Durawa, M. V. | Search bioRxiv

bioRxiv2026

The La Crosse virus M segment determines virus isolate cell-to-cell spread and virulence

La Crosse virus (LACV) is an orthobunyavirus spread by mosquitoes in North America and can cause severe neurological disease. Despite this burden, there is a lack of LACV antiviral treatments as our fundamental understanding of how LACV spreads and causes disease remains incomplete. To investigate LACV biology, we took advantage of two genetically similar LACV lineage I isolates (LACV1960 and LACV1978) where we found that LACV1960 infects and replicates at a higher rate than LACV1978, while LACV1978 exhibits enhanced cell-cell spread and in vivo virulence and dissemination. To investigate the genomic determinants behind these phenotypes, we generated reassortants between each isolate. We found that each genomic segment contributed to LACV pathogenesis with the M segment as a dominant determinant for LACV pathogenesis in vivo and plaque size and replication in vitro. To address which M segment protein contributes to plaque size and infectivity, we generated M segment chimeric viruses using a LACV1978 background and swapping in regions of the LACV1960 M segment. We found that the Gc head domain determined plaque size and infectivity, with the LACV1960 Gc head chimera producing small plaques but having increased infectivity over the wild-type LACV1978. Finally, using natural LACV lineage isolates, we showed that plaque size is variable across and within lineages suggesting changes in the M segment may impact LACV in nature. In future studies, we will continue to investigate the mechanisms behind how cell-to-cell spread influences dissemination to better understand how LACV genome segments affect spread, infectivity, virulence, and viral fitness.

microbiology↗

bioRxiv2025

The orthobunyavirus Gc glycoprotein head and stalk drives an infectious virion assembly pathway that is specific for the insect host

The Orthobunyavirus genus of arthropod-borne segmented RNA viruses comprises several important pathogens including the human-infecting Oropouche virus and animal-infecting Schmallenberg virus (SBV). The prototypical Bunyamwera orthobunyavirus (BUNV) possesses envelope-embedded glycoprotein spikes, with Gc head and stalk domains forming distinctive tripods covering the envelope-proximal Gn. Spike ectodomains mediate virus entry, while endodomains interact with nucleoprotein (NP) enwrapped genome segments to mediate virion assembly. Interestingly, BUNV Gc head/stalk domains are redundant for virus growth in mammalian cells, consistent with isolations of SBV from animals bearing head/stalk deletions. However, orthobunyavirus isolations from arthropods in nature appear to maintain these domains strictly. To investigate this discrepancy, we compared the multiplication characteristics of wildtype BUNV (BUNV-WT) with a Gc head/stalk deleted BUNV (BUNV-{Delta}7). In mammalian cells BUNV-WT and BUNV-{Delta}7 grew to equivalent titres, whereas BUNV-{Delta}7 titres from insect cells were 1000-fold lower than BUNV-WT and strikingly produced no virions following blood meal infection of Aedes mosquitoes. To understand this insect-specific restriction in virion production, we showed the intracellular abundance of BUNV-WT and BUNV-{Delta}7 Gc and NP components were equivalent, suggesting {Delta}7-Gc was assembly-deficient. To explore this, we investigated Gc and {Delta}7-Gc interactions during BUNV-WT and BUNV-{Delta}7 infections of both insect and mammalian cells by co-immunoprecipitation and multiplex mass spectrometry, revealing {Delta}7-Gc exhibited markedly reduced NP interactions in insect cells. We propose Gc recruits genome segments during virion assembly and that the complete Gc head/stalk assembly is maintained in nature due to its essential role in the insect host. IMPORTANCEOrthobunyaviruses are arthropod-borne viruses that cause severe disease in humans and animals, including congenital malformations and abortions. Orthobunyavirus Gn and Gc glycoproteins form spikes, which mediate entry and genome recruitment during assembly. In nature, OBVs bearing large deletions within Gc head/stalk domains have been isolated from animals, yet the head/stalk domains appear to be strictly maintained within insects. To investigate this discrepancy, we compared the multiplication of Bunyamwera orthobunyavirus (BUNV-WT) with head/stalk-deleted variant (BUNV-{Delta}7). In mammalian cells both viruses reached similar titres, but strikingly BUNV-{Delta}7 failed to produce virions in both insect cells and Aedes mosquitoes. We showed this was because BUNV-{Delta}7 failed to assemble new virions, revealing its head/stalk-deleted Gc was deficient in interactions with genome components. We propose that Gc drives species-specific interactions with genome segments during virion assembly, explaining why Gc head/stalk domains are conserved in nature due to their essential role in the insect host.

microbiology↗