Emergence of Toscana Virus in Europe

In southern Europe, Toscana virus is one of the three leading causes of aseptic meningitis

Yaoundé-like virus in resident wild bird, Ghana

Tissue and swab samples from 551 wild birds collected in Ghana (October-November 2007) were assayed for alphaviruses, flaviviruses, and influenza A viruses using polymerase chain (PCR) techniques. One pool sample tested positive for Flavivirus RNA; further testing revealed that the amplified sequence was Yaoundé virus (YAOV), or closely related to it. YAOV is an apparently rare Flavivirus closely related to medically important human pathogens Japanese Encephalitis virus and West Nile virus. It is known only from West Africa. This is the first detection from Ghana, and only the second detection from a bird. Samples were negative for alphaviruses and Influenza A virus.We thank field companions including E. Bonaccorso, M. Robbins, and M. Thompson; laboratory companions, including A. Negredo and N. Reyes; Dr. F. Pozo and I. Casas from ISCIII for their collaboration; and L. Benitez for introduction to ISCIII. This study was supported by the US National Biological Information Infrastructure, the GAINS program of the Wildlife Conservation Society, and the Centers for Disease Control and Prevention, grants FIS PI07/1308 of the Red de Investigacion de Centros de Enfermedaes Tropicales RD06/0021, and the agreement signed between the Institute of Health Carlos III and the Spanish Ministry of Health and Social Policy for the surveillance of imported viral hemorrhagic fevers. Influenza work was supported by grant GR09/0040 (MPY-1440/09) ISCIII. RW is supported by grant CGL2010-15734/BOS, Ministerio de Ciencia e Innovación, Spain

Putative New Lineage of West Nile Virus, Spain

To ascertain the presence of West Nile virus (WNV), we sampled mosquitoes in 2006 in locations in southern Spain where humans had been infected. WNV genomic RNA was detected in 1 pool from unfed female Culex pipiens mosquitoes. Phylogenetic analysis demonstrated that this sequence cannot be assigned to previously described lineages of WNV

Comparative Evaluation of Indirect Immunofluorescence and NS-1-Based ELISA to Determine Zika Virus-Specific IgM

Differential diagnosis of the Zika virus (ZIKV) is hampered by cross-reactivity with other flaviviruses, mainly dengue viruses. The aim of this study was to compare two commercial methods for detecting ZIKV immunoglobulin M (IgM), an indirect immunofluorescence (IIF) and an enzyme immunoassay (ELISA), using the non-structural (NS) 1 protein as an antigen, both from EuroImmun, Germany. In total, 255 serum samples were analyzed, 203 of which showed laboratory markers of ZIKV infections (PCR-positive in serum and/or in urine and/or positive or indeterminate specific IgM). When tested with IIF, 163 samples were IgM-positive, while 13 samples were indeterminate and 78 were negative. When IIF-positive samples were tested using ELISA, we found 61 positive results, 14 indeterminate results, and 88 negative results. Among the indeterminate cases tested with IIF, ELISA analysis found two positive, two indeterminate, and nine negative results. Finally, 74 of the 78 IIF-negative samples proved also to be negative using ELISA. For the calculations, all indeterminate results were considered to be positive. The agreement, sensitivity, and specificity between ELISA and IIF were 60.2%, 44.9%, and 94.9%, respectively. Overall, 101 samples showed discrepant results; these samples were finally classified on the basis of other ZIKV diagnostic approaches (PCR-positive in serum and/or in urine, IgG determinations using IIF or ELISA, and ZIKV Plaque Reduction Neutralization test-positive), when available. A final classification of 228 samples was possible; 126 of them were positive and 102 were negative. The corresponding values of agreement, sensitivity, and specificity of IIF were 86.0%, 96.8%, and 72.5%, respectively. The corresponding figures for ELISA were 81.1%, 65.9%, and 100%, respectively. The ELISA and IIF methods are both adequate approaches for detecting ZIKV-specific IgM. However, considering their respective weaknesses (low sensitivity in ELISA and low specificity in IIF), serological results must be considered jointly with other laboratory results.The study was partially financed by a contract between the ISCIII and Euroimmun Diagnostics España SLU (MVP216/17), and by ISCIII, project RD16CIII/0003/0003, “Red de Enfermedades Tropicales”, Subprogram RETICS Plan Estatal de I+D+I 2013-2016, and co-funded by FEDER “Una manera de hacer Europa” and project PI16CIII/00037.S

Mutations in Coding and Non-Coding Regions in Varicella-Zoster Virus Causing Fatal Hemorrhagic Fever Without Rash in an Immunocompetent Patient: Case Report

Introduction: We report the case of a fatal hemorrhagic varicella primary infection in an immunocompetent man and whole-genome characterization of the virus for the investigation of biomarkers of virulence. Case: A 38-year-old patient born in Nigeria presented to the emergency department with abdominal pain and subsequently developed fatal hemorrhagic disease without skin rash. Extensive laboratory tests including serology and PCR for arenaviruses, bunyaviruses and ebolaviruses were negative. Varicella-zoster virus (VZV) PCR of sera, liver and spleen tissue samples from autopsy revealed the presence of VZV DNA. Primary infection by varicella-zoster virus with hemorrhagic manifestations was diagnosed after virological testing. The VZV genome was sequenced using a mWGS approach. Bioinformatic analysis showed 53 mutations across the genome, 33 of them producing non-synonymous variants affecting up to 14 genes. Some of them, such as ORF11 and ORF 62, encoded for essential functions related to skin or neurotropism. To our knowledge, the mutations reported here have never been described in a VZV causing such a devastating outcome. Discussion: In immunocompetent patients, viral factors should be considered in patients with uncommon symptoms or severe diseases. Some relevant mutations revealed by using whole genome sequencing (WGS) directly from clinical samples may be involved in this case and deserves further investigation. Conclusion: Differential diagnosis of varicella-zoster virus in immunocompetent adults should be considered among patients with suspected VHF, even if the expected vesicular rash is not present at admission and does not arise thereafter. Whole genome sequencing of strains causing uncommon symptoms and/or mortality is needed for epidemiological surveillance and further characterization of putative markers of virulence. Additionally, this report highlights the recommendation for a VZV vaccination policy in non-immunized migrants from developing countries.This work was supported by a grant from Instituto de Salud Carlos III. Project code MPY1372/12. The journal’s Rapid Service fee was paid by Consorcio Centro de Investigación en Red (CIBER). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Untargeted metagenomic sequencing identifies Toscana virus in patients with idiopathic meningitis, southern Spain, 2015 to 2019

Background: Various pathogens, including bacteria, fungi, parasites, and viruses can lead to meningitis. Among viruses causing meningitis, Toscana virus (TOSV), a phlebovirus, is transmitted through sandfly bites. TOSV infection may be suspected if patients with enterovirus- and herpesvirus-negative aseptic (non-bacterial) meningitis recall recent insect bites. Other epidemiological factors (season, rural area) may be considered. The broad range of possible meningitis aetiologies poses considerable diagnosis challenges. Untargeted metagenomic next-generation sequencing (mNGS) can potentially identify pathogens, which are not considered or detected in routine diagnostic panels. Aim: In this retrospective, single-centre observational study, we investigated mNGS usefulness to understand the cause of meningitis when conventional approaches fail. Methods: Cerebrospinal fluid (CSF) samples from patients hospitalised in southern Spain in 2015-2019 with aseptic meningitis and no aetiology found by conventional testing, were subjected to mNGS. Patients' demographic characteristics had been recorded and physicians had asked them about recent insect bites. Obtained viral genome sequences were phylogenetically analysed. Results: Among 23 idiopathic cases, TOSV was identified in eight (all male; median age:  39 years, range: 15-78 years). Five cases lived in an urban setting, three occurred in autumn and only one recalled insect bites. Phylogenetic analysis of TOSV segment sequences supported one intra-genotype reassortment event. Conclusions: Our study highlights the usefulness of mNGS for identifying viral pathogens directly in CSF. In southern Spain, TOSV should be considered regardless of recalling of insect bites or other epidemiological criteria. Detection of a disease-associated reassortant TOSV emphasises the importance of monitoring the spread and evolution of phleboviruses in Mediterranean countries.ESL laboratory received funding from Institut Pasteur, from the INCEPTION programme (Investissements d’Avenir grant ANR-16-CONV-0005), from the NIH PICREID program (Award Number U01AI151758) and from the Labex IBEID (ANR-10-LABX-62-IBEID). MDFG acknowledges funding from the Junta de Andalucia (PI-0216-2019) and Instituto de Salud Carlos III (Acción Estratégica en Salud Intramural PI20CIII/00005).S

Genetic Diversity of Toscana Virus

Distribution of Toscana virus (TOSV) is evolving with climate change, and pathogenicity may be higher in nonexposed populations outside areas of current prevalence (Mediterranean Basin). To characterize genetic diversity of TOSV, we determined the coding sequences of isolates from Spain and France. TOSV is more diverse than other well-studied phleboviruses (e.g.,Rift Valley fever virus)

Toscana Virus in Spain

Toscana virus (TOSV, Phlebovirus, family Bunyaviridae) infection is one of the most prevalent arboviruses in Spain. Within the objectives of a multidisciplinary network, a study on the epidemiology of TOSV was conducted in Granada, in southern Spain. The overall seroprevalence rate was 24.9%, significantly increasing with age. TOSV was detected in 3 of 103 sandfly pools by viral culture or reverse transcription–polymerase chain reaction from a region of the L gene. Nucleotide sequence homology was 99%–100% in TOSV from vectors and patients and 80%–81% compared to the Italian strain ISS Phl.3. Sequencing of the N gene of TOSV isolates from patients and vectors indicated 87%–88% and 100% homology at the nucleotide and amino acid levels, respectively, compared to the Italian strain. These findings demonstrate the circulation of at least 2 different lineages of TOSV in the Mediterranean basin, the Italian lineage and the Spanish lineage.Grant sponsor was Red EVITAR, Fondo de Investigaciones Sanitarias, Spanish Ministry of Health, grant no. G03/059. Ximena Collao has a research grant from Valparaiso University (MECESUP project, Chile). The study of vectors, i.e., capture of phlebotomines and taxonomic classification, was supported by the Junta de Andalucía, research grant CVI 176. Dr Sanbonmatsu-Gámez is a microbiologist on a fellowship from the EVITAR network (Fondo de Investigaciones Sanitarias, Spanish Ministry of Health; grant no. G03/059). Her research interest focuses on viral infectious diseases, especially arthropodborne viral diseases