Santos, P. D. | Search bioRxiv

bioRxiv2024

The absence of superinfection exclusion of Borna disease virus 2 maintains genomic polymorphisms in persistently infected cells

Viruses belonging to the genus Orthobornavirus within the family Bornaviridae are known to infect various vertebrate species, including mammals and birds. Within the genus, the species Orthobornavirus bornaense includes two mammalian viruses: Borna disease virus 1 (BoDV-1), the prototype of the family, and its closely related virus, BoDV-2. BoDV-1 was identified as the causative agent of Borna disease (BD) in horses, sheep, humans, and other mammals. BoDV-2 was first detected in a pony in eastern Austria in 1999 that exhibited severe and incurable neurological symptoms. Although BoDV-2 shares approximately 80% nucleotide identity with BoDV-1, its virological properties, including host range, replication ability, and pathogenicity, remain unclear. In this study, we aimed to investigate the virological properties of BoDV-2 by re-evaluating its whole-genome sequence using RNA sequencing. Compared to the published reference sequence, we identified two nonsynonymous nucleotide substitutions in the large (L) gene. One of these substitutions was found to be critical for the restoration of polymerase activity, enabling the successful recovery of recombinant BoDV-2 (rBoDV-2) through reverse genetics. We also identified two nonsynonymous single-nucleotide polymorphisms (SNPs) in the L gene and one in the phosphoprotein (P) gene. Substitution of these SNPs significantly enhanced the growth ability of rBoDV-2. In addition, our studies showed that BoDV-2 does not induce superinfection exclusion in cells, allowing persistence of low-fitness genome variants for an extended period of time. These findings help to characterize the virological properties of BoDV-2 and shed light on how bornaviruses maintain genetic diversity in infected cells. ImportanceMammalian bornaviruses are a general term for viruses belonging to the genus Orthobornavirus that infect mammalian species, e.g., Borna disease virus 1 and 2 (BoDV-1 and -2) and variegated squirrel bornavirus 1 (VSBV-1). Although BoDV-1 and VSBV-1 are reported to be associated with fatal encephalitis in humans, the infectivity and pathogenicity of BoDV-2 in humans remain unclear. To determine the virological properties of BoDV-2, we developed a reverse genetics system for BoDV-2. By using recombinant BoDV-2s, we identified several nucleotides that affect the growth ability of BoDV-2 and revealed the molecular mechanisms through which BoDV-2 maintains genetic heterogeneity in persistently infected cells. This reverse genetics system will accelerate the biological studies of BoDV-2 and contribute to the development of countermeasures against mammalian bornaviruses.

microbiology↗

bioRxiv2022

An advanced sequence clustering and designation workflow reveals the enzootic maintenance of a dominant West Nile virus subclade in Germany

West Nile virus (WNV) is the most widespread arthropod-borne (arbo) virus and the primary cause of arboviral encephalitis globally. Members of WNV species genetically diverged and are classified into different hierarchical groups below species rank. However, the demarcation criteria for allocating WNV sequences into these groups remain individual, inconsistent, and the use of names for different levels of the hierarchical levels is unstructured. In order to have an objective and comprehensible grouping of WNV sequences, we developed an advanced grouping workflow using the "affinity propagation clustering"-algorithm and newly included the "agglomerative hierarchical clustering"-algorithm for the allocation of WNV sequences into different groups below species rank. In addition, we propose to use a fixed set of terms for the hierarchical naming of WNV below species level and a clear decimal numbering system to label the determined groups. For validation, we applied the refined workflow to WNV sequences that have been previously grouped into various lineages, clades, and clusters in other studies. Although our workflow regrouped some WNV sequences, overall, it generally corresponds with previous groupings. We employed our novel approach to the sequences from the WNV circulation in Germany 2020, primarily from WNV-infected birds and horses. Besides two newly defined minor (sub)clusters comprising only of three sequences each, subcluster 2.5.3.4.3c was the predominant WNV sequence group detected in Germany from 2018-20. This predominant subcluster was also associated with at least five human WNV-infections in 2019-20. In summary, our analyses imply that the genetic diversity of the WNV population in Germany is shaped by enzootic maintenance of the dominant WNV subcluster accompanied by sporadic incursions of other rare clusters and subclusters. Moreover, we show that our refined approach for sequence grouping yields meaningful results. Although we primarily aimed at a more detailed WNV classification, the presented workflow can also be applied to the objective genotyping of other virus species.

genetics↗