Vitamin D and the Ability to Produce 1,25(OH)2D Are Critical for Protection from Viral Infection of the Lungs.

Vitamin D supplementation is linked to improved outcomes from respiratory virus infection, and the COVID-19 pandemic renewed interest in understanding the potential role of vitamin D in protecting the lung from viral infections. Therefore, we evaluated the role of vitamin D using animal models of pandemic H1N1 influenza and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. In mice, dietary-induced vitamin D deficiency resulted in lung inflammation that was present prior to infection. Vitamin D sufficient (D+) and deficient (D-) wildtype (WT) and D+ and D- Cyp27B1 (Cyp) knockout (KO, cannot produce 1,25(OH)2D) mice were infected with pandemic H1N1. D- WT, D+ Cyp KO, and D- Cyp KO mice all exhibited significantly reduced survival compared to D+ WT mice. Importantly, survival was not the result of reduced viral replication, as influenza M gene expression in the lungs was similar for all animals. Based on these findings, additional experiments were performed using the mouse and hamster models of SARS-CoV-2 infection. In these studies, high dose vitamin D supplementation reduced lung inflammation in mice but not hamsters. A trend to faster weight recovery was observed in 1,25(OH)2D treated mice that survived SARS-CoV-2 infection. There was no effect of vitamin D on SARS-CoV-2 N gene expression in the lung of either mice or hamsters. Therefore, vitamin D deficiency enhanced disease severity, while vitamin D sufficiency/supplementation reduced inflammation following infections with H1N1 influenza and SARS-CoV-2.

Genome rearrangement of influenza virus for anti-viral drug screening.

Rearrangement of the influenza A genome such that NS2 is expressed downstream of PB1 permits the insertion of a foreign gene in the NS gene segment. In this report, the genome rearranged strategy was extended to A/California/04/2009 (pH1N1), and Gaussia luciferase (GLuc) or GFP was expressed downstream of the full-length NS1 gene (designated GLucCa04 and GFPCa04, respectively). In growth kinetics studies, culture of amantadine sensitive GLucCa04 (Sens/GlucCa04) in the presence of amantadine significantly decreased GLuc expression and viral titers for 48 h post-infection (hpi). When Sens/GlucCa04 was subsequently used in an in vitro anti-viral screening assay, amantadine treatment significantly decreased GLuc expression from amantadine sensitive compared to amantadine resistant GLucCa04 (Res/GlucCa04) as early as 16 hpi. In in vivo screening studies, DBA mice were treated daily with amantadine from 1 day prior to infection and inoculated with either Sens/GlucCa04 or Res/GlucCa04 alone or as a co-infection with the parental strain. On days 3 and 5 post-infection, lung samples were collected and amantadine treatment was shown to decrease GLuc expression by two orders of magnitude (p<0.05) in Sens/GlucCa04 infected mice. Furthermore, while both Sens and Res/GlucCa04 were highly attenuated, addition of the parental strain to the inoculum yielded clinical disease indicative of GLuc expression and pulmonary viral titers. These findings indicate that the use of GLucCa04 can potentially accelerate in vitro and in vivo anti-viral screening by shortening the time required for virus detection.