virology assays
Recorded terms: plaque and TCID50 titration; plaque assay; TCID50 titration
Archaeal members of the L30 protein family, but not their orthologues from any other domain of life, block production of the spliced influenza A virus, influenza B virus and isavirus transcripts without measurably disturbing host splicing, identifying a shared and apparently noncanonical feature of orthomyxovirus splicing.
In golden hamsters, productive SARS-CoV-2 replication in the airways generates circulating type I and III interferon that primes every organ against infection, and blunting or bypassing that airway response permits viremia and productive infection of liver, kidney, spleen and brain.
An iterative, scarless and biallelic method for overwriting large mammalian genomic segments in mouse embryonic stem cells, used to build a recoded Trp53 locus and mice carrying the human ACE2 and TMPRSS2 loci in place of their mouse counterparts.
Intranasal SARS-CoV-2 infection of golden hamsters deposits viral RNA without infectious virus in dorsal root ganglia and spinal cord, producing a milder but longer-lasting mechanical hypersensitivity than influenza A virus and a neuropathic dorsal root ganglion transcriptome at 31 days, from which ILF3 emerges as an analgesic target validated in mouse pain models.
Comparison of young and older golden hamsters infected with SARS-CoV-2 shows that age reduces the magnitude and duration of the innate response and of tissue repair, expands suppressor T cells and IL-17-driven neutrophil recruitment, and lowers germinal centre B cell frequency and neutralizing antibody potency without raising lung virus titres.
Kinase substrate specificity profiling assigns the phosphorylation cluster in the SARS-CoV-2 nucleocapsid SR-rich domain to a cascade initiated by SRPK1 and SRPK2 and extended by GSK-3 and casein kinase 1, whose inhibition suppresses coronavirus replication.
Benchmarked against pandemic influenza in golden hamsters, SARS-CoV-2 uniquely sustains interferon signaling, chemokine production and myeloid activation in olfactory bulb and epithelium a month after clearance, alongside altered behavior and matching signatures in recovered human olfactory tissue.
Uncoupling the two steps of influenza A virus genome replication with viral promoter mutations shows that ANP32A is required for synthesis of both the complementary RNA intermediate and progeny genomic RNA, and that it acts on the actively replicating polymerase rather than the encapsidating one.
In golden hamsters, influenza A virus infection reduces SARS-CoV-2 replication during coinfection, after preinfection, and even one to two weeks after influenza has been cleared, while SARS-CoV-2 leaves influenza replication in vivo unchanged.
A microfluidic bronchial airway chip lined with differentiated human airway epithelium and pulmonary endothelium reproduces strain-dependent influenza virulence, cytokine output and neutrophil recruitment, and when drugs are delivered at clinically achievable blood concentrations under flow it separates candidates that work in cell lines from those that also work in hamsters challenged with SARS-CoV-2.
A genome-scale CRISPR loss-of-function screen in ACE2-expressing human alveolar epithelial cells ranks every protein-coding gene by the effect of its loss on SARS-CoV-2 infection, converging on endosomal machinery, and links several top hits to increased cholesterol biosynthesis and, for RAB7A, to intracellular sequestration of ACE2.
Longitudinal tracking of antigen-specific lymphocytes in golden hamsters shows that memory from the founder strain of SARS-CoV-2 clears a beta variant rechallenge and that transferred memory T cells alone lower viral load, yet protected animals still infected every cohoused naive partner.
Longitudinal transcriptional and histological profiling of SARS-CoV-2 infected golden hamsters maps a wave of inflammation that reaches tissues with little or no productive replication, and shows that intranasal type I interferon given before or after challenge lowers viral load and disease burden.
Human ocular surface tissue carries SARS-CoV-2 entry machinery and supports productive replication, with the limbus most permissive in both adult donor cells and stem cell derived whole-eye cultures, where infection drives NF-kB chemokine induction and blunted interferon signaling.
A library of 135 barcoded coxsackievirus B3 clones shows that orally inoculated virus reaches systemic tissues within 20 minutes and replicates as a diverse population, after which fewer than three variants come to dominate every tissue in the animal without any detectable adaptive mutation.
A time-resolved phosphoproteomic survey of SARS-CoV-2-infected cells showing that infection acts mainly through signalling rather than protein abundance, activating casein kinase II and the p38 cascade while shutting down mitotic kinases, and converting that kinase profile into inhibitors with antiviral activity.
A library of 107 barcoded influenza A viruses differing only in their NS1 sequence, competed in dog cells, human cells, chicken eggs and mice, resolves NS1-driven fitness as a set of divergent and partly convergent evolutionary trajectories rather than a single ordered adaptation gradient.
Applying one uniform small RNA-based selective pressure to four virus families in vertebrate cells shows that the ability to escape it tracks with the capacity for polymerase template switching rather than with genome polarity as such, since positive-strand viruses excise the targeted sequence while negative-strand viruses are cleared and a recombination-defective poliovirus cannot escape.
A one-step recombineering strategy that inserts hematopoietic-specific miR-142 target sites into the untranslated region of the human cytomegalovirus IE2 transcript permits virus rescue in fibroblasts while silencing IE2 selectively in myeloid cells, revealing that IE2 loss raises rather than abolishes replication in macrophages.
Recreating a small RNA antiviral response in mice, using either host microRNAs repurposed as virus-specific guides or a virus-encoded artificial small interfering RNA, attenuates influenza A virus by more than five logs and prevents disease without any requirement for type I interferon signaling.
An attenuated influenza A virus engineered to deliver individual artificial small interfering RNAs enables a fitness-based loss-of-function screen inside an infected mouse, and that screen identifies MDA5 as a contributor to the antiviral response to influenza A virus despite the established role of RIG-I as the sensor that induces interferon beta.
Loss of the nuclear RNase III enzyme Drosha, but not of Dicer, increases RNA virus replication in mammalian fibroblasts, and diverse RNA viruses drive Drosha into the cytoplasm by CRM1-dependent export in a manner that does not require new protein synthesis, RIG-I, TBK1 or type I interferon signaling.
Genetically barcoded influenza A virus libraries tracked through cell culture, embryonated eggs, guinea pigs, ferrets and mice show that transmission bottlenecks differ by route and recipient, with airborne transmission reducing a diverse inoculum to as few as two founder clones.
Sustained IRF7 activity induces the kinase MAP3K8, which phosphorylates the proline-rich hinge of IRF3 and redirects it from homodimers into IRF3 and IRF7 heterodimers, broadening the antiviral transcriptome and scaling the cellular response to the persistence of the viral threat.
Engineering vesicular stomatitis virus to eliminate RISC-loaded small RNAs attenuates rather than enhances replication in mice, and confers no replication advantage even when interferon signaling is removed, arguing that small RNA silencing does not contribute to mammalian antiviral defense.
Engineering target sites for miR-192, a microRNA abundant in human and mouse respiratory tissue but absent from the ferret respiratory tract, into the influenza A virus hemagglutinin segment attenuates the virus in mice while leaving replication and transmission in ferrets intact, providing a genetic layer of biocontainment for transmission studies.
Influenza A virus engineered to be silenced by the hematopoietic microRNA miR-142 shows that replication inside antigen presenting cells is dispensable for CD8 T cell priming and clearance but required for much of the RIG-I-dependent type I interferon response in vivo.
Inserting hematopoietic-specific miR-142 target sites into the dengue virus 3-prime untranslated region excludes replication from macrophages and dendritic cells in vivo and abolishes spread to other tissues, identifying hematopoietic cells as the dominant amplification compartment.
Engineering microRNA response elements for a mammalian-ubiquitous microRNA directly into the influenza A virus nucleoprotein coding sequence attenuates the virus in mice while leaving growth in embryonated chicken eggs intact, yielding live attenuated vaccine candidates for H1N1 and H5N1.
Mice lacking IKKε produce normal interferon-β but fail to induce roughly a third of interferon-stimulated genes, because interferon activates IKKε, which phosphorylates STAT1 at Ser708 and thereby determines whether ISGF3 occupies a subset of response elements.
The IKK-related kinases IKKepsilon and TBK1 are identified as components of the virus-activated kinase activity that phosphorylates the C-terminal serine cluster of IRF-3 and IRF-7, linking pathogen sensing to type I interferon gene induction and to establishment of an antiviral state.