Biology subjects

Laloli, L.

Publications and source records attributed to Laloli, L..

7 recordsLinked to original sources

bioRxiv2026

Genetic modification of primary airway epithelial cell cultures defines MX1 as a barrier for zoonotic influenza A viruses in the human respiratory epithelium

The antiviral innate immune response is a major species barrier that emerging viruses must overcome to infect and transmit efficiently in new hosts. In humans, the interferon-stimulated MX1 gene encodes the antiviral MX1 protein, which restricts zoonotic influenza A viruses (IAVs). Resistance to MX1 is considered a key feature of human-adapted IAVs and a potential determinant of zoonotic and pandemic risk. However, MX1-mediated restriction and viral escape have mostly been investigated in cell lines and animal models, which incompletely recapitulate virus-host interactions in the human airway epithelium, representing the primary site of IAV replication. Although primary human airway epithelial cell (hAEC) cultures, such as airway organoids and air-liquid interface (ALI) cultures, provide a physiologically relevant model, their use in studying MX1 restriction has been limited by challenges in achieving efficient and reliable genetic modification. Here, we used engineered virus-like particles (eVLPs) to deliver Cas9-sgRNA ribonucleoprotein complexes into primary hAECs and disrupt MX1 expression. This approach generated MX1-knockout airway organoids and organoid-derived hAEC ALI cultures with >90% genomic-level editing efficiency and near-complete loss of MX1 protein, while preserving epithelial differentiation at the air-liquid interface. MX1 knockout in hAEC ALI cultures had only a minor effect on the replication kinetics of the human-adapted H1N1pdm09 strain. By contrast, MX1 knockout markedly impaired interferon lambda-mediated restriction of the zoonotic bovine H5N1/Texas and avian H7N9/Anhui strains, resulting in viral titers up to 3-4 logs TCID50 higher than in MX1-competent cultures. These findings provide direct evidence that MX1 restricts zoonotic IAVs in primary human airway epithelium. Our study establishes a biologically relevant platform for assessing the sensitivity of emerging IAVs to a key antiviral host factor and thereby informs evaluations into their zoonotic and pandemic risk.

microbiology↗

bioRxiv2021

Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, but not B.1.351, in animal models

Emerging variants of concern (VOCs) drive the SARS-CoV-2 pandemic. We assessed VOC B.1.1.7, now prevalent in several countries, and VOC B.1.351, representing the greatest threat to populations with immunity to the early SARS-CoV-2 progenitors. B.1.1.7 showed a clear fitness advantage over the progenitor variant (wt-S614G) in ferrets and two mouse models, where the substitutions in the spike glycoprotein were major drivers for fitness advantage. In the "superspreader" hamster model, B.1.1.7 and wt-S614G had comparable fitness, whereas B.1.351 was outcompeted. The VOCs had similar replication kinetics as compared to wt-S614G in human airway epithelial cultures. Our study highlights the importance of using multiple models for complete fitness characterization of VOCs and demonstrates adaptation of B.1.1.7 towards increased upper respiratory tract replication and enhanced transmission in vivo. Summary sentenceB.1.1.7 VOC outcompetes progenitor SARS-CoV-2 in upper respiratory tract replication competition in vivo.

microbiology↗

bioRxiv2021

Establishment of a reverse genetic system from a bovine derived Influenza D virus isolate.

The ruminant-associated Influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5' and 3' rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5' and 3' NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D/CN286) into the bidirectional pHW-2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5' and 3' non-coding regions (NCR) highlighted that the terminal 3' end of all segments harbours an Uracil instead of a Cytosine nucleotide, similar to other Influenza viruses. Subsequent analysis on the functionality of the 5' and 3' NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5' and 3' NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin by use of an in vitro model recapitulating the natural replicate site of IDV in bovine and swine. This revealed that the reverse genetic clone D/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.

microbiology↗

bioRxiv2020

Susceptibility of well-differentiated airway epithelial cell cultures from domestic and wildlife animals to SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally, and the number of cases continues to rise all over the world. Besides humans, the zoonotic origin, as well as intermediate and potential spillback host reservoirs of SARS-CoV-2 are unknown. To circumvent ethical and experimental constraints, and more importantly, to reduce and refine animal experimentation, we employed our airway epithelial cell (AEC) culture repository composed of various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. In this study, we inoculated well-differentiated animal AEC cultures of monkey, cat, ferret, dog, rabbit, pig, cattle, goat, llama, camel, and two neotropical bat species with SARS-CoV-2. We observed that SARS-CoV-2 only replicated efficiently in monkey and cat AEC culture models. Whole-genome sequencing of progeny virus revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat epithelial airway cells. Our findings, together with the previously reported human-to-animal spillover events warrants close surveillance to understand the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

microbiology↗

bioRxiv2020

Disparate temperature-dependent virus - host dynamics for SARS-CoV-2 and SARS-CoV in the human respiratory epithelium

Since its emergence in December 2019, SARS-CoV-2 has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human respiratory tract have been shown to affect the replication kinetics of several viruses, as well as host immune response dynamics, we investigated the impact of temperatures during SARS-CoV-2 and SARS-CoV infection in the human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated more efficiently at temperatures encountered in the upper respiratory tract, and displayed higher sensitivity to type I and type III IFNs. Time-resolved transcriptome analysis highlighted a temperature-dependent and virus-specific induction of the IFN-mediated antiviral response. These data reflect clinical features of SARS-CoV-2 and SARS-CoV, as well as their associated transmission efficiencies, and provide crucial insight on pivotal virus - host interaction dynamics.

microbiology↗

bioRxiv2020

Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis.

Respiratory viruses, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), target cells found in the human respiratory epithelium. These cells, which form a pseudostratified epithelial layer along the airways, constitute the first line of defence against respiratory pathogens and play a crucial role in the host antiviral response. However, despite their key role in host defence, it remains unknown how distinct cell types in the respiratory epithelium respond to IAV infection and how these responses may contribute to IAV-induced pathogenesis and overall disease outcome. Here, we used single cell RNA-sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to a respiratory virus infection in its natural target cells - namely, the human respiratory epithelium.

microbiology↗

bioRxiv2020

Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate in E. coli hosts due to size and occasional instability1-3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Paramyxoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24 in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.

microbiology↗