Biology subjects

Thannickal, S. A.

Publications and source records attributed to Thannickal, S. A..

6 recordsLinked to original sources

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↗

bioRxiv2024

La Crosse virus reassortants highlight genomic determinants of infection and pathogenesis

The genomic determinants that contribute to orthobunyavirus infection and pathogenesis are not well-defined. In this study, we harnessed the process of reassortment to understand which viral factors drive change in the replication and pathogenesis of La Crosse virus (LACV). We systematically reassorted the genomic segments of two genetically similar Lineage I LACV isolates into six unique reassortants. Despite the parental isolates having high levels of RNA and protein consensus, the reassortants demonstrate how minimal changes in RNA and protein structure can have significant changes in viral growth and reproduction in vitro in mammalian and insect models. We observed that swapping the S segment between isolates led to differences in replication and assembly resulting in one non-rescuable reassortant and one viable reassortant that exhibited an increase in viral growth dynamics. Switching the M segment led to changes in viral plaque phenotype and growth kinetics. L segment reassortants similarly differed in changes in viral growth dynamics. We further explored the M segment reassortants in a neonate mouse model and observed a role for the M segment in neuroinflammation and virulence. Through reassortment of the La Crosse virus genomic segments, we are able to further understand how genomic determinants of infection and pathogenesis operate in orthobunyaviruses. Future investigations will focus on identifying the specific molecular elements that govern the observed phenotypes in vitro and in vivo. ImportanceLa Crosse virus is the leading cause of pediatric arboviral encephalitis in the United States, yet it is largely unknown how each of the three genomic segments contribute to pathogenesis and disease. Our study utilizes genomic reassortment between two similar Lineage I LACV isolates to understand genomic determinants for differences in infection and pathogenesis phenotypes in vitro and in vivo. By identifying roles for each segment in observed outcomes, we are able to plan further studies for molecular characterization of these phenotypes. Additionally, it is imperative to continue to characterize orthobunyavirus function since climate change will expand the range and prevalence of arthropod-borne diseases such as LACV in the United States.

microbiology↗

bioRxiv2023

The chikungunya virus E1 glycoprotein fusion loop and hinge alter glycoprotein dynamics leading to cell and host specific changes in infectivity.

Alphaviruses infect both mammals and insects, yet the distinct mechanisms that alphaviruses use to infect different hosts are not well defined. In this study, we characterize CHIKV E1 variants in the fusion loop (E1-M88L) and hinge region (E1-N20Y) in vitro and in vivo to understand how these regions of the E1 glycoprotein contribute to host-specific infection. Through cell culture assays, we found that CHIKV E1-N20Y enhanced infectivity in mosquito cells while the CHIKV E1-M88L variant enhanced virus binding and infectivity in both BHK-21 and C6/36 cells, and led to changes in the virus cholesterol-dependence in BHK-21 cells. Given these in vitro results and that residue E1-M88L is in a defined Mxra8 interacting domain, we hypothesized that this residue may be important for receptor usage. However, while the CHIKV E1-M88L variant increased replication in Mxra8-deficient mice compared to WT CHIKV, it was attenuated in vitro in mouse fibroblasts, suggesting that residue E1-M88 may function in a cell-type dependent manner to alter entry. Finally, using molecular dynamics to understand how potential changes in the E1 glycoprotein may impact the CHIKV glycoprotein E1-E2 complex, we found that E1-M88L and other E1 domain II variants lead to changes in both E1 and E2 dynamics. Taken together, these studies show that key residues in the CHIKV E1 fusion loop and hinge region function through changes in E1-E2 dynamics to facilitate cell- and host-dependent entry. ImportanceArthropod-borne viruses (arboviruses) are significant global public health threats, and their continued emergence around the world highlights the need to understand how these viruses replicate at the molecular level. The alphavirus class II glycoproteins are critical for virus entry in mosquitoes and mammals, yet how these proteins function is not completely understood. Therefore, to address these gaps in our knowledge, it is critical to dissect how distinct glycoprotein domains function in vitro and in vivo. Here, we show that changes in the CHIKV E1 fusion loop and hinge contribute to host-specific entry and E1-E2 dynamics, furthering our knowledge of how alphaviruses infect mammals and insects.

microbiology↗

bioRxiv2023

The La Crosse virus class II fusion glycoprotein ij loop contributes to infectivity and cholesterol-dependent entry.

Arthropod-borne viruses (arboviruses) are an emerging and evolving global public health threat with little to no antiviral treatments. La Crosse virus (LACV) from the Bunyavirales order is responsible for pediatric encephalitis cases in the United States, yet little is known about the infectivity of LACV. Given the structural similarities between class II fusion glycoproteins of LACV and chikungunya virus (CHIKV), an alphavirus from the Togaviridae family, we hypothesized that LACV would share similar entry mechanisms to CHIKV. To test this hypothesis, we performed cholesterol-depletion and repletion assays and used cholesterol modulating compounds to study LACV entry and replication. We found that LACV entry was cholesterol-dependent while replication was less affected by cholesterol manipulation. In addition, we generated single point mutants in the LACV ij loop that corresponded to known CHIKV residues important for virus entry. We found that a conserved histidine and alanine residue in the Gc ij loop impaired virus infectivity and attenuate LACV in vitro and in vivo. Finally, we took an evolution-based approach to explore how the LACV glycoprotein evolution in mosquitoes and mice. We found multiple variants that cluster in the Gc glycoprotein head domain, supporting the Gc glycoprotein as a target for LACV adaptation. Together, these results begin to characterize the mechanisms of LACV infectivity and how the LACV glycoprotein contributes to infectivity and pathogenesis. ImportanceVector-borne arboviruses are significant health threats leading to devastating disease worldwide. This emergence and the fact that there are little to no vaccines or antivirals targeting these viruses highlights the need to study how arboviruses replicate at the molecular level. One potential antiviral target is the class II fusion glycoprotein. Alphaviruses, flaviviruses, and bunyaviruses encode a class II fusion glycoprotein that contain strong structural similarities in the tip of domain II. Here we show that the bunyavirus La Crosse virus uses similar mechanisms to entry as the alphavirus chikungunya virus and residues in the ij loop are important for virus infectivity. These studies show that genetically diverse viruses use similar mechanisms through concerned structure domains, suggesting these may be a target for broad-spectrum antivirals to multiple arbovirus families.

microbiology↗

bioRxiv2021

Two-dimensional multiplexed assay for rapid and deep SARS-CoV-2 serology profiling and for machine learning prediction of neutralization capacity

Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype-antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.

immunology↗

bioRxiv2021

Structurally conserved domains between flavivirus and alphavirus fusion glycoproteins contribute to replication in mammals and infectious virion production.

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants including an envelope glycoprotein variant in {beta}-strand c (V114M) of domain II. We have previously shown that the analogous {beta}-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E {beta}-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice, and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that structurally analogous residues in the alphavirus and flavivirus class II fusion proteins contribute to virus infection in vivo and highlight these shared domains as targets for broad-spectrum arbovirus therapies. Author SummaryArboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge on the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

microbiology↗