Biology subjects

Brennan, B.

Publications and source records attributed to Brennan, B..

10 recordsLinked to original sources

bioRxiv2026

Reverse genetics and comparative pathogenesis of Lone star virus.

Lone star virus (LSV) is a bandavirus first isolated from Amblyomma americanum ticks in the United States (U.S.) and is phylogenetically related to severe fever with thrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV), and Bhanja virus, each of which has been associated with severe human disease. In contrast to these better-characterized bandaviruses, LSV remains poorly studied, and its pathogenic potential is not well defined. Recent detection of LSV RNA in cerebrospinal fluid from an immunocompromised patient in Idaho, U.S., with fatal meningoencephalitis further highlights the need for experimental systems to investigate LSV biology. Here, we rescued recombinant (r) LSV from cloned cDNA and used it to characterize LSV. rLSV replicated similarly to the parental isolate in mammalian cells and caused rapid, systemic, and lethal disease in IFNAR-/- mice, with widespread detection of viral (v) RNA across multiple tissues, hepatic and splenic pathology, and induction of inflammatory cytokines. In contrast, C57BL/6J mice controlled infection and exhibited no clinical disease. To place LSV within a broader comparative framework, we generated rSFTSV from cloned cDNA and compared rLSV, rSFTSV, and HRTV in cell culture and IFNAR-/- mice. Our studies revealed distinct disease kinetics among these related tick-borne bandaviruses and showed that HRTV-induced immunity protected against homologous HRTV rechallenge and heterologous rSFTSV challenge, but not rLSV challenge. Together, these findings establish reverse-genetics platforms and small-animal models for comparative bandavirus studies, define key features of LSV pathogenesis, and place this neglected virus within a framework of related bandaviruses that differ in virulence and immunological overlap. ImportanceTick-borne bandaviruses include several viruses associated with severe human disease, yet many related viruses remain poorly characterized. Lone star virus (LSV) was first isolated from Amblyomma americanum ticks decades ago, but experimental tools and animal models to study LSV infection have been lacking. Here, we generated a recombinant LSV and used it to define the outcome of infection in immunocompromised and immunocompetent mice. We show that LSV can cause rapid, systemic, and lethal disease when type I interferon signaling is absent, whereas immunocompetent mice restrict infection and remain clinically normal. By comparing LSV with Heartland virus and severe fever with thrombocytopenia syndrome virus, we also show that related tick-borne bandaviruses differ in disease kinetics and immune protection. These findings provide foundational tools for studying LSV and highlight the importance of experimentally characterizing neglected tick-borne viruses before their pathogenic potential is fully understood.

microbiology↗

bioRxiv2026

Functional divergence of the NSs protein defines interferon antagonism and viral fitness across Bhanja virus lineages.

Bhanja virus (BHAV) is a tick-borne bandavirus (Family Phenuiviridae) with a broad geographic distribution and documented neuroinvasive capacity, yet its molecular biology remains poorly understood. Within the genus Bandavirus, the non-structural protein NSs functions as the primary antagonist of innate immune responses, although mechanistic details for BHAV remain unknown. We established a reverse genetics platform for BHAV by combining virion RNA sequencing with terminal untranslated region mapping, enabling generation of recombinant viruses and systematic investigation of viral determinants. Using this system, we characterized recombinant BHAV replication in mammalian and arthropod cell lines and demonstrated that interferon competence is a critical determinant of viral replication in mammalian cells. Notably, we observed substantial amino acid divergence within the NSs protein across geographically distinct BHAV isolates, despite higher conservation of other viral proteins. To investigate the functional significance of this variation, we generated recombinant viruses expressing heterologous NSs proteins from African and European isolates. Viruses expressing NSs from ibAr2709 or R1819 isolates exhibited enhanced capacity to suppress interferon-beta induction compared to the prototype IG690 strain, correlating with increased viral protein accumulation in interferon-competent cells. Mechanistic studies revealed that BHAV NSs proteins inhibit interferon induction upstream of IRF3, with ibAr2709 and R1819 NSs showing selective inhibition of TBK1 phosphorylation. However, unlike highly pathogenic bandaviruses such as severe fever with thrombocytopenia syndrome virus (SFTSV), BHAV NSs proteins exhibit comparatively weak antagonism of downstream interferon signalling. In vivo studies using interferon alpha/beta receptor-deficient mice demonstrated that NSs sequence variation influences viral replication in splenic tissue without substantially altering disease phenotype. Together, these findings establish BHAV reverse genetics tools for future investigation and reveal how naturally-occurring NSs divergence modulates innate immune antagonism and viral fitness while maintaining an overall attenuated disease phenotype. Author SummaryBhanja virus is a tick-borne virus distributed across Asia, Africa, and Europe that causes neurological disease, yet little is known about how it replicates or evades immunity. We developed the laboratory tools to create recombinant Bhanja viruses and investigate viral functions. We discovered that the virus replicates efficiently in cells lacking interferon responses but is severely restricted in normal immune-competent cells, revealing interferon as a major barrier to viral growth. We observed that Bhanja virus isolates from distinct geographic regions have different versions of a viral protein called NSs that suppresses interferon responses. Isolates from Africa and Europe were better at blocking interferon production than the original Asian strain, correlating with increased viral growth in immune-competent cells. However, unlike related viruses that cause more serious disease such as SFTSV, BHAV NSs proteins only partially antagonise interferon. While this weak antagonism likely contributes to the attenuated phenotype observed in our experimental systems, the natural pathogenesis of Bhanja virus in human hosts and tick vectors remains to be determined. This study provides tools for examining Bhanja virus biology and demonstrates that small viral differences in viral sequence can meaningfully influence evasion of innate immunity and viral fitness without triggering severe disease.

microbiology↗

bioRxiv2025

Tracking blaKPC Plasmid Dissemination within and between Enterobacterales across Michigan Over a Decade

AbstractblaKPC is endemic among Enterobacterales in the USA. While present on diverse plasmids, blaKPC burden is often associated with the clonal spread of multi-drug resistant (MDR) epidemic lineages. In this study we sought to determine the relative contributions of clonal spread and plasmid transfer to blaKPC burden across Michigan healthcare facilities over a decade. To this end we performed whole-genome sequencing of 1,058 KPC-producing isolates collected from 47 Michigan healthcare facilities between 2013 and 2022, including long-read sequencing for 527 isolates to enable precise plasmid tracking. Analysis with MOB-suite identified 64 distinct KPC plasmid types ("secondary clusters"), with the AK975 broad-host range plasmid being the most prevalent, found in 27% of isolates, spanning 20 species and 92 sequence types. Among genomes with AK975, 30% were from epidemic and 70% non-epidemic lineages, highlighting its broad role in regional blaKPC spread. Epidemic lineages of various species constituted 46% of the study population. Epidemic lineages differed in their primary plasmids, and even within epidemic lineages there were clonal expansions with distinct blaKPC plasmids, including in some cases AK975. These findings highlight two patterns of KPC spread: transmission of epidemic lineages harboring broad-range and lineage-specific KPC plasmids; and broader spread of AK975 among diverse species. Traditional surveillance studies often focus on common MDR lineages, potentially overlooking rare species and lineages that mediate the spread of plasmid-borne antimicrobial resistance (AMR) genes. Here we show how longitudinal studies tracking plasmids across species are essential to understand the pathways leading to AMR infections in hospitals. ImportanceThis decade-long longitudinal study highlights the persistence and spread of key KPC- carrying plasmid across multiple bacterial species in the region, including some uncommon ones. It also emphasizes the differences in KPC plasmids across lineages within the same species. While some lineages acquire multiple plasmids with resistance, they are unable to successfully maintain the plasmids. In contrast, clonal sub-populations of KPC-producing bacteria disseminate selected plasmids, establishing a stable host-plasmid combination. Comprehensive genomic surveillance that includes all pathogenic species and plasmids is crucial to understanding the regional transmission dynamics of plasmid-borne antimicrobial resistance (AMR). While outbreak studies define the blowup of a successful lineage and associated plasmids, longitudinal studies identify the reservoir-species and circulating plasmids in the context of plasmid- borne AMR.

genomics↗

bioRxiv2025

SFTSV NSs protein is a novel tick antiviral RNAi response suppressor

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne phenuivirus causing high mortality in humans. While the non-structural protein NSs is dispensable for replication in interferon-deficient mammalian cells, we demonstrate for the first time that NSs is essential for viral replication in tick cells. SFTSV infection triggers canonical Dicer-2-mediated antiviral RNA interference (RNAi) in tick cells, producing virus-derived small interfering RNAs (siRNAs) that target viral transcripts for degradation. We show that NSs functions as a viral suppressor of RNAi by selectively engaging and depleting single-stranded RNAs derived from 22-nucleotide siRNAs, likely limiting their incorporation into RNA-induced silencing complexes (RISC). Complementation with a heterologous RNAi suppressor (p19 protein) partially rescues replication of NSs-deficient virus, validating the RNAi suppressive function of NSs. These findings reveal that successful tick-borne viral replication requires host-specific immune evasion strategies and establish NSs-mediated RNAi suppression as essential for SFTSV persistence in arthropod vectors. Significance StatementThis study reveals a critical requirement for SFTSV infection that differs between hosts. While the viral NSs protein is dispensable in interferon deficient mammalian cells, it is essential for replication in tick cells, the natural vectors of the virus. We show that NSs functions as a viral RNA silencing suppressor by associating with virus derived small RNAs and limiting their availability to the tick antiviral RNA interference machinery. This provides mechanistic insight into how tick-borne viruses evade arthropod immune defences through engagement of functional siRNAs rather than broad inhibition of RNA interference pathways. Together, these findings demonstrate that SFTSV employs distinct immune evasion strategies in different hosts and identify NSs as a key determinant of vector cell infection.

microbiology↗

bioRxiv2025

From prototype to outbreak: conserved pathogenesis of Oropouche virus in a novel murine pregnancy model highlights its public health implications

Oropouche virus (OROV) is an emerging orthobunyavirus responsible for widespread outbreaks across South and Central America. The recent surge in congenital infections has raised urgent concerns about OROVs threat to maternal and fetal health. Here, we establish an in vivo model of OROV vertical transmission using the ancestral (prototype) strain BeAn19991 in immunocompetent C57BL/6J mice. We demonstrate that OROV efficiently replicates in maternal tissues, crosses the maternal-fetal interface, and infects both placental and fetal tissues. Parallel infections in human trophoblast-derived cell lines confirm conserved placental tropism across the ancestral strain and a contemporary (outbreak) isolate from the current outbreak. Importantly, we show that vertical transmission is not a recently acquired trait but a long-standing feature of OROV biology. Offspring born to infected dams mount neutralizing antibody responses and exhibit partial protection upon challenge. These findings conclusively confirm OROV as a vertically transmissible arbovirus, highlighting the urgent need to integrate OROV into surveillance, diagnostic, and vaccine preparedness efforts.

microbiology↗

bioRxiv2024

Embryonically generated interneurons (EGINs) exhibit a distinctive developmental program and lamination in the olfactory bulb

Interneurons (INs) in the mouse olfactory bulb (OB) develop during an extensive period of time that begins in the embryo and continues throughout the lifetime of an individual. The connectivity and location of these neurons is profoundly affected by the time of generation, making these developmental windows critical to understand the circuitry of the OB. Here, we focus on the OB embryonic generated interneurons (or EGINs), which have generally received less attention than those generated in the adult. Birthdates of EGINs were differentiated by embryonic injections of thymidine analogs and their final destinations and phenotypes analyzed by immunohistochemistry. We found that EGINs were retained in the adult and were distributed across all layers of the OB. However, a lateral-to-medial neurogenic gradient is seen only in the earliest generated EGINs. Within the granule cell layer (GCL), EGINs predominantly accumulated in the superficial region at almost all ages and remained there during adulthood. Immunostaining for calbindin, parvalbumin, tyrosine hydroxylase, and calretinin were largely negative suggesting that EGINs may represent subpopulations of OB INs that are not yet fully characterized. Using in utero electroporation to label EGIN progenitors, we demonstrated that they reach the primordial OB as early as E13 and begin to differentiate apical dendrites by E15. At E16, EGIN progenitors clustered into a chain of migrating neuroblasts denoting the embryonic rostral migratory stream (RMS). Collectively, our data highlight the importance of studying OB INs in isolated time windows to better understand the formation of circuits that define the olfactory responses.

neuroscience↗

bioRxiv2024

Uukuniemi virus infection causes a pervasive remodelling of the RNA-binding proteome in tick cell cultures

Cellular RNA-binding proteins (RBPs) are pivotal for the viral lifecycle, mediating key host-virus interactions that promote or repress virus infection. While these interactions have been largely studied in the vertebrate host, no comprehensive analyses of protein-RNA interactions occurring in cells of arbovirus vectors, in particular ticks, have been performed to date. Here we systematically identified the responses of the RNA-binding proteome (RBPome) to infection with a prototype bunyavirus (Uukuniemi virus; UUKV) in tick cells and discovered changes in RNA-binding activity for 283 proteins. In an orthogonal approach, we analysed the composition of the viral ribonucleoprotein by immunoprecipitation of UUKV nucleocapsid protein (N) in infected cells. We found many tick RBPs that are regulated by UUKV infection and associate with viral nucleocapsid protein complexes. We confirmed experimentally that these RBPs impact UUKV infection. This includes the tick homolog of topoisomerase 3B (TOP3B), a protein able to manipulate the topology of RNA, which showed an effect on viral particle production. Our data thus reveals the first protein-RNA interaction map for infected tick cells. Research highlightsO_LIUUKV RNAs interact with nearly three hundred tick cell RBPs. C_LIO_LIDemonstrated an enrichment of N protein interactors within the upregulated RIC data suggesting a direct involvement in viral RNA metabolism and translation. C_LIO_LIDeveloped a robust methodology to silence gene expression in tick cell cultures. C_LIO_LIThe TOP3B complex facilitates efficient packaging of UUKV virions. C_LI

microbiology↗

bioRxiv2024

The PAZ domain of Aedes aegypti Dicer 2 is critical for accurate and high-fidelity size determination of virus-derived small interfering RNAs.

The exogenous siRNA (exo-siRNA) pathway is a critical RNA interference response involved in controlling arbovirus replication in mosquito cells. It is initiated by the detection of viral long double-stranded RNA (dsRNA) by the RNase III enzyme Dicer 2 (Dcr2), which is processed into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs, or vsiRNAs that are taken up by the Argonaute 2 (Ago2) protein to target viral single-stranded RNAs. The detailed understanding of Dicer structure, function and domains owes much to studies outside the context of viral infection, and how Dcr2 domains contribute to detecting viral dsRNA to mount antiviral responses in infected mosquito cells remains much less understood. Here, we used a Dcr2 reconstitution system in Aedes aegypti derived Dcr2 KO cells to assess the contribution of the PAZ domain to induction of the exo-siRNA pathway following infection with Semliki Forest virus (SFV; Togaviridae, Alphavirus). Amino acids critical for PAZ activity were identified, and loss of PAZ function affected the production of 21 nt vsiRNAs -though not the overall ability of Dcr2 to process viral dsRNA- and silencing activity. This study establishes the importance of correct vsiRNA size in mosquito exo-siRNA antiviral responses, as well as the PAZ domains functional contribution to Dcr2 processing of viral dsRNA to 21 nt vsiRNAs.

microbiology↗

bioRxiv2023

Temperature elevation synergises with and enhances the type-I IFN-mediated restriction of MPXV

Fever is an evolutionary conserved host pro-inflammatory immune response that governs the regulation of multiple biological processes to control the outcome of infection. In January 2022, the World Health Organization (WHO) reported a global outbreak in mpox cases with a high incidence of human-to-human transmission. A frequent prodromal symptom of monkeypox virus (MPXV) infection is fever, with a febrile temperature range of 38.3 to 40.5 {degrees}C. However, the outcome of temperature elevation on MPXV infection remains poorly defined. Here, we isolated a circulating strain of MPXV from a patient who presented with fever (38.5 {degrees}C) and rash from the 2022 outbreak. Genomic sequencing identified this isolate to belong to the epidemic Clade IIb.B1. Transcriptomic analysis of infected cells demonstrated this virus to induce a strong IL6 pro-inflammatory immune response, consistent with a role for this pyrogen in the regulation of fever. We identify host-cell temperature at both physiological skin (33 {degrees}C) and clinical febrile temperatures (38.5 and 40 {degrees}C) to be a key determinant in the outcome of infection through the differential regulation of MPXV transcription and associated amplitude of host cytokine response to infection. Incubation of infected cells at 38.5 or 40 {degrees}C led to a restriction or ablation in MPXV replication, respectively. Importantly, this thermal inhibition was reversible upon temperature downshift to 37 {degrees}C without detriment to viral replication fitness. Co-stimulation of the type-I interferon (IFN) response led to a dose- and temperature-dependent inhibition in MPXV replication that restricted the re-establishment of infection upon temperature downshift and withdrawal of IFN as an immune stimulus. Our data identify febrile temperatures associated with mpox disease to be a critical component of the host pro-inflammatory immune response to infection which can synergise with the type-I IFN response to enhance the host-cell mediated restriction of MPXV.

microbiology↗

bioRxiv2021

An outbreak of Rift Valley fever among peri-urban dairy cattle in northern Tanzania, 2018

Undetected Rift Valley fever (RVF) outbreaks are expected in endemic countries but little is known about their size or frequency. We describe a previously unreported RVF outbreak involving dairy cattle that appeared to be limited to the edge of the town of Moshi, Tanzania and occurred from May through August, 2018. The outbreak was detected retrospectively using samples collected as part of a cohort study investigating the causes of livestock abortion across northern Tanzania. A total of 14 RVF-associated cattle abortions were identified using a combination of serology and quantitative reverse transcription PCR (RT-qPCR). Milk samples from three (21%) of 14 cases were also RT-qPCR positive. Genotyping revealed circulation of RVF viruses from two lineages. The occurrence of an RVF outbreak among peri-urban dairy cattle, and evidence for RVF viral shedding in milk, highlights the potential for emerging zoonotic risks associated with the growth of urban and peri-urban livestock populations.

ecology↗