SARS-CoV-2 REPLICONS REVEAL SPIKE-INDEPENDENT ATTENUATION OF THE OMICRON VARIANT

Background: The SARS-CoV-2 Omicron variant is highly immune evasive but is attenuated in cell and animal models of infection, which many reports attribute to spike mutations. However, the phenotype and contribution to viral fitness of Omicron non-spike mutations remain unknown. Method(s): To study mutations across the entire genome independent of spike, we developed a novel cloning and replicon system capable of generating mutants within 6 hours and obtaining phenotypic results within 3-4 days. Result(s): Using a series of Omicron replicons, we found that ORF1ab harbors critical mutations, especially in the nonstructural protein 6 (NSP6), which lower viral fitness and are currently evolving in Omicron subvariants. In addition, Omicron mutations in several NSPs epistatically interact and are critical for viral replication and polyprotein processing. Conclusion(s): Collectively, we describe a robust replicon technology to study mutations across the genome and our data highlight the need to vigilantly study and monitor non-spike mutations in emerging Omicron subvariants.

SARS-CoV-2 BA.5 CAUSES HIGHER REPLICATION AND LETHAL INFECTION IN K18-hACE2 MICE

Background: The continuous evolution of SARS-CoV-2 in the diverse immune landscape (natural, vaccine, hybrid) is giving rise to novel immune escape mutations. So far, the resulting new variants (BA.1, BA.2, BA.2.12.1) were observed to cause mild infections, however, BA.5 infections are associated with an increased risk of hospitalization.1 Therefore it is essential to investigate the pathogenesis of BA.5. Method(s): Here we compared the pathogenicity of Pre-Omicron (B.1.351) and Omicron (BA.1, BA.2.12.1, and BA.5) variants in wild-type C57BL/6J mice and K18-hACE2 mice. The virus replication kinetics was also studied in human Calu3, pulmonary alveolar type 2 (AT2) cells, and airway organoids (HAO). Cell-to-cell spread of virus was measured by syncytia formation assay and immunohistochemistry (IHC) of infected lungs. Result(s): In the results, infection in C57BL/6J mice showed severe weight loss ( >15%) for B.1.351 infected mice and moderate ( >5%) for BA.5 infected. C57BL/6J mice showed higher virus replication of B.1.351 followed by BA.5, BA.1, and BA.2.12.1. At the peak of virus replication (2 days) plaque-forming units from lung extract of BA.5 infected mice were two, and three logs higher compared to BA.1 and BA.2.12.1 respectively. BA.5 infection was lethal to 80% of infected K18-hACE2 mice, whereas the mice looked normal after infection with BA.1 and BA.2.12.1. BA.5 infected mice showed high virus replication in brain tissue. Surprisingly the syncytia formation assay and IHC for BA.5 was comparable to that of B.1.351, indicating the higher cell-to-cell spread of BA.5 and B.1.351 compared to BA.1 and BA.2.12.1, which is one of the measures of pathogenicity. Calu3 and HAO showed the same trend of virus replication as was observed in-vivo experiments however AT2 cells were found to be resistant to BA.5 replication. Conclusion(s): These results suggest that the BA.5 variant (lineage) of Omicron has the potential to regain the pathogenicity as it shows increased virulence compared to other Omicron sub-variants. Lethal infection of BA.5 in K18-hACE2 mice may be attributed to catastrophic encephalitis and increased cell-to-cell spread.

111. Adaptation and Delivery of a Partially Virtual Curriculum for Pre-Exposure Prophylaxis Peer Mentors in Kenya: Lessons Learned

Purpose: While Pre-Exposure Prophylaxis (PrEP) is highly effective at preventing HIV, uptake is low among adolescents. In low- and middle-income countries (LMIC), peer mentors (PMs) are considered best practice to increase PrEP acceptability and uptake. Globally, COVID19 has shifted much education and training to virtual formats. Most young people in LMIC have cell phones. Our objective is to describe our experiences developing and delivering a mixed virtual/physical curriculum for training PrEP PMs. Methods: IRB and local research ethics committee approval was obtained. A literature search (PubMed, EBSCO, USAID website, and MedEd Portal) yielded one published curriculum for PrEP PMs. This curriculum was combined with locally developed HIV PM education modules to create a new curriculum, with planned virtual and physical sessions. Curriculum materials were reviewed and agreed upon by all authors. All sessions were delivered by authors, with the majority delivered by Americans. The first 4 hours were done virtually via Zoom, covering the basics of HIV, detailed information on PrEP, adolescent development, and confidentiality. The remaining sessions were held in person and covered expectations of PMs, basic family planning, research ethics, action planning, role playing, and a review of virtual topics. Feedback was solicited from the PMs after virtual training. A debriefing session was held with the five facilitators involved in training: 1 research staff and 1 physician investigator from Kenya, 1 research staff and 2 physician investigators from the US - all female. Results: All five PMs (aged 21 – 27) participated. One identified as female, and one as LGBTQ. Feedback was solicited via anonymous survey (n=3) after the virtual sessions and debriefing with Kenyan research staff. Respondents strongly agreed that the virtual training was worth their time. Although PMs felt they were able to learn in the virtual format, facilitators noted more engagement during in-person sessions. Facilitators noted the importance of introductions and challenges of building cohesiveness for virtual sessions, particularly with sensitive content and internet limitations restricting video use (eg. low bandwidth, use of cell phones). Kenyan investigators noted that the Kenyan educational system is hierarchical, with students largely learning passively. They felt that this, combined with the newness of virtual learning and minimal dedicated time for introductions, may have hampered active virtual participation. While no PMs pointed to race or accent as limitations, facilitators noted differences between American and Kenyan English idioms, cadence, speed, and pronunciation that may have caused difficulty. Given Kenya's history of colonialism, all raised concerns that PMs may have been more deferential to light-skinned, foreign facilitators. Facilitators who observed both virtual and in-person sessions felt it was easier to break barriers of colonialism and assess for differences in spoken English in person. Facilitators felt that if virtual training were to be used in the future, it would be beneficial to have physical sessions first to set an interactive, educational tone and allow participants to build rapport. Conclusions: Virtual delivery of PM educator training in a LMIC setting is difficult and requires careful consideration or technological limitations and culture. Sources of Support: Indiana CTSI;Grant Number UL1RR025761-01.

94. Implementing Online Public Deliberations on Minor Consent for Biomedical HIV Prevention Research in Diverse Cities

Purpose: Adolescents/young adults (AYA) from racial/ethnic communities have high rates of HIV but little access to biomedical research, due to complexities around consent. Requirement of parental consent for participation in biomedical research is protective and strongly supported by parents, but in biomedical HIV prevention, minors are less likely to participate in research because of concerns about disclosure. Public deliberation (PD) is a process to obtain community input on complex policy issues, by bringing together AYA and adults, who have an investment in an issue, but with potentially opposing views, to provide education, clarify values, and facilitate discussion, reflection, and recommendations. To inform institutional review boards, institutions, and investigators, PDs were held with the goal of obtaining community perspectives and recommendations on minor consent for biomedical HIV prevention research from communities affected by youth HIV. Due to COVID-19 pandemic restrictions, we used an online format and conducted PDs across four evenings. We then conducted post-deliberation interviews to describe participants' experiences in the online PD. Methods: As part of an IRB approved PD, we conducted semi-structured interviews with youth and adult community members who had participated in the deliberations, held in Tampa and Baltimore. The interviews, which were conducted over Zoom, queried deliberants about their experiences voicing their perspectives, their comfort level, their degree of trust in the deliberation process, and ideas for how to better engage future deliberants. Interviews were audio-recorded, transcribed, and field notes were generated. Data were analyzed using thematic analysis. Results: We interviewed 13 community members: seven from Tampa (African American=3, White=3, Latinax=1;AYA=2) and six from Baltimore (African American=6;AYA=1). Facilitators: Deliberants from both communities indicated that personal connections were important for building consensus and understanding. When other participants shared personal stories and perspectives, deliberants were more receptive to hearing and accepting new ideas and opinions that differed from their own. Challenges: Tampa deliberants reported that they preferred an online deliberation because it helped overcome practical barriers to in-person deliberations, such as access to transportation and long commutes. Baltimore participants indicated they would have preferred in-person interactions to build trust, increase comfort, and augment engagement. Participants from both communities discussed distrust in research due to the historical legacy of racism in research and medicine. Due to this legacy, they reported that distrust influenced their views of minor-self-consent and impacted the deliberation process around building consensus. For example, concerns about coercion of minor human subjects influenced their views on minor consent. Recommendations: Participants recommend that strategies be developed to increase engagement in the virtual space. These strategies include use of (a) breakout sessions to increase comfort with sharing;(b) personal storytelling and reviewing group agreements to increase trust, (c) early polling activities to ensure engagement, (d) and asking adults to provide space for youth to voice their perspectives. Conclusions: While online public deliberation on sensitive topics with a vulnerable population is possible, it is important for researchers to focus on providing a safe environment, to acknowledge historical racism in research, and to use methods to maximally engage participants. Sources of Support: PCORI.

142. The Reach, Implementation, and Effectiveness of Virtual Sex Education for Youth in Foster Care

Purpose: Youth in foster care have high rates of adverse sexual health outcomes and are important targets for evidence-based sex education. With the COVID-19 pandemic, sexual health programming was moved to a virtual format. However, few data existed to guide this transition. While it lowers expenses and can potentially broaden geographic reach, it is unclear if virtual programming meets the needs of youth in foster care or other vulnerable populations. We conducted a mixed-methods analysis comparing the reach, implementation, and effectiveness of virtual vs in-person sex education for youth in foster care before and during the COVID-19 pandemic. Methods: Indiana Proud and Connected Teens (IN-PACT) provides evidenced-based sex education programs to system-involved youth. The data used in this study focused exclusively on foster-care programming and includes attendance records, facilitator session reviews (n=64) from 2020-2021 virtual programs, and youth surveys from 2018-2020 in-person (n=965) and virtual (n=50) programs. Reach was measured using youth baseline survey demographics and sexual behaviors;implementation by free responses from facilitators on challenges and adaptation for virtual teaching;and effectiveness by attendance records and youth behavior intention on follow-up surveys. Results: Reach: Youth demographic diversity was maintained for virtual programming in ethnicity, race, sex, and sexual orientation. However, youth in virtual programs had lower rates of self-reported risk behaviors including lower rates of involvement with juvenile justice (35.0% vs 59.4%, p<0.01) to have ever had sex (44.4% vs 78.8%, p<0.001) or contributed to a pregnancy (4.4% vs 23.4%, p<0.05). And though not statistically significant, virtual youth reported higher rates of condom use (44.4% vs 30.4%, p=0.371) and lower rates of substance use before sex in the past 3 months (15.6% vs 28.5%, p=0.114) as compared to in-person youth. Implementation: Technical challenges included connection difficulties and learning curves to using Zoom. Virtual facilitators incorporated more technology than they did in-person by playing videos on complicated topics such as conception and STIs. In terms of curriculum, hands-on condom demonstrations were changed to facilitator-run experiments such as having youth use socks at home to simulate condoms on their arms. Breakout rooms were utilized to maintain small group work but were cumbersome. Relational challenges included awkward silences, disengagement, and a decrease in group trust due to cameras being turned off during sensitive topics and less connection between youth and facilitators. Effectiveness: Attendance records show that fewer virtual youth completed 100% of programming, as compared to in-person youth (23% vs 54%). More virtual youth answered yes to the question "As a result of this program, will you abstain from sex for the next three months?” as compared to in-person youth (55% vs 45%, p=0.462). However, virtual youth were significantly less likely to have baseline sexual experience. Conclusions: In-person sexual health programming had a wider reach, experienced fewer implementation challenges, and was potentially more effective than virtual programming for youth in foster care. If virtual programming becomes necessary again, sex educators and researchers can use these data to redesign virtual programming that maximizes reach, implementation, and effectiveness. Sources of Support: HHS 90AK0041-02-00 to Health Care Education and Training Inc.

Effect of Covid-19 Diagnosis on Organ Donation and Transplantation

Purpose: The coronavirus disease 2019 (COVID-19) pandemic has challenged many aspects of healthcare, including organ donation and transplantation. The purpose of this study is to demonstrate that utilization of COVID-positive organs can be carried out safely. Method(s): De-identified data from 569 organ donors processed through an organ procurement organization (OPO) from March 24, 2020, through September 30, 2021, was collected from the OPO's database and retrospectively analyzed. Demographics, clinical measures, transplant numbers, and outcomes were recorded. Result(s): 25 COVID-positive (study) and 544 COVID-negative (control) organ donors were analyzed. There was no significant difference between the mean ages of the study group (43.12+/-11.08, p = 0.665) and the control group (44.15+/-17.94, p = 0.665). The COVID-positive group achieved donor management goals at a significantly lower rate than the COVID-negative group (4.0% vs 48.7%, p = 0.000012). The COVID-positive group required significantly more continuous renal replacement therapy (16.0% vs 1.8%, p < 0.00001), and extracorporeal membrane oxygenation (24.0% vs 0.7%, p < 0.00001). Significantly fewer organs were transplanted from the COVID-positive donors (1.12+/-1.013, p < 0.00001) than from the COVIDnegative donors (2.56+/-1.671, p < 0.00001). The mean observed to expected ratio for the study group (0.5372+/-0.47434, p < 0.00001) was significantly lower than that of the control group (0.9489+/-0.55041, p < 0.00001). The study group donors were significantly more likely to be categorized as donation after circulatory death (DCD) donors (96.0% vs 27.8%, p < 0.00001). There was no significant difference between the groups regarding delayed graft function in the recipient (18.2% vs 26.8%, p = 0.522561) nor regarding the need for dialysis post-transplant (9.1% vs 11.6%, p = 0.795292). Conclusion(s): Fewer organs from COVID-positive donors were utilized for transplantation than organs from COVID-negative donors over the study period. COVID-positive organs have been of no detriment to recipients, as there is no evidence of increased delayed graft function nor the need for dialysis. Though no short-term COVID-19 transmission has been identified, we will continue to monitor for this and to track non-renal transplant outcomes. A larger multi-center study is warranted to further delineate the safety and efficacy of implementing protocols to utilize these organs.

Persistent Changes in the Gut Microbiome of Covid-19 Survivors

SARS-CoV-2 is detectable in gastrointestinal secretions and has been associated with changes to the gut microbiome during severe infection. However, the majority of individuals with COVID-19 will develop mild infection and remain outpatient for the duration of infection, with a relative paucity of information on the gut microbiome of this group. Furthermore, symptoms can persist for 6 months or longer in some patients, which has presented additional health care burdens given incomplete understanding of the long term impact of SARS-CoV-2 infection. To fill these knowledge gaps, we longitudinally sampled the gut microbial community of subjects infected with SARS-CoV-2 and their household contacts living in San Francisco at varying lengths of time after infection. We report an association between COVID-19 and the gut microbiome. COVID-19 positive subjects exhibited greater variability in the gut microbiome over time. In concordance with this finding, COVID-19 positive gut microbial communities were more self-distinct when compared to COVID-19 negative individuals across sample collections. Population level social distancing practices varied during the time of sample collection in our cohort, and we found an association between population level movement and gut microbial community variation. To better define the impacts of SARS-Cov-2 independent of genetic and environmental variables, are utilizing five SARS-CoV-2 variants (Alpha, Beta, Washington, Delta, and Omicron) in separate mouse models to test the impact of SARSCoV- 2 on the gut microbiome in severe and mild infections to define the impacts of SARS-CoV-2 on gut microbial ecology independent of genetic and environmental variables. We conclude that even mild cases of COVID-19 result in months-long disruption in the gut microbial community, with additional perturbations linked to massive shifts in population level social distancing practices.

NEUROPILIN-1 MEDIATES SARS-CoV-2 INFECTION of ASTROCYTES PROMOTING NEURON DYSFUNCTION

Background: SARS-CoV-2 primarily infects the lung but may also damage other organs including the brain, heart, kidney, and intestine. Central nervous system (CNS) disorders include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Methods: We used iPSC-derived brain organoids and primary human astrocytes from cerebral cortex to study direct SARS-CoV-2 infection, as confirmed by Spike and Nucleocapsid immunostaining and RT-qPCR. siRNAs, blocking antibodies, and small molecule inhibitors were used to assess SARS-CoV-2 receptor candidates. Bulk RNA-seq, DNA methylation seq, and Nanostring GeoMx digital spatial profiling were utilized to identify virus-induced changes in host gene expression. Results: Astrocytes were robustly infected by SARS-CoV-2 in brain organoids while neurons and neuroprogenitor cells supported only low-level infection. Based on siRNA knockdowns, Neuropilin-1, not ACE2, functioned as the primary receptor for SARS-CoV-2 in astrocytes. The endolysosomal two-pore channel protein, TPC, also facilitated infection likely through its regulatory effects on endocytosis. Other alternative receptors, including the AXL tyrosine kinase, CD147, and dipeptidyl protease 4 (DPP4), did not function as SARS-CoV-2 receptors in astrocytes. SARS-CoV-2 infection dynamically induced type I, II, and III interferons, and genes involved in Toll-like receptor signaling, MDA5 and RIG-I sensing of double-stranded RNA, and production of inflammatory cytokines. Genes activating apoptosis were also increased. Down-regulated genes included those involved in water, ion and lipid transport, synaptic transmission, and formation of cell junctions. Epigenetic analyses revealed transcriptional changes related to DNA methylation states, particularly decreased DNA methylation in interferon-related genes. Long-term viral infection of brain organoids resulted in progressive neuronal degeneration and death. Conclusion: Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. Deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

A Critical Discourse Analysis to Prevent the Second Wave of COVID-19 Pandemic in Indonesia

The government's choice of complicated language style in communicating issues or cases of Covid-19 risks bringing misunderstandings among the public. The choice of terms which are very elitist will only target certain circles. The government's communication strategy during this pandemic certainly creates new problems. One of the impacts arising from the government's elitist communication approach is the emergence of an information gap between the upper middle class and lower middle class which has a systemic impact, for example the panic buying phenomenon ahead of the implementation of the Social Restrictions Large-Scale (PSBB). In linguistics, an information (discourse) can be framed with topicalization techniques. This technique is a strategy for promoting information that will be highlighted. The part of the information that has a higher negative meaning burden tends to be not highlighted. The issue of the increase in positive cases of Covid-19 does not seem too prominent. © 2021. All Rights Reserved.

Thromboelastography utilization for dabigatran reversal in a patient with acute kidney injury.

PURPOSE: This case report describes utilization of thromboelastography (TEG) in the setting of an acute major bleed in a patient on dabigatran who had concomitant acute kidney injury. SUMMARY: An 80-year-old female presented to the emergency department after a fall with complaints of pain in her knee, shoulder, and hip. Her medical history was significant for coronary artery disease, for which she took clopidogrel 75 mg daily, and atrial fibrillation, for which she took dabigatran 150 mg twice daily. The physical exam was remarkable for pain within the shoulder, hip, and knee, which had swelling and ecchymosis that extended into the right thigh. Given the possibility of compartment syndrome with multiple possible etiologies of coagulopathy, TEG and computed tomography angiography (CTa) of the right lower extremity were performed. The initial TEG showed prolonged R time and activated clotting time, indicating clotting factor dysfunction with no additional coagulopathy noted, including antiplatelet effects. On the basis of the TEG and CTa findings, it was decided to reverse dabigatran with 5 grams of idarucizumab. Approximately 1 hour after administration of idarucizumab, the patient was taken to interventional radiology where a limited angiogram of the right lower extremity showed no active extravasation. Because of the patient's renal dysfunction and the possibility of rebound hypercoaguability, repeat TEG tests were ordered at 4 and 8 hours after the initial reversal to ensure clearance of idarucizumab-dabigatran complexes. The repeat TEG values showed complete reversal of the initial coagulopathy noted. During the admission, the patient required no blood transfusions or surgical interventions and all her initial laboratory results improved. CONCLUSION: Serial TEG testing was successful at managing multiple coagulopathies in a patient at risk for trauma-induced compartment syndrome.