In 2012, cases of lethal pneumonia among Chinese miners prompted the isolation of a rat-borne henipavirus (HNV), Mòjiāng virus (MojV). Although MojV is genetically related to highly pathogenic bat-borne henipaviruses, the absence of a conserved ephrin receptor-binding motif in the MojV attachment glycoprotein (MojV-G) indicates a differing host-cell recognition mechanism. Here we find that MojV-G displays a six-bladed β-propeller fold bearing limited similarity to known paramyxoviral attachment glycoproteins, in particular at host receptor-binding surfaces. We confirm the inability of MojV-G to interact with known paramyxoviral receptors in vitro, indicating an independence from well-characterized ephrinB2/B3, sialic acid and CD150-mediated entry pathways. Furthermore, we find that MojV-G is antigenically distinct, indicating that MojV would less likely be detected in existing large-scale serological screening studies focused on well-established HNVs. Altogether, these data indicate a unique host-cell entry pathway for this emerging and potentially pathogenic HNV.

The on-going coronavirus disease 2019 (COVID-19) pandemic has mobilized a global effort to develop vaccines and therapeutics that inhibit viral entry by inducing or transferring antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). Phase I/II vaccine clinical trials, monoclonal antibodies, and convalescent sera have all shown promise. However, these efforts often require extensive screening with the live virus under onerous high biocontainment conditions (BSL-3). Virus neutralization assays (VNAs) remain the gold standard for evaluating the anti-viral potency of antibodies and entry inhibitors. The proliferation of pseudotyped virus systems that can be used in BSL-2 compatible VNAs is a positive development. Yet, there is marked variability between VNAs and how the findings are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale. We used our CoV2pp to interrogate the role of exogenous and endogenous proteases in CoV-2-S mediated entry and standardized our VNA based on that understanding. Our CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in a validated set of patient sera. Our system was subsequently validated by three independent groups as an out-of-the-box VNA. More than 120 patient sera were screened, and we report descriptive statistics for absolute (abs) IC50, IC80, and IC90 values from all positive patient sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

Hepatitis C virus (HCV) is the leading cause of liver cancer in the Western Hemisphere. HCV infection requires miR-122, which is expressed only in liver cells, and thus is one reason that replication of this virus occurs efficiently only in cells of hepatic origin. To understand how HCV genetics impact miR-122 usage, we knocked out miR-122 using clustered regularly interspaced short palindromic repeat (CRISPR) technology and adapted virus to replicate in the presence of noncognate miR-122 RNAs. In doing so, we identified viral mutations that allow replication in the complete absence of miR-122. This work provides new insights into how HCV genetics influence miR-122 requirements and proves that replication can occur without this miRNA, which has broad implications for how HCV tropism is maintained.

Polyomaviruses (PyVs) are associated with tumors including Merkel cell carcinoma (MCC). Several PyVs encode microRNAs (miRNAs) but to date no abundant PyV miRNAs have been reported in tumors. To better understand the function of the Merkel cell PyV (MCPyV) miRNA, we examined phylogenetically-related viruses for miRNA expression. We show that two primate PyVs and the more distantly-related raccoon PyV (RacPyV) encode miRNAs that share genomic position and partial sequence identity with MCPyV miRNAs. Unlike MCPyV miRNA in MCC, RacPyV miRNA is highly abundant in raccoon tumors. RacPyV miRNA negatively regulates reporters of early viral (T antigen) transcripts, yet robust viral miRNA expression is tolerated in tumors. We also identify raccoon miRNAs expressed in RacPyV-associated neuroglial brain tumors, including several likely oncogenic miRNAs (oncomiRs). This work describes the first PyV miRNA abundantly expressed in tumors and is consistent with a possible role for both host and viral miRNAs in RacPyV-associated tumors.

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