic region. VP1 phylogenetic analysis revealed that this strain is closely related to VDPV2 strains circulating in Senegal in 2021. According to Bayesian phylogenetics, the most recent common ancestor of the imported VDPV2 could date back 2.6 years (95% HPD: 1.7-3.7) in Senegal. We suggest that all VDPV2s circulating in 2020-21 in Senegal, Guinea, Gambia, and Mauritania have an ancestral origin in Senegal estimated around 2015. All 50 stool samples from healthy case contacts collected in Spain (n = 25) and Senegal (n = 25) and four wastewater samples collected in Spain were poliovirus negative. Discussion: By using a whole-genome sequencing protocol with unbiased metagenomics from the clinical sample and viral isolate with high sequence coverage, efficiency, and throughput, we confirmed the classification of VDPV as a circulating type. The close genomic linkage with strains from Senegal was consistent with their classification as imported. Given the scarce number of complete genome sequences for NPEV-C in public databases, this protocol could help expand poliovirus and NPEV-C sequencing capacity worldwide.This study was partially supported by two grants from the Instituto de Salud Carlos III [grant numbers PI18CIII/00017 and PI20CIII/00005].S

Diverse Large HIV-1 Non-subtype B Clusters Are Spreading Among Men Who Have Sex With Men in Spain

In Western Europe, the HIV-1 epidemic among men who have sex with men (MSM) is dominated by subtype B. However, recently, other genetic forms have been reported to circulate in this population, as evidenced by their grouping in clusters predominantly comprising European individuals. Here we describe four large HIV-1 non-subtype B clusters spreading among MSM in Spain. Samples were collected in 9 regions. A pol fragment was amplified from plasma RNA or blood-extracted DNA. Phylogenetic analyses were performed via maximum likelihood, including database sequences of the same genetic forms as the identified clusters. Times and locations of the most recent common ancestors (MRCA) of clusters were estimated with a Bayesian method. Five large non-subtype B clusters associated with MSM were identified. The largest one, of F1 subtype, was reported previously. The other four were of CRF02_AG (CRF02_1; n = 115) and subtypes A1 (A1_1; n = 66), F1 (F1_3; n = 36), and C (C_7; n = 17). Most individuals belonging to them had been diagnosed of HIV-1 infection in the last 10 years. Each cluster comprised viruses from 3 to 8 Spanish regions and also comprised or was related to viruses from other countries: CRF02_1 comprised a Japanese subcluster and viruses from 8 other countries from Western Europe, Asia, and South America; A1_1 comprised viruses from Portugal, United Kingom, and United States, and was related to the A1 strain circulating in Greece, Albania and Cyprus; F1_3 was related to viruses from Romania; and C_7 comprised viruses from Portugal and was related to a virus from Mozambique. A subcluster within CRF02_1 was associated with heterosexual transmission. Near full-length genomes of each cluster were of uniform genetic form. Times of MRCAs of CRF02_1, A1_1, F1_3, and C_7 were estimated around 1986, 1989, 2013, and 1983, respectively. MRCA locations for CRF02_1 and A1_1 were uncertain (however initial expansions in Spain in Madrid and Vigo, respectively, were estimated) and were most probable in Bilbao, Spain, for F1_3 and Portugal for C_7. These results show that the HIV-1 epidemic among MSM in Spain is becoming increasingly diverse through the expansion of diverse non-subtype B clusters, comprising or related to viruses circulating in other countries.This work was funded through Acción Estratégica en Salud Intramural (AESI), Instituto de Salud Carlos III, project “Estudios sobre vigilancia epidemiológica molecular del VIH-1 en España,” PI16CIII/00033; Red de Investigación en SIDA (RIS), Instituto de Salud Carlos III, Subdirección General de Evaluación y Fondo Europeo de Desarrollo Regional (FEDER), Plan Nacional I+D+I, project RD16ISCIII/0002/0004; scientific agreements with Consellería de Sanidade, Government of Galicia (MVI 1004/16) and Osakidetza-Servicio Vasco de Salud, Government of Basque Country (MVI 1001/16); European Research Infrastructures for Poverty Related Diseases (EURIPRED). Seventh Framework Program: FP7-Capacities-infrastructures-2012-1, grant agreement 312661; and Dirección General de Farmacia, Ministerio de Sanidad, Servicios Sociales e Igualdad, Government of Spain (grant EC11-272).S

Longer intervals between SARS-CoV-2 infection and mRNA-1273 doses improve the neutralization of different variants of concern

The humoral immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern elicited by vaccination was evaluated in COVID-19 recovered individuals (Rec) separated 1-3 months (Rec2m) or 4-12 months (Rec9m) postinfection and compared to the response in naïve participants. Antibody-mediated immune responses were assessed in 66 participants by three commercial immunoassays and a SARS-CoV-2 lentiviral-based pseudovirus neutralization assay. Immunoglobulin (Ig) levels against SARS-CoV-2 spike were lower in naïve participants after two doses than in Rec after a single dose (p < 0.05). After two doses in Rec, levels of total Ig to receptor-binding domain were significantly increased in Rec9m compared to Rec2m (p < 0.001). The neutralizing potency observed in Rec9m was consistently higher than in Rec2m against variants of concern (VOCs) Alpha, Beta, Delta, and BA.1 sublineage of Omicron with 2.2-2.8-fold increases. Increasing the interval between SARS-CoV-2 infection and the vaccination with messenger RNA-based vaccines to more than 3 months generates a more efficient heterologous humoral immune response against VOCs by allowing enough time to mount a strong recall memory B cell response.This work is funded by Instituto de Salud Carlos III, a Spanish public body assigned to the Ministry of Science and Innovation that manages and promotes public clinical research related to Public Health, by Grants PI19CIII/00004 (José Alcamí and Francisco Díez‐Fuertes) and PI21CIII/00025 (Javier García‐Pérez and Mayte Pérez‐Olmeda), COVID‐19 Fund (Grants COV20/00679 (Javier García‐Pérez, Mayte Pérez‐Olmeda, José Alcamí, and Francisco Díez‐Fuertes) and COV20/00072 (Javier García‐Pérez, Mayte Pérez‐Olmeda, Almudena Ramírez‐García, María Castillo de la Osa, Rocio Layunta Acero, Laura Vicente‐Izquierdo, Cristina Avendaño‐Solá, and José Alcamí), and CIBERINFEC, co‐financed by the European Regional Development Fund (FEDER) “A way to make Europe.”S

Immune response and reactogenicity after immunization with two-doses of an experimental COVID-19 vaccine (CVnCOV) followed by a third-fourth shot with a standard mRNA vaccine (BNT162b2): RescueVacs multicenter cohort study

Background: There is no evidence to date on immunogenic response among individuals who participated in clinical trials of COVID-19 experimental vaccines redirected to standard national vaccination regimens. Methods: This multicentre, prospective controlled cohort study included subjects who received a COVID-19 experimental vaccine (CVnCoV)(test group, TG) - and unvaccinated subjects (control group, CG), selected among individuals to be vaccinated according to the Spanish vaccination program. All study subjects received BNT162b2 as a standard national vaccination schedule, except 8 (from CG) who received mRNA-1273 and were excluded from immunogenicity analyses. Anti-RBD antibodies level and neutralising titres (NT50) against G614, Beta, Mu, Delta and Omicron variants were analysed. Reactogenicity was also assessed. Findings: 130 participants (TG:92; CG:38) completed standard vaccination. In TG, median (IQR) of anti-RBD antibodies after first BNT162b2 dose were 10740·0 BAU/mL (4466·0-12500) compared to 29·8 BAU/mL (14·5-47·8) in CG (p <0·0001). Median NT50 (IQR) of G614 was 2674·0 (1865·0-3997·0) in TG and 63·0 (16·0-123·1) in CG (p <0·0001). After second BNT162b2 dose, anti-RBD levels increased to ≥12500 BAU/mL (11625·0-12500) in TG compared to 1859·0 BAU/mL (915·4-3820·0) in CG (p <0·0001). NT50 was 2626·5 (1756·0-5472·0) and 850·4 (525·1-1608·0), respectively (p <0·0001). Variant-specific (Beta, Mu, Omicron) response was also assessed. Most frequent adverse reactions were headache, myalgia, and local pain. No severe AEs were reported. Interpretation: Heterologous BNT162b2 as third and fourth doses in previously suboptimal immunized individuals elicit stronger immune response than that obtained with two doses of BNT162b2. This apparent benefit was also observed in variant-specific response. No safety concerns arose.This work is partially funded by Institute of Health Carlos III (Instituto de Salud Carlos III − ISCIII −), (grants PI19CIII/00004 −JA- and PI21CIII/00025 −MPO, JG-), and COVID-19 FUND (grants COV20/00679 −MPO- and COV20/00072 −JA-) and CIBERINFEC, co-financed by the European Regional Development Fund (FEDER) “A way to make Europe”. Instituto de Salud Carlos III is a Spanish public body assigned to the Ministry of Science and Innovation that manages and promotes public clinical research related to public health. The authors thank Esther Prieto, MD (cited with consent) for editorial assistance and writing support (funded by the Research Foundation of HCSC).S

Genetic Study of SARS-CoV-2 Non Structural Protein 12 in COVID-19 Patients Non Responders to Remdesivir

Remdesivir (RDV) was the first antiviral drug approved by the FDA to treat severe coronavirus disease-2019 (COVID-19) patients. RDV inhibits SARS-CoV-2 replication by stalling the non structural protein 12 (nsp12) subunit of the RNA-dependent RNA polymerase (RdRp). No evidence of global widespread RDV-resistance mutations has been reported, however, defining genetic pathways to RDV resistance and determining emergent mutations prior and subsequent antiviral therapy in clinical settings is necessary. This study identified 57/149 (38.3%) patients who did not respond to one course (5-days) (n = 36/111, 32.4%) or prolonged (5 to 20 days) (n = 21/38, 55.3%) RDV therapy by subgenomic RNA detection. Genetic variants in the nsp12 gene were detected in 29/49 (59.2%) non responder patients by Illumina sequencing, including the de novo E83D mutation that emerged in an immunosuppressed patient after receiving 10 + 8 days of RDV, and the L838I detected at baseline and/or after prolonged RDV treatment in 9/49 (18.4%) non responder subjects. Although 3D protein modeling predicted no interference with RDV, the amino acid substitutions detected in the nsp12 involved changes on the electrostatic outer surface and in secondary structures that may alter antiviral response. It is important for health surveillance to study potential mutations associated with drug resistance as well as the benefit of RDV retreatment, especially in immunosuppressed patients and in those with persistent replication. IMPORTANCE This study provides clinical and microbiologic data of an extended population of hospitalized patients for COVID-19 pneumonia who experienced treatment failure, detected by the presence of subgenomic RNA (sgRNA). The genetic variants found in the nsp12 pharmacological target of RDV bring into focus the importance of monitoring emergent mutations, one of the objectives of the World Health Organization (WHO) for health surveillance. These mutations become even more crucial as RDV keeps being prescribed and new molecules are being repurposed for the treatment of COVID-19. The present article offers new perspectives for the clinical management of non responder patients treated and retreated with RDV and emphasizes the need of further research of the benefit of combinatorial therapies and RDV retreatment, especially in immunosuppressed patients with persistent replication after therapy.This work was financed by a Gilead Sciences grant (IN-ES-540-6089) and CIBER Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, España (CB21/13/00081). This work was financed by ad hoc patronage funds for research on COVID-19 from donations from citizens and organizations to the Hospital Clínic de Barcelona-Fundació Clínic per a la Recerca Biomèdica.S

Non-motor symptom burden in patients with Parkinson's disease with impulse control disorders and compulsive behaviours : results from the COPPADIS cohort

The study was aimed at analysing the frequency of impulse control disorders (ICDs) and compulsive behaviours (CBs) in patients with Parkinson's disease (PD) and in control subjects (CS) as well as the relationship between ICDs/CBs and motor, nonmotor features and dopaminergic treatment in PD patients. Data came from COPPADIS-2015, an observational, descriptive, nationwide (Spain) study. We used the validated Questionnaire for Impulsive-Compulsive Disorders in Parkinson's Disease-Rating Scale (QUIP-RS) for ICD/CB screening. The association between demographic data and ICDs/CBs was analyzed in both groups. In PD, this relationship was evaluated using clinical features and treatment-related data. As result, 613 PD patients (mean age 62.47 ± 9.09 years, 59.87% men) and 179 CS (mean age 60.84 ± 8.33 years, 47.48% men) were included. ICDs and CBs were more frequent in PD (ICDs 12.7% vs. 1.6%, p < 0.001; CBs 7.18% vs. 1.67%, p = 0.01). PD patients had more frequent previous ICDs history, premorbid impulsive personality and antidepressant treatment (p < 0.05) compared with CS. In PD, patients with ICDs/CBs presented younger age at disease onset, more frequent history of previous ICDs and premorbid personality (p < 0.05), as well as higher comorbidity with nonmotor symptoms, including depression and poor quality of life. Treatment with dopamine agonists increased the risk of ICDs/CBs, being dose dependent (p < 0.05). As conclusions, ICDs and CBs were more frequent in patients with PD than in CS. More nonmotor symptoms were present in patients with PD who had ICDs/CBs compared with those without. Dopamine agonists have a prominent effect on ICDs/CBs, which could be influenced by dose

Diverse Large HIV-1 Non-subtype B Clusters Are Spreading Among Men Who Have Sex With Men in Spain

In Western Europe, the HIV-1 epidemic among men who have sex with men (MSM) is dominated by subtype B. However, recently, other genetic forms have been reported to circulate in this population, as evidenced by their grouping in clusters predominantly comprising European individuals. Here we describe four large HIV-1 non-subtype B clusters spreading among MSM in Spain. Samples were collected in 9 regions. A pol fragment was amplified from plasma RNA or blood-extracted DNA. Phylogenetic analyses were performed via maximum likelihood, including database sequences of the same genetic forms as the identified clusters. Times and locations of the most recent common ancestors (MRCA) of clusters were estimated with a Bayesian method. Five large non-subtype B clusters associated with MSM were identified. The largest one, of F1 subtype, was reported previously. The other four were of CRF02_AG (CRF02_1; n = 115) and subtypes A1 (A1_1; n = 66), F1 (F1_3; n = 36), and C (C_7; n = 17). Most individuals belonging to them had been diagnosed of HIV-1 infection in the last 10 years. Each cluster comprised viruses from 3 to 8 Spanish regions and also comprised or was related to viruses from other countries: CRF02_1 comprised a Japanese subcluster and viruses from 8 other countries from Western Europe, Asia, and South America; A1_1 comprised viruses from Portugal, United Kingom, and United States, and was related to the A1 strain circulating in Greece, Albania and Cyprus; F1_3 was related to viruses from Romania; and C_7 comprised viruses from Portugal and was related to a virus from Mozambique. A subcluster within CRF02_1 was associated with heterosexual transmission. Near full-length genomes of each cluster were of uniform genetic form. Times of MRCAs of CRF02_1, A1_1, F1_3, and C_7 were estimated around 1986, 1989, 2013, and 1983, respectively. MRCA locations for CRF02_1 and A1_1 were uncertain (however initial expansions in Spain in Madrid and Vigo, respectively, were estimated) and were most probable in Bilbao, Spain, for F1_3 and Portugal for C_7. These results show that the HIV-1 epidemic among MSM in Spain is becoming increasingly diverse through the expansion of diverse non-subtype B clusters, comprising or related to viruses circulating in other countries

Differentiation of type-I porcine reproductive and respiratory syndrome virus vaccines and field strains by restriction fragment length polymorphism analysis

The use of modified live virus (MLV) vaccines is a common procedure to control porcine reproductive and respiratory syndrome virus (PRRSV) infection in the great majority of countries from America, Asia and Europe, including Spain. Current discriminatory techniques allow the detection of different MLV type-II vaccine strains. Herein we report a rapid and accurate technique aimed to discriminate between MLV type-I vaccine strains and Spanish field strains. This technique comprises a reverse transcription (RT) and nested polymerase chain reaction (nPCR) amplification of PRRSV ORF5 followed by a digestion of RT-nPCR products with two specific endonucleases, ItaI and AccI. Combined utilization of ItaI and AccI generates restriction fragments length polymorphisms (RFLP) patterns adequate for the differentiation of 30 Spanish field isolates, of which 12 were isolated between 1991 and 1995 and 18 between 2000 and 2003. These different RFLP patterns can be used to distinguish unequivocally between Spanish field strains of PRRSV and the three MLV type-I vaccines used in Spain: AmervacPRRS®, Pyrsvac-183® and PorcilisPRRS®

Genetic and phenotypic analyses of sequential vpu alleles from HIV-infected IFN-treated patients

International audienceTreatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties