High-throughput split-GFP antiviral screening assay against fusogenic paramyxoviruses

Several paramyxoviruses are of great public health importance causing decades of pandemics (such as Measles virus) and dangerous zoonosis (such as Nipah virus). In addition, new paramyxoviruses are continuously been discovered but no antiviral drug has been established yet. Their distinctive viral characteristic of syncytia formation in host cells was exploited to develop a novel split-green fluorescent screening assay, where non-functional fragments of a fluorescent protein attached to host cells, recombine and fluoresce when a syncytia is formed. The viability of the assay was confirmed through detecting infection and fusion by representative paramyxoviruses as well as testing against known broad-spectrum antiviral drugs. This protocol was subsequently used to screen for antiviral inhibitors in existing repurposed library of nearly 3000 compounds against Cedar virus, a non-pathogenic model Henipavirus. Two molecules, Cathepsin Inhibitor 1 and PF-543 were confirmed as potent inhibitors of Cedar virus, acting by blocking virus-induced cell-cell fusion. The assay is versatile as its mechanism targets the consequence of fusion rather than the specific viral protein which may enable application in other virus families that cause syncytia formation.
(SWM)

Lrp1 is a host entry factor for Rift Valley fever virus (RVFV)

Lrp1 is a member of the low-density lipoprotein (LDL) receptor family, and they play roles in lipid metabolism and in several endocytic and inflammatory signalling pathways. Screening of genome-wide CRISPR/Cas9 libraries revealed that Lrp1, together with its modulators, RAP and GRP94, are essential host factors for the virulence of RVFV. Lrp1 is conserved across species and different cell types, which could explain the wide host range of the zoonotic virus. Knockout of Lrp1, RAP and GRP94 significantly reduced infection by both the pathogenic and attenuated strains of RVFV. Reduced infection was observed in both cell lines and in primary mouse embryonic fibroblasts. The direct interaction between Lrp1 and glycoprotein was analyzed and found to play an important role in the infection process. Targeting Lrp1 offers a promising therapeutic target against RVFV, which has a pandemic potential.
(MRM)

Human Respiratory Syncytial Virus Infection in a Human T Cell Line is Hampered at Multiple Steps

Human Respiratory Syncytial Virus (HRSV) is a leading cause of acute respiratory infections in humans, more so in infants, the elderly and immunocompromised individuals. HRSV-cell interactions have been studied using epithelial cells. HRSV-cell interaction was studied using lymphoid cells. Although the viral-cell attachment of HRSV to Hep-2 and A3.01 cells is only slightly different, viral genome replication and protein production is greatly reduced in A3.01 cells compared to Hep-2 cells. While there is a clear colocalization between HRSV F protein and Golgi markers in Hep-2 cells, HRSV F protein shows partial colocalization with Golgi markers in A3.01 cells. Although HRSV proteins were readily detected in intracellular compartments of A3.01 cells, very little accumulation of HRSV products was seen at the plasma membrane, which resulted in a lower filament production in these cells. This study suggests that viral replication in A3.01 cells is altered at several stages, including virus-cell fusion, formation of defective inclusion bodies, impaired viral protein production and lack of effective trafficking of viral proteins to virus assembly sites.
(MRM)

The nucleocapsid protein of Crimean–Congo hemorrhagic fever virus interacts with eIF4A to promote the translation of viral mRNA in cells

Crimean–Congo hemorrhagic fever virus (CCHFV), a tick-borne nairovirus of the Bunyavirales order, differs from many viruses in that it does not suppress host cell protein synthesis. This raises the question of how the virus’s mRNAs are able to be efficiently translated despite competition from host transcripts. This study demonstrates that the CCHFV nucleocapsid protein (N protein) boosts the translation of luciferase reporter mRNA, but only when assisted by the viral S-segment mRNA-derived 5’ untranslated region (5’ UTR). Inhibiting eIF4E, a typical cap-binding factor, did not disrupt this enhanced translation. However, when eIF4G was cleaved or eIF4A was chemically inhibited, the N protein’s ability to promote translation was lost. These results reveal that both eIF4A and eIF4G are essential for N protein-driven translation, and this process specifically relies on the viral mRNA 5’ UTR. If the 5’ UTR was randomized, the translation efficiency of the viral S-segment mRNA dropped sharply. Wild-type (WT) S-segment mRNA was found to strongly associate with ribosomes, while the N protein remained bound to the WT 5’ UTR, likely helping ribosomes load repeatedly, encouraging polysome formation and thus enhancing protein synthesis. In contrast, the randomized UTR version of the S-segment mRNA was mostly not associated with ribosomes, leading to reduced protein output. The data indicate that the N protein physically interacts with eIF4A and probably sequesters eIF4A–eIF4G complexes for dedicated use in translating viral S-segment mRNAs, thereby giving viral RNA an advantage over cellular mRNAs for access to the host translation machinery.
(MKO)

Highly specific serological diagnosis of Borna disease virus 1 (BoDV-1) and variegated squirrel bornavirus 1 (VSBV-1) encephalitis by novel antibody isotype assay with multiple viral antigens

Bornavirus encephalitis is a critical and deadly emerging disease in humans in Germany, caused by Borna disease virus 1 (BoDV-1) and variegated squirrel bornavirus 1 (VSBV-1). The disease typically progresses rapidly, but diagnostic efforts, particularly serological tests, are constrained by issues with sensitivity and specificity, often leading to delayed care. This study aimed to create and evaluate a novel spot immunoassay capable of detecting IgG, IgM and IgA antibodies that target four recombinant BoDV-1 proteins: nucleoprotein (N), phosphoprotein (P), accessory protein X (X), and glycoprotein (GP). The study conducted a retrospective analysis on a substantial cohort compromising 15 patients with confirmed bornavirus encephalitis. These were analyzed together with samples from 241 patients suffering from encephalitis of unknown origin, 58 interference samples, and 40 healthy blood donors. The assay demonstrated a highly specific diagnosis (97-100%) for both forms of bornavirus encephalitis. The method also achieved a diagnostic sensitivity of up to 92%. The newly developed spot immunoassay offers a user-friendly, specific, and sensitive tool for the serological diagnosis of human bornavirus encephalitis.
(MKO)

Prediction and characterisation of the human B cell response to a heterologous two-dose Ebola vaccine

Given the increasing Ebola virus disease (EVD) outbreaks and the critical importance of protecting frontline health workers, this study comprehensively characterizes the human B cell immune responses elicited by the heterologous two-dose Ebola vaccine regimen of Ad26.ZEBOV (prime) and MVA-BN-Filo (boost). Utilizing single-cell RNA-sequencing and ELISpot assays, the research revealed robust plasma cell and lasting B cell memory responses post-vaccination. Total IgG-secreting plasma cell (IgG-ASC) frequencies peaked at days 9-11 post-prime and days 5-7 post-boost, correlating with extensive differential gene regulation, particularly of immunoglobulin gene segments. Memory B cell (BMEM) responses were observed after the first dose and significantly enhanced by the second, persisting for over four years post-vaccination with minimal waning. A unique B cell receptor CDRH3 sequence, resembling Orthoebolavirus zairense (EBOV) glycoprotein-binding antibodies, was detected exclusively post-vaccination. Notably, machine-learning models trained on early blood gene expression successfully predicted the magnitude of subsequent antibody responses, identifying key genes such as IGHV3-15, associated with EBOV-GP binding antibodies. These findings underscore the predictive utility of early immune responses and provide crucial mechanistic insights for future EVD preparedness strategies.
(MKO)

Identification of CCZ1 as an essential lysosomal trafficking regulator in Marburg and Ebola virus infections

CCZ1 is a guanine nucleotide exchange factor involved in endolysosomal trafficking of viruses. Haploid murine embryonis stem cells were subjected to transposon mutagenesis  so as to identify genes essential for filoviruses infection. The knockout of CCZ1 from both halploid and diploid cells resulted in a significant drop in infection by the vesicular stomatitis virus pseudotyped with the Marburg virus glycoprotein, confirming its importance in the entry of filoviruses into cells. In addition, the knockout of both CCZ1 and RAB7 results into a complte resistance to Marburg virus  infection in human cells. The CCZ1 knockdown in human hepatocytes and human blood-vessel organoidsalso resulted in  reduction in viral infection. The role of CCZ1 in endosomal trafficking ahead of lysosomal fusion was also confirmed. Ccz1 is also involved in the entro of SARS-CoV-2 infection through the endosomal pathway, but is not involved in Lassa viruses. Hence, CCZ1 is a potential target for drugs targeting viral infections caused by Marburg, ebola and sars-cov-2 viruses.
(MRM)