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Background The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference. Methods We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage. Results Consensus sequences from households had limited diversity (mean pairwise distance, 3.06 single-nucleotide polymorphisms [SNPs]; range, 0–40). Most (83.1%, 255 of 307) samples harbored at least 1 intrahost single-nucleotide variant ([iSNV] median, 117; interquartile range [IQR], 17–208), above a minor allele frequency threshold of 0.2%. Pairs in the same household shared significantly more iSNVs (mean, 1.20 iSNVs; 95% confidence interval [CI], 1.02–1.39) than did pairs in different households infected with the same viral clade (mean, 0.31 iSNVs; 95% CI, .28–.34), a signal that decreases with increasingly stringent minor allele frequency thresholds. The number of shared iSNVs was significantly associated with an increased odds of household membership (adjusted odds ratio, 1.35; 95% CI, 1.23–1.49). However, the poor concordance of iSNVs detected across sequencing replicates (24.8% and 35.0% above a 0.2% and 1% threshold) confirms technical concerns that current sequencing and bioinformatic workflows do not consistently recover low-frequency within-host variants. Conclusions Shared within-host variation may augment the information in consensus sequences for predicting transmission linkages. Improving sensitivity and specificity of within-host variant identification will improve the informativeness of within-host variation.

Background As wild poliovirus is eradicated, preventing circulation of vaccine-derived poliovirus is top priority. Our lab developed real-time multiplex PCR assays and deep sequencing methodology to detect and characterize OPV strains from stool samples. The method requires gel purification of PCR product created from viral RNA in stool samples. However, the process filters out a significant portion of samples. Here, we compare gel- vs. DNA- purification and sample retainment for downstream analysis. Methods 554 stool samples qPCR positive for at least one OPV serotype from a previous study were used. There are 268 serotype 1 (S1) isolates, 405 serotype 2 (S2) isolates, and 318 serotype 3 (S3) isolates. PCR amplicons created from viral RNA ran through a 0.08% agarose gel to identity presence of the ∼3.5kb amplicon of interest. PCR amplicons then underwent either a gel-purification spin column kit or a DNA purification spin column kit. Samples with DNA concentration >10ng/uL in the elution product is required for NGS. Purification Methods Flowchart Graph 1:Gel-purification, DNA-purification, and suggested purification workflow charts Results Comparing 168 S2 samples gel vs. DNA-purified, of 90 bands identified with both protocols, 100% of DNA-purified samples and 35.6% of the gel-purified samples had DNA concentration >10ng/ul. 41.3% of banded samples with < =34CT had >10ng/ul for gel-purified samples vs. 100% for DNA-purified samples. Of 43 pediatric participants, prior OPV and IPV did not impact proportion of number of individuals with viable samples for NGS (16/31 (51.6%) vs 25/43 (58.1%) respectively). Among samples processed via gel purification, the median number of samples per positive participant dropped from 2 (IQR=1-3) to 1 (IQR=1-1). From the same set of samples, gel purification reduced the number of OPV vaccinated children 38 (82.6%), their household contacts 2 (4.3%), and community contacts 6 (13%) to 21 (80.7%) OPV vaccinated children, 1 (3.8%) household contacts, and 4 (15.3%) community contacts. Table 1:Purification outcome of 168 S2 unique-participant-day (UPD) samples via gel-purification and DNA-purificationGraph 2:Demographic information of patient sample loss form the gel-purification process on 168 S2 samples via gel-purification workflow 2a. Sample collection across different days for 90 unique-participant-day (UPD) samples pre gel-purification vs 32 UPD samples post gel-purification. 2b. OPV and IPV does spread per each 43 pediatric unique-participant (UP) from the 90 UPD samples pre gel-purification vs 25 pediatric UP from the 32 UPD post gel-purification. 2c. Vaccination status (OPV vaccinated child, household contact, unvaccinated community contact) of each 43 unique-participant (UP) from the 90UPD samples pre gel-purification vs 25 UP from the 32 UPD post gel-purification. 2d. Number of samples collected per UP fron the 90 UPD samples pre gel-purification vs 32 UPD samples post gel-purification. Graph 3:Demographic information of patient sample loss form the gel-purification process on 168 S2 samples via suggested purification workflow 2a. Sample collection across different days for 75 unique-participant-day (UPD) samples pre gel-purification vs 31 UPD samples post gel-purification. 2b. OPV and IPV does spread per each 38 pediatric unique-participant (UP) from the 75 UPD samples pre gel-purification vs 24 pediatric UP from the 31 UPD post gel-purification. 2c. Vaccination status (OPV vaccinated child, household contact, unvaccinated community contact) of each 41 unique-participant (UP) from the 75UPD samples pre gel-purification vs 25 UP from the 31 UPD post gel-purification. 2d. Number of samples collected per UP fron the 75 UPD samples pre gel-purification vs 31 UPD samples post gel-purification. Conclusion Conclusions: Gel-purification causes a reduction in sample numbers and variation, causing an incomplete data set and data misrepresentation for downstream analysis, particularly a reduction of samples for within host variation analysis. DNA purification of banded samples with < =34CT may be most optimal for NGS. Analysis of all isolates are in process. Disclosures Yvonne A. Maldonado, MD, Pfizer: Grant/Research Support|Pfizer: Member, DSMB, Pfizer Meningococcal Vaccine clinical trial.

Background Serological tests directed against SARS-CoV-2 can provide information about the timing of infection and immunity against the virus. However, the kinetics of the host immune response to SARS-CoV-2 remain poorly understood. We established a household transmission study to analyze the serological responses within households, to determine longitudinal immune responses to infection. Methods From April 2020 to April 2022, we prospectively enrolled 76 households with at least one RT-PCR confirmed case of COVID-19. Participants were asked to provide blood samples at three time points: at baseline within 2 weeks of the index’s diagnosis of COVID-19, and at one- and three-months post-enrollment. Samples were tested for the presence of IgG antibodies against SARS-CoV-2 spike protein via an FDA EUA approved ELISA. Demographics, medical history, and symptomatology were also collected. Results To date, we have analyzed 238 serologic samples from 135 participants, including 82 baseline samples, 89 one-month samples, and 67 three-month samples. At baseline, 67.8% (n=40/59) of all confirmed cases tested positive for SARS-CoV-2 antibodies, which increased to 86.4% (n=57/66) at the one month, and 85.1% at three months (n=40/47). Of those confirmed infected participants that failed to seroconvert at baseline, almost all reported symptoms (n=14/19, 73.7%) and did not have chronic medical conditions (n=17/19, 89.5%). Of the 19, 3 failed to seroconvert by their third visit. All individuals who were fully vaccinated at the time of each visit tested positive for antibodies at baseline (n=26), one-month (n=27), and three-months (n=20). Of those who were not fully vaccinated, 56 (41.1%) were positive for antibodies at baseline, 62 (59.7%) were positive at one -month, and 47 (63.8%) at three-months. Differences in seropositivity rates between pediatric and adult participants, as well as between index cases and household contacts, at each visit were also identified (Table 1). Conclusion Identifying differences in seroprevalence in various demographic groups can provide insight into longitudinal immune responses post-infection. Future analyses on seropositivity among previously infected individuals who received therapeutics may be of interest. Disclosures Andra L. Blomkalns, MD, MBA, Eli Lilly and Company: Grant/Research Support Yvonne A. Maldonado, MD, Pfizer: Grant/Research Support|Pfizer: Member, DSMB, Pfizer Meningococcal Vaccine clinical trial.

BackgroundThe limited variation observed among SARS-CoV-2 consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference.MethodsWe performed tiled amplicon sequencing on 307 SARS-CoV-2 samples from four prospective studies and combined sequence data with household membership data, a proxy for transmission linkage.ResultsConsensus sequences from households had limited diversity (mean pairwise distance, 3.06 SNPs; range, 0-40). Most (83.1%, 255/307) samples harbored at least one intrahost single nucleotide variant (iSNV; median: 117; IQR: 17-208), when applying a liberal minor allele frequency of 0.5% and prior to filtering. A mean of 15.4% of within-host iSNVs were recovered one day later. Pairs in the same household shared significantly more iSNVs (mean: 1.20 iSNVs; 95% CI: 1.02-1.39) than did pairs in different households infected with the same viral clade (mean: 0.31 iSNVs; 95% CI: 0.28-0.34), a signal that increases with increasingly liberal thresholds.ConclusionsAlthough only a subset of within-host variation is consistently shared across likely transmission pairs, shared iSNVs may augment the information in consensus sequences for predicting transmission linkages.

Bronchopulmonary dysplasia (BPD) is a disease prevalent in preterm babies with a need for supplemental oxygen, resulting in impaired lung development and dysregulated vascularization. Epidemiologic studies have shown that males are more prone to BPD and have a delayed recovery compared with females, for reasons unknown. Herein, we tried to recapitulate mild, moderate, and severe BPD, using two different strains of mice, in males and females: CD1 (outbred) and C57BL/6 (inbred). Aside from higher body weight in the CD1 strain, there were no other gross morphologic differences with respect to alveolar development between the two strains. With respect to lung morphology after oxygen exposure, females had less injury with better preservation of alveolar chord length and decreased alveolar protein leak and inflammatory cells in the bronchoalveolar lavage fluid. In addition, housekeeping genes, which are routinely used as loading controls, were expressed differently in males and females. In the BPD mouse model, gonadotropin-releasing hormone was increased in females compared with males. Specific miRNAs (miR-146 and miR-34a) were expressed differently in the sexes. In the severe BPD mouse model, administering miR-146 mimic to males attenuated lung damage, whereas administering miR-146 inhibitor to females increased pulmonary injury.

Background Currently, the primary mechanism for poliovirus detection is acute flaccid paralysis (AFP) surveillance, with environmental sampling serving as a complement. However, as AFP cases drop, environmental surveillance will become increasingly critical for poliovirus detection. Mexico provides a natural environment to study oral polio vaccine (OPV) transmission, as it provides routine injected polio vaccine immunization and biannual OPV campaigns in February and May. Methods As part of a study of OPV transmission in which 155 children were vaccinated with OPV, monthly sewage samples were collected from rivers leading from 3 indigenous Mexican villages (Capoluca, Campo Grande, and Tuxpanguillo) from February to May 2015. Samples were also collected from October 2015 to October 2017, during which time there were standard OPV campaigns. Samples were analyzed for the presence of OPV serotypes, using a real-time qualitative polymerase chain reaction assay capable of detecting as few as 9, 12, and 10 copies/100 µL of viral ribonucleic acid for OPV serotypes 1, 2, and 3 (OPV-1, -2, and -3), respectively. Included here are 54 samples, taken up to November 2016. Results Of the 54 samples, 13 (24%) were positive for OPV. After the vaccination of 155 children in February 2015, OPV was found 2 months after vaccination. After unrestricted OPV administration in February 2016, OPV was detected in sewage up to 8 months after vaccination. OPV-3 was found in 11 of the 13 positive samples (85%), OPV-2 was found in 3 positive samples (23%), and OPV-1 was found in 1 sample (8%). Conclusions OPV can be detected even when small amounts of the vaccine are introduced into a community, as shown by OPV-positive sewage samples even when only 155 children were vaccinated. When OPV vaccination was unrestricted, sewage samples were positive up to 8 months after vaccination, implying community OPV circulation for at least 8 months. OPV-3 was the serotype most found in these samples, indicating prolonged transmission of OPV-3 when compared to the other serotypes. Future work could compare the phylogenetic variance of OPV isolates from sewage after OPV vaccinations.

Background Global polio eradication efforts rely in part on molecular methods of detecting polioviruses, both wild and vaccine strains, from human and environmental samples. Previous assays used for detection of Sabin oral polio vaccine (OPV) in fecal samples have been labor and time intensive and vary in their sensitivity and specificity. Methods We developed a high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay able to detect all 3 OPV strains in fecal samples. The assay used a KingFisher Duo Prime system for viral RNA isolation and extraction. Positive samples were retested and Sanger sequenced for verification of Sabin serotype identity. Results The 95% lower limit of detection was determined to be 3 copies per reaction for Sabin 1 and 3 and 4 copies per reaction for Sabin 2, with no cross-reactivity between the 3 serotypes and their primers. A total of 554 samples (3.6%) were positive, with 304 positive samples (54.9%) containing >1 serotype. Of the positive samples, 476 (85.9%) contained enough RNA to be sequenced, and of these all sequences were Sabin serotypes. The previous assay we used could process 48 samples in a 10-hour period, whereas the new assay processed >100 samples in 6 hours. Conclusions The new high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay allowed for sensitive and specific detection of OPV serotypes while greatly decreasing sample handling and processing time. We were able to sequence 72.4% of the 210 positive samples in the cycle threshold range of 35–37.

Background The World Health Assembly 2012 Polio Eradication and Endgame Strategic Plan calls for the eventual cessation of all oral polio vaccines (OPVs), to be replaced with inactivated polio vaccine (IPV); however, IPV induces less robust mucosal immunity than OPV. This study characterized household and community OPV shedding and transmission after OPV vaccination within primarily IPV-vaccinated communities. Methods Households in 3 IPV-vaccinated Mexican communities were randomized to receive 3 levels of OPV vaccination coverage (70%, 30%, or 10%). Ten stool samples were collected from all household members over 71 days. Analysis compared vaccinated subjects, household contacts of vaccinated subjects, and subjects in unvaccinated households. Logistic and Cox regression models were fitted to characterize transmission of OPV by coverage and household vaccination status. Results Among 148 vaccinated children, 380 household contacts, and 1124 unvaccinated community contacts, 78%, 18%, and 7%, respectively, shed OPV. Community and household contacts showed no differences in transmission (odds ratio [OR], 0.67; 95% confidence interval [CI], .37–1.20), in shedding trajectory (OR, 0.61; 95% CI, .35–1.07), or in time to shedding (hazard ratio, 0.68; 95% CI, .39–1.19). Transmission began as quickly as 1 day after vaccination and persisted as long as 71 days after vaccination. Transmission within unvaccinated households differed significantly across vaccination coverage communities, with the 70% community experiencing the most transmissions (15%), and the 10% community experiencing the least (4%). These trends persisted over time and in the time to first shedding analyses. Conclusions Transmission did not differ between household contacts of vaccinees and unvaccinated households. Understanding poliovirus transmission dynamics is important for postcertification control.

Bronchopulmonary dysplasia (BPD) is a multifactorial disease of preterm infants characterized by alveolar simplification, dysregulated pulmonary vasculature, and pulmonary hypertension. Invasive mechanical ventilation, hyperoxia, infection, and various other contributing factors acting on the immature lung result in permanent damage to the pulmonary architecture. Animal models have played a critical role in our understanding of the pathogenesis behind this very complex disease, and this chapter will attempt to describe the uses, advantages, and limitations of the mouse, rat, guinea pig, rabbit, lamb, baboon, and rhesus monkey models of BPD.

Background As wild poliovirus is nearing global eradication and countries switch from Oral Polio Vaccine (OPV) to Inactivated Polio Vaccine (IPV), preventing circulating vaccine-derived poliovirus is a top priority. However, the circulation of OPV serotypes remains a concern in undervaccinated communities. We sought to examine the relationship between pediatric nutritional status and OPV shedding based on length-for-age categorizations. Mexico provides a natural environment to study these patterns as it provides routine IPV immunization and bi-annual OPV campaigns. Methods We enrolled 466 households with children eligible for OPV before the February 2015 national health week from 3 semi-rural Indigenous communities near Orizaba, Mexico. In each community, a different proportion of eligible children received OPV (10%, 30%, 70%), with a total of 155 vaccinated children. OPV shedding was measured by RT-qPCR detection of OPV in samples collected serially over 10 weeks. Anthropometric measurements were collected and compared with the WHO Multicenter Growth Reference Study growth curves to assign stunting. Associations between stunting, OPV shedding, and shedding duration were tested by Fisher exact test and Wilcoxon-Man-Whitney Test (α = 0.05). Results Samples of fecal OPV isolates were collected over time and analyzed from 148 vaccinees. 25 (17%) of the vaccinees were stunted. There was no relationship between pediatric stunting and likelihood of ever shedding any serotype of OPV (P = 0.82). The mean duration of OPV shedding by stunted and non-stunted children differed, but not significantly (10.9 days vs. 9.3 days, respectively, P = 0.32). We did not find any statistically significant differences between stunting status and shedding of any individual OPV serotype. Conclusion Further understanding of factors related to OPV shedding is necessary to approach efficient worldwide poliovirus control. We found no relationship between stunting status and both OPV shedding and shedding duration post-vaccination, suggesting that nutritional status does not play a role in OPV shedding. The ongoing analysis includes longitudinal analysis of OPV shedding patterns by nutritional status, and the impact of stunting on viral load and reversion of OPV to vaccine-associated paralytic polio mutants. Disclosures All authors: No reported disclosures.