Growth media affects susceptibility of air-lifted human nasal epithelial cell cultures to SARS-CoV2, but not Influenza A, virus infection.

Primary differentiated human epithelial cell cultures have been widely used by researchers to study viral fitness and virus-host interactions, especially during the COVID19 pandemic. These cultures recapitulate important characteristics of the respiratory epithelium such as diverse cell type composition, polarization, and innate immune responses. However, standardization and validation of these cultures remains an open issue. In this study, two different expansion medias were evaluated and the impact on the resulting differentiated culture was determined. Use of both Airway and Ex Plus media types resulted in high quality, consistent cultures that were able to be used for these studies. Upon histological evaluation, Airway-grown cultures were more organized and had a higher proportion of basal progenitor cells while Ex Plus- grown cultures had a higher proportion terminally differentiated cell types. In addition to having different cell type proportions and organization, the two different growth medias led to cultures with altered susceptibility to infection with SARS-CoV-2 but not Influenza A virus. RNAseq comparing cultures grown in different growth medias prior to differentiation uncovered a high degree of differentially expressed genes in cultures from the same donor. RNAseq on differentiated cultures showed less variation between growth medias but alterations in pathways that control the expression of human transmembrane proteases including TMPRSS11 and TMPRSS2 were documented. Enhanced susceptibility to SARS-CoV-2 cannot be explained by altered cell type proportions alone, rather serine protease cofactor expression also contributes to the enhanced replication of SARS-CoV-2 as inhibition with camostat affected replication of an early SARS-CoV-2 variant and a Delta, but not Omicron, variant showed difference in replication efficiency between culture types. Therefore, it is important for the research community to standardize cell culture protocols particularly when characterizing novel viruses.

SARS-CoV-2 infection alters mitochondrial and cytoskeletal function in human respiratory epithelial cells mediated by expression of spike protein.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SCV2), which has resulted in higher morbidity and mortality rate than other respiratory viral infections, such as Influenza A virus (IAV) infection. Investigating the molecular mechanisms of SCV2-host infection vs IAV is vital in exploring antiviral drug targets against SCV2. We assessed differential gene expression in human nasal cells upon SCV2 or IAV infection using RNA sequencing. Compared to IAV, we observed alterations in both metabolic and cytoskeletal pathways suggestive of epithelial remodeling in the SCV2-infected cells, reminiscent of pathways activated as a response to chronic injury. We found that spike protein interaction with the epithelium was sufficient to instigate these epithelial responses using a SCV2 spike pseudovirus. Specifically, we found downregulation of the mitochondrial markers SIRT3 and TOMM22. Moreover, SCV2 spike infection increased extracellular acidification and decreased oxygen consumption rate in the epithelium. In addition, we observed cytoskeletal rearrangements with a reduction in the actin-severing protein cofilin-1 and an increase in polymerized actin, indicating epithelial cytoskeletal rearrangements. This study revealed distinct epithelial responses to SCV2 infection, with early mitochondrial dysfunction in the host cells and evidence of cytoskeletal remodeling that could contribute to the worsened outcome in COVID-19 patients compared to IAV patients. These changes in cell structure and energetics could contribute to cellular resilience early during infection, allowing for prolonged cell survival and potentially paving the way for more chronic symptoms. IMPORTANCE COVID-19 has caused a global pandemic affecting millions of people worldwide, resulting in a higher mortality rate and concerns of more persistent symptoms compared to influenza A. To study this, we compare lung epithelial responses to both viruses. Interestingly, we found that in response to SARS-CoV-2 infection, the cellular energetics changed and there were cell structural rearrangements. These changes in cell structure could lead to prolonged epithelial cell survival, even in the face of not working well, potentially contributing to the development of chronic symptoms. In summary, these findings represent strategies utilized by the cell to survive the infection but result in a fundamental shift in the epithelial phenotype, with potential long-term consequences, which could set the stage for the development of chronic lung disease or long COVID-19.

Early Transcriptional Responses of Human Nasal Epithelial Cells to Infection with Influenza A and SARS-CoV-2 Virus Differ and Are Influenced by Physiological Temperature.

Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an understudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insight into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affected SCV2, but not IAV, viral replicative fitness and that SCV2-infected cultures were slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changed the basal transcriptomic landscape of epithelial cells, but that it also impacted the response to infection. The induction of interferon and other innate immune responses was not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remained consistent, there may be metabolic or signaling changes that affect how well the cultures were able to adapt to new pressures, such as infection. Finally, we show that hNECs responded differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.

Early transcriptional responses of human nasal epithelial cells to infection with Influenza A and SARS-CoV-2 virus differ and are influenced by physiological temperature.

Influenza A (IAV) and SARS-CoV-2 (SCV2) viruses represent an ongoing threat to public health. Both viruses target the respiratory tract, which consists of a gradient of cell types, receptor expression, and temperature. Environmental temperature has been an un-derstudied contributor to infection susceptibility and understanding its impact on host responses to infection could help uncover new insights into severe disease risk factors. As the nasal passageways are the initial site of respiratory virus infection, in this study we investigated the effect of temperature on host responses in human nasal epithelial cells (hNECs) utilizing IAV and SCV2 in vitro infection models. We demonstrate that temperature affects SCV2, but not IAV, viral replicative fitness and that SCV2 infected cultures are slower to mount an infection-induced response, likely due to suppression by the virus. Additionally, we show that that temperature not only changes the basal transcriptomic landscape of epithelial cells, but that it also impacts the response to infection. The induction of interferon and other innate immune responses were not drastically affected by temperature, suggesting that while the baseline antiviral response at different temperatures remains consistent, there may be metabolic or signaling changes that affect how well the cultures are able to adapt to new pressures such as infection. Finally, we show that hNECs respond differently to IAV and SCV2 infection in ways that give insight into how the virus is able to manipulate the cell to allow for replication and release. Taken together, these data give new insight into the innate immune response to respiratory infections and can assist in identifying new treatment strategies for respiratory infections.

HA and M2 sequences alter the replication of 2013-16 H1 live attenuated influenza vaccine infection in human nasal epithelial cell cultures.

From 2013 to 2016, the H1N1 component of live, attenuated influenza vaccine (LAIV) performed very poorly in contrast to the inactivated influenza vaccine. We utilized a primary, differentiated human nasal epithelial cell (hNEC) culture system to assess the replication differences between isogenic LAIVs containing the HA segment from either A/Bolivia/559/2013 (rBol), which showed poor vaccine efficacy, and A/Slovenia/2903/2015 (rSlov), which had reasonable vaccine efficacy. There were minimal differences in infectious virus production in Madin-Darby Canine Kidney (MDCK) cells, but the rSlov LAIV showed markedly improved replication in hNEC cultures at both 32 °C and 37 °C, demonstrating that the HA segment alone could impact LAIV replication in physiologically relevant systems. The rSlov-infected hNEC cultures showed stronger production of interferon and proinflammatory chemokines which might also be contributing to the increased overall vaccine effectiveness through enhanced recruitment and activation of immune cells. An M2-S86A mutation had no positive effects on H1 LAIV replication in hNEC cultures, in contrast to the increased infectious virus production seen in an H3 LAIV. No obvious defects in viral RNA packaging were detected, suggesting that HA function, rather than defective particle production, may be driving the differential infectious virus production in hNEC cultures. Overall, we have shown that not all H1 HA segments can be successfully used in LAIV, and this phenotype cannot be fully explained by segment incompatibilities. Physiologically relevant temperatures and primary cell cultures should be used to demonstrate that candidate LAIVs can replicate efficiently, which is a necessary property for effective vaccines.

Ultrasound does not detect early blood loss in healthy volunteers donating blood.

BACKGROUND: Ultrasound has been suggested as a useful non-invasive tool for the detection of early blood loss. Two possible sonographic markers for hypovolemia are the diameter of the inferior vena cava (IVC) and the thickness of the left ventricle (LV). STUDY OBJECTIVES: The goal of the study was to evaluate the utility of ultrasound to detect signs of early hemorrhagic shock in healthy volunteers, compared with changes in vital signs. METHODS: In the current study, healthy volunteers from blood donation drives were used as models for early hemorrhage. Changes in vital signs, IVC diameter, and LV wall thickness were recorded after approximately 500 cc of blood loss. RESULTS: Thirty-eight subjects were enrolled and completed the study. After blood donation, there was a 7-mm Hg (8%) decrease in mean arterial pressure without a significant change in heart rate. There was a decrease in maximum IVC diameter (IVCmax) (12% decrease [95% confidence interval (CI) -6 to -19] in short axis and 20% decrease [95% CI -12 to -27] in long axis), but no change was seen in the respiratory caval index ((IVCmax - IVCmin)/IVCmax) × 100). There was no change in LV wall thickness. CONCLUSION: In this study, serial changes in vital signs, IVC diameter, and LV wall thickness were clinically insignificant after approximately 500 cc of blood loss in healthy volunteers.

Success of ultrasound-guided peripheral intravenous access with skin marking.

OBJECTIVES: The most effective technique for ultrasound-guided peripheral intravenous access (USGPIVA) is unknown. In the traditional short-axis technique (locate, align, puncture [LAP]), the target vessel is aligned in short axis with the center of the transducer. The needle is then directed toward the target under real-time ultrasound (US) guidance. Locate, align, mark, puncture (LAMP) requires the extra step of marking the skin at two points over the path of the vein and proceeding with direct visualization as in LAP. The difference in success between these two techniques was compared among variably experienced emergency physician and emergency nurse operators. METHODS: Subjects in an urban academic emergency department (ED) were randomized to obtain intravenous (IV) access using either LAP or LAMP after two failed blind attempts. Primary outcomes were success of the procedure and time to complete the procedure in variably experienced operators. RESULTS: A total of 101 patients were enrolled. There was no difference in success between LAP and LAMP, even among the least experienced operators. Of successful attempts, LAMP took longer than LAP (median 4 minutes, interquartile range [IQR] 4-10.5 vs. median 2.9 minutes, IQR 1.6-7; p = 0.004). Only the most experienced operators were associated with higher levels of success (first attempt odds ratio [OR] 6.64; 95% confidence interval [CI] = 2 to 22). Overall success with up to two attempts was 73%. Complications included a 2.8% arterial puncture rate and 12% infiltration rate. CONCLUSIONS: LAMP did not improve success of USGPIVA in variably experienced operators. Experience was associated with higher rates of success for USGPIVA.

Comparison of two transducers for ultrasound-guided vascular access in long axis.

The long-axis technique for ultrasound-guided vascular access may benefit users attempting deeper targets. The purpose of this study was to determine if a difference exists in the difficulty experienced by novice ultrasound users in obtaining vascular access in long axis with linear vs. curvilinear transducers. Subjects obtained access on simulated peripheral veins in a gel model. Time to successful cannulation, number of surface breaks, and number of needle redirects was recorded. Statistical methods used include analysis of variance, regression analysis, and negative binomial regression. The study population was a convenience sample of 24 4th-year medical students, Emergency Medicine residents, attendings, and nurses, and off-service residents rotating in the Emergency Department who had performed less than three ultrasound-guided vascular access procedures. The difference between the number of surface breaks and redirects and the perceived difficulty between the linear and curvilinear transducers was statistically significant (p = 0.002, p = 0.049, p = 0.04). The difference in time to cannulation with the linear and curvilinear transducers was not statistically significant. Novice ultrasound users found the curvilinear transducer easier to use for simulated vascular access in the long axis. Studies utilizing live patients and more experienced ultrasonographers could determine whether the preference for the curvilinear transducer amounts to clinically meaningful shorter times to needle entry and more successful first attempts.