Animal models recapitulating COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Intranasally inoculated transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. We evaluated the clinical and virological dynamics of SARS-CoV-2 using two intranasal doses (104 and 106 PFUs), with a detailed spatiotemporal pathologic analysis of the 106 dose cohort. Despite generally mild-to-moderate pneumonia, clinical decline resulting in euthanasia or death was commonly associated with hypothermia and viral neurodissemination independent of inoculation dose. Neuroinvasion was first observed at 4 days post-infection, initially restricted to the olfactory bulb suggesting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. Absence of viremia suggests neuroinvasion occurs independently of transport across the blood-brain barrier. SARS-CoV-2 tropism was neither restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), nor inclusive of some ACE2-positive cell lineages (e.g., bronchiolar epithelium and brain vasculature). Absence of detectable ACE2 protein expression in neurons but overexpression in neuroepithelium suggest this as the most likely portal of neuroinvasion, with subsequent ACE2 independent lethal neurodissemination. A paucity of epidemiological data and contradicting evidence for neuroinvasion and neurodissemination in humans call into question the translational relevance of this model.

Animal models recapitulating the distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. However, the cause(s) and mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate inflammation in the lungs, lethality was invariably associated with viral neuroinvasion and neuronal damage (including spinal motor neurons). Neuroinvasion occurred following virus transport through the olfactory neuroepithelium in a manner that was only partially dependent on hACE2. Interestingly, SARS-CoV-2 tropism was overall neither widespread among nor restricted to only ACE2-expressing cells. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis.SUMMARYCOVID-19 is a respiratory disease caused by SARS-CoV-2, a betacoronavirus. Here, we show that in a widely used transgenic mouse model of COVID-19, lethality is invariably associated with viral neuroinvasion and the ensuing neuronal disease, while lung inflammation remains moderate.

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Escherichia coli is a gram-negative bacillus that colonizes the gastrointestinal tract of humans and animals. 46 Although some strains are considered commensals, various intestinal and extraintestinal pathogenic E. coli pathotypes are associated with a wide range of clinical disease states in the host; 16,41 these strains are responsible for the deaths of more than 2 million humans annually. 65 Specific pathotypes often harbor similar virulence factors and correspond to distinct clinical and histologic lesions. Intestinal pathotypes include enteropathogenic E. coli, enterohemorrhagic E. coli, enteroinvasive E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, diffusely adhering E. coli, and adherent-invasive E. coli. 65 Extraintestinal pathotypes include uropathogenic E. coli and neonatal meningitis E. coli, both of which have an enhanced ability to translocate through the intestinal epithelium and cause severe clinical disease.E. coli strains typically are classified into 1 of the 4 major phylogenetic groups: A, B1, B2, and D. 10,14,60 Groups B2 and D are often associated with pathogenicity, whereas fecal strains belonging to groups A and B1 generally lack virulence factors. 22,60 Strains belonging to pathogroup B2 have been isolated from the feces of persons from developed countries with increasing frequency. 52,70 These pathogenic strains encode various combinations of virulence genes and pathogenicity islands which promote invasion and colonization, evasion of host defenses, and damage to host tissues. Associated virulence factors include cytotoxins such as genotoxic cyclomodulins, cytotoxic necrotizing factors (cnf), cytolethal distending toxin (cdt), and the genotoxin colibactin (pks). These virulence factors modulate host cellular differentiation, proliferation, and apoptosis and promote cytopathic effects. 7,21,69 CNF is a 115-kDa cyclomodulin protein that induces cell-cycle alterations and cytoskeletal changes by activating rho GTPases, which leads to a variety of aberrant phenotypic effects including micropinocytosis, megalocytosis, and multinucleation. 62 cnf1 is chromosomally encoded, 23 whereas cnf2 is plasmid-encoded. 20 cnf-producing E. coli are considered necrotoxigenic and are associated with intestinal, urinary, 23 and meningeal infection of humans. 41 cnf + E. coli have previously been isolated from clinically

C57BL/6 (B6) mice from Taconic Sciences (Tac) and the Jackson Laboratory (Jax) were infected with H. pylori PMSS1 (Hp) for 16 week; there was no significant difference in the gastric histologic activity index between Hp infected Tac and Jax B6. However, the degree of gastric mucous metaplasia and Th1-associated IgG2c levels in response to Hp infection were increased in Tac mice over Jax mice, whereas the colonization levels of gastric Hp were higher by 8-fold in Jax B6 compared with Tac B6. Additionally, mRNA expression of gastric Il-1β, Il-17A and RegIIIγ were significantly lower in the infected Tac compared to the infected Jax mice. There were significant differences in the microbial community structures in stomach, colon, and feces between Jax and Tac B6 females. Differences in gastric microbial communities between Jax and Tac B6 females are predicted to affect the metagenome. Moreover, Hp infection perturbed the microbial community structures in the stomach, colon and feces of Jax mice, but only altered the colonic microbial composition of Tac mice. Our data indicate that the GI microbiome of Tac B6 mice is compositionally distinct from Jax B6 mice, which likely resulted in different pathological, immunological, and microbial responses to Hp infection.