Ecological and clinical evidence of the establishment of West Nile virus in a large urban area in Europe, Berlin, Germany, 2021 to 2022.

BackgroundWest Nile virus (WNV), found in Berlin in birds since 2018 and humans since 2019, is a mosquito-borne virus that can manifest in humans as West Nile fever (WNF) or neuroinvasive disease (WNND). However, human WNV infections and associated disease are likely underdiagnosed.AimWe aimed to identify and genetically characterise WNV infections in humans and mosquitoes in Berlin.MethodsWe investigated acute WNV infection cases reported to the State Office for Health and Social Affairs Berlin in 2021 and analysed cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown aetiology (n = 489) for the presence of WNV. Mosquitoes were trapped at identified potential exposure sites of cases and examined for WNV infection.ResultsWest Nile virus was isolated and sequenced from a blood donor with WNF, a symptomatic patient with WNND and a WNND case retrospectively identified from testing CSF. All cases occurred in 2021 and had no history of travel 14 days prior to symptom onset (incubation period of the disease). We detected WNV in Culex pipiens mosquitoes sampled at the exposure site of one case in 2021, and in 2022. Genome analyses revealed a monophyletic Berlin-specific virus clade in which two enzootic mosquito-associated variants can be delineated based on tree topology and presence of single nucleotide variants. Both variants have highly identical counterparts in human cases indicating local acquisition of infection.ConclusionOur study provides evidence that autochthonous WNV lineage 2 infections occurred in Berlin and the virus has established an endemic maintenance cycle.

Potential of avian and mammalian species A rotaviruses to reassort as explored by plasmid only-based reverse genetics.

Species A rotavirus (RVA) is an important gastrointestinal pathogen that is widely distributed in humans, mammalian animals and birds. The RVA genome consists of eleven double-stranded RNA segments, enabling the generation of novel strains with new pathogenic or antigenic features by genetic reassortment. While reassortants between human and mammalian animal RVAs have been repeatedly described, data on the reassortment potential of avian RVA strains are rare. To investigate genome segment exchanges between avian and mammalian RVA strains, a plasmid-based reverse genetics strategy originally developed for the simian RVA strain SA11 was used here. All eleven genome segments of the chicken RVA strain 02V0002G3 were cloned into similar plasmids as in the SA11 system. However, in contrast to SA11, no infectious virus could be generated by transfection of the eleven 02V0002G3 plasmids into cell culture under the same conditions. In another series of experiments, each of the genome segments of 02V0002G3 was transfected together with the remaining ten genome segments of SA11. Viable mono-reassortants were only retrieved for the avian VP3 and VP4 genes. The reassortant viruses were structurally indistinguishable from their parental viruses, but grew to slightly lower titers in cell culture. The results indicate that the VP3 and VP4 genes, but not the other genes of avian RVA, can functionally substitute their mammalian homologs and create viable reassortants. Further research should focus on the reasons behind the reassortment incompatibility and on the optimization of the system for the generation of viable avian RVA rescued entirely from cloned avian RVA genome segments.

Whole genome sequence analysis of cell culture-adapted rotavirus A strains from chicken.

Rotavirus A (RVA) is a major cause of gastroenteritis in humans and mammalian animals, and has also been abundantly detected in avian species. Avian RVA infection is associated with diarrhea, reduced growth and increased mortality, leading to economic losses in the poultry industry. Avian RVA forms a unique genetic clade within the whole RVA species. However, up to now, only a few full-length avian RVA genomes have been published and only a small number of avian RVA strains have been adapted to grow in cell culture for subsequent studies. Here, the four cell culture-adapted chicken RVA strains 02V0002G3, 04V0027G6, 05V0500F6 and 06V0661G1 were characterized in more detail. Transmission electron microscopy of the viruses derived from culture supernatant showed a typical triple-layered morphology of rotavirus particles; in addition, strain 06V0661G1 showed a high proportion of double-layered particles. The (nearly) complete genome sequences of the viruses were determined using next-generation sequencing (NGS). The resulting sequences were compared to full-length or partial sequences of the strains previously determined using Sanger sequencing; and a few nucleotide mismatches, some of them resulting in amino acid substitutions, were identified. The genomes of strains 02V0002G3, 04V0027G6 and 05V0500F6 were closely related to each other showing a G19-P[30]-I11-R6-C6-M7-A16-N6-T8-E10-H8 genotype constellation. Strain 06V0661G1 carries the VP4 genotype P[31] in the same genetic backbone like the other strains. However, further sequence analysis showed that the genes of this strain, especially that encoding NSP3, clustered more separately from the other strains in phylogenetic trees. The characterized cell culture-adapted chicken RVA strains may be useful for future studies investigating genetic diversity and replication of avian rotaviruses, as well as for the development of vaccines and diagnostic tools.

Generation of Simian Rotavirus Reassortants with VP4- and VP7-Encoding Genome Segments from Human Strains Circulating in Africa Using Reverse Genetics.

Human rotavirus A (RVA) causes acute gastroenteritis in infants and young children. The broad use of two vaccines, which are based on RVA strains from Europe and North America, significantly reduced rotavirus disease burden worldwide. However, a lower vaccine effectiveness is recorded in some regions of the world, such as sub-Saharan Africa, where diverse RVA strains are circulating. Here, a plasmid-based reverse genetics system was used to generate simian RVA reassortants with VP4 and VP7 proteins derived from African human RVA strains not previously adapted to cell culture. We were able to rescue 1/3 VP4 mono-reassortants, 3/3 VP7 mono-reassortants, but no VP4/VP7 double reassortant. Electron microscopy showed typical triple-layered virus particles for the rescued reassortants. All reassortants stably replicated in MA-104 cells; however, the VP4 reassortant showed significantly slower growth compared to the simian RVA or the VP7 reassortants. The results indicate that, at least in cell culture, human VP7 has a high reassortment potential, while reassortment of human VP4 from unadapted human RVA strains with simian RVA seems to be limited. The characterized reassortants may be useful for future studies investigating replication and reassortment requirements of rotaviruses as well as for the development of next generation rotavirus vaccines.

Distantly Related Rotaviruses in Common Shrews, Germany, 2004-2014.

We screened samples from common shrews (Sorex araneus) collected in Germany during 2004-2014 and identified 3 genetically divergent rotaviruses. Virus protein 6 sequence similarities to prototype rotaviruses were low (64.5% rotavirus A, 50.1% rotavirus C [tentative species K], 48.2% rotavirus H [tentative species L]). Shrew-associated rotaviruses might have zoonotic potential.

Generation of simian rotavirus reassortants with diverse VP4 genes using reverse genetics.

Species A rotaviruses (RVAs) are a major cause of gastroenteritis in animals and humans. Their genome consists of 11 segments of dsRNA, and reassortment events between animal and human strains can contribute to the high genetic diversity of RVAs. We used a plasmid-based reverse genetics system to investigate the reassortment potential of the genome segment encoding the viral outer capsid protein VP4, which is a major antigenic determinant, mediates viral entry and plays an important role in host cell tropism. We rescued reassortant viruses containing VP4 from porcine, bovine, bat, pheasant or chicken RVA strains in the backbone of simian strain SA11. The VP4 reassortants could be stably passaged in MA-104 cells and induced cytopathic effects. However, analysis of growth kinetics revealed marked differences in replication efficiency. Our results show that the VP4-encoding genome segment has a high reassortment potential, even between virus strains from highly divergent species. This can result in replication-competent reassortants with new genomic, growth and antigenic features.