The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis.

Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein µ1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was nonlethal in wild-type mice, whereas more than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, as well as extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 than T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a nonlethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis. IMPORTANCE Reovirus is a nonenveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the nonlethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.

Lymphatic Type 1 Interferon Responses Are Critical for Control of Systemic Reovirus Dissemination.

Mammalian orthoreovirus (reovirus) spreads from the site of infection to every organ system in the body via the blood. However, mechanisms that underlie reovirus hematogenous spread remain undefined. Nonstructural protein σ1s is a critical determinant of reovirus bloodstream dissemination that is required for efficient viral replication in many types of cultured cells. Here, we used the specificity of the σ1s protein for promoting hematogenous spread as a platform to uncover a role for lymphatic type 1 interferon (IFN-1) responses in limiting reovirus systemic dissemination. We found that replication of a σ1s-deficient reovirus was restored to wild-type levels in cells with defective interferon-α receptor (IFNAR1) signaling. Reovirus spreads systemically following oral inoculation of neonatal mice, whereas the σ1s-null virus remains localized to the intestine. We found that σ1s enables reovirus spread in the presence of a functional IFN-1 response, as the σ1s-deficient reovirus disseminated comparably to wild-type virus in IFNAR1-/- mice. Lymphatics are hypothesized to mediate reovirus spread from the intestine to the bloodstream. IFNAR1 deletion from cells expressing lymphatic vessel endothelium receptor 1 (LYVE-1), a marker for lymphatic endothelial cells, enabled the σ1s-deficient reovirus to disseminate systemically. Together, our findings indicate that IFN-1 responses in lymphatics limit reovirus dissemination. Our data further suggest that the lymphatics are an important conduit for reovirus hematogenous spread.IMPORTANCE Type 1 interferons (IFN-1) are critical host responses to viral infection. However, the contribution of IFN-1 responses to control of viruses in specific cell and tissue types is not fully defined. Here, we identify IFN-1 responses in lymphatics as important for limiting reovirus dissemination. We found that nonstructural protein σ1s enhances reovirus resistance to IFN-1 responses, as a reovirus mutant lacking σ1s was more sensitive to IFN-1 than wild-type virus. In neonatal mice, σ1s is required for reovirus systemic spread. We used tissue-specific IFNAR1 deletion in combination with the IFN-1-sensitive σ1s-null reovirus as a tool to test how IFN-1 responses in lymphatics affect reovirus systemic spread. Deletion of IFNAR1 in lymphatic cells using Cre-lox technology enabled dissemination of the IFN-1-sensitive σ1s-deficient reovirus. Together, our results indicate that IFN-1 responses in lymphatics are critical for controlling reovirus systemic spread.

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression.

Reovirus nonstructural protein σ1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of σ1s during the reovirus replication cycle is not known. In this study, we found that σ1s was required for efficient reovirus replication in simian virus 40 (SV40)-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild-type reovirus produced substantially higher viral titers than a σ1s-deficient mutant. The σ1s protein was not required for early events in reovirus infection, as evidenced by the fact that no difference in infectivity between the wild-type and σ1s-null viruses was observed. However, the wild-type virus produced markedly higher viral protein levels than the σ1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the σ1s protein was dispensable for cell killing and the induction of type I interferon responses. In the absence of σ1s, viral factory (VF) maturation was impaired but sufficient to support low levels of reovirus replication. Together, our results indicate that σ1s is not absolutely essential for viral protein production but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that σ1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.IMPORTANCE Hematogenous dissemination is a critical step in the pathogenesis of many viruses. For reovirus, nonstructural protein σ1s is required for viral spread via the blood. However, the mechanism by which σ1s promotes reovirus dissemination is unknown. In this study, we identified σ1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which σ1s is required for efficient reovirus replication. In these cells, wild-type virus produced substantially higher levels of viral protein than a σ1s-deficient mutant. The σ1s protein was not required for viral mRNA transcription or viral protein stability. Since reduced levels of viral protein were synthesized in the absence of σ1s, the maturation of viral factories was impaired, and significantly fewer viral progeny were produced. Taken together, our findings indicate that σ1s is required for optimal reovirus protein production, and thereby viral replication, in cells required for hematogenous reovirus dissemination.

Differential Delivery of Genomic Double-Stranded RNA Causes Reovirus Strain-Specific Differences in Interferon Regulatory Factor 3 Activation.

Serotype 3 (T3) reoviruses induce substantially more type 1 interferon (IFN-I) secretion than serotype 1 (T1) strains. However, the mechanisms underlying differences in IFN-I production between T1 and T3 reoviruses remain undefined. Here, we found that differences in IFN-I production between T1 and T3 reoviruses correlate with activation of interferon regulatory factor 3 (IRF3), a key transcription factor for the production of IFN-I. T3 strain rsT3D activated IRF3 more rapidly and to a greater extent than the T1 strain rsT1L, in simian virus 40 (SV40) immortalized endothelial cells (SVECs). Differences in IRF3 activation between rsT1L and rsT3D were observed in the first hours of infection and were independent of de novo viral RNA and protein synthesis. NF-κB activation mirrored IRF3 activation, with rsT3D inducing more NF-κB activity than rsT1L. We also found that IRF3 and NF-κB are activated in a mitochondrial antiviral-signaling protein (MAVS)-dependent manner. rsT1L does not suppress IRF3 activation, as IRF3 phosphorylation could be induced in rsT1L-infected cells. Transfected rsT1L and rsT3D RNA induced IRF3 phosphorylation, indicating that genomic RNA from both strains has the capacity to activate IRF3. Finally, bypassing the normal route of reovirus entry by transfecting in vitro-generated viral cores revealed that rsT1L and rsT3D core particles induced equivalent IRF3 activation. Taken together, our findings indicate that entry-related events that occur after outer capsid disassembly, but prior to deposition of viral cores into the cytoplasm, influence the efficiency of IFN-I responses to reovirus. This work provides further insight into mechanisms by which nonenveloped viruses activate innate immune responses.IMPORTANCE Detection of viral nucleic acids by the host cell triggers type 1 interferon (IFN-I) responses, which are critical for containing and clearing viral infections. Viral RNA is sensed in the cytoplasm by cellular receptors that initiate signaling pathways, leading to the activation of interferon regulatory factor 3 (IRF3) and NF-κB, key transcription factors required for IFN-I induction. Serotype 3 (T3) reoviruses induce significantly more IFN-I than serotype 1 (T1) strains. In this work, we found that differences in IFN-I production by T1 and T3 reoviruses correlate with differential IRF3 activation. Differences in IRF3 activation are not caused by a blockade of the IRF3 activation by a T1 strain. Rather, differences in events during the late stages of viral entry determine the capacity of reovirus to activate host IFN-I responses. Together, our work provides insight into mechanisms of IFN-I induction by nonenveloped viruses.

Endothelial JAM-A promotes reovirus viremia and bloodstream dissemination.

Viruses that cause systemic disease often spread through the bloodstream to infect target tissues. Although viremia is an important step in the pathogenesis of many viruses, how viremia is established is not well understood. Reovirus has been used to dissect mechanisms of viral pathogenesis and is being evaluated in clinical trials as an oncolytic agent. After peroral entry into mice, reovirus replicates within the gastrointestinal tract and disseminates systemically via hematogenous or neural routes. Junctional adhesion molecule-A (JAM-A) is a tight junction protein that serves as a receptor for reovirus. JAM-A is required for establishment of viremia and viral spread to sites of secondary replication. JAM-A also is expressed on the surface of circulating hematopoietic cells. To determine contributions of endothelial and hematopoietic JAM-A to reovirus dissemination and pathogenesis, we generated strains of mice with altered JAM-A expression in these cell types and assessed bloodstream spread of reovirus strain type 1 Lang (T1L), which disseminates solely by hematogenous routes. We found that endothelial JAM-A but not hematopoietic JAM-A facilitates reovirus T1L bloodstream entry and egress. Understanding how viruses establish viremia may aid in development of inhibitors of this critical step in viral pathogenesis and foster engineering of improved oncolytic viral vectors.

An ITAM in a nonenveloped virus regulates activation of NF-κB, induction of beta interferon, and viral spread.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: µ2, σ2, and λ2. We demonstrate for the first time that µ2 is phosphorylated, contains a functional ITAM, and activates NF-κB. Specifically, µ2 and µNS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the µ2 ITAM. Moreover, both the µ2 ITAM and Syk are required for maximal µ2 activation of NF-κB. A mutant virus lacking the µ2 ITAM activates NF-κB less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-ß) than does wild-type virus despite similar replication. Notably, the consequences of these µ2 ITAM effects are cell type specific. In fibroblasts where NF-κB is required for reovirus-induced apoptosis, the µ2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the µ2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the µ2 ITAM on viral spread reflects the cell type-specific effects of NF-κB and IFN-ß. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.

Nonstructural protein σ1s mediates reovirus-induced cell cycle arrest and apoptosis.

Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.

Genetic determinants of reovirus pathogenesis in a murine model of respiratory infection.

Many viruses invade mucosal surfaces to establish infection in the host. Some viruses are restricted to mucosal surfaces, whereas others disseminate to sites of secondary replication. Studies of strain-specific differences in reovirus mucosal infection and systemic dissemination have enhanced an understanding of viral determinants and molecular mechanisms that regulate viral pathogenesis. After peroral inoculation, reovirus strain type 1 Lang replicates to high titers in the intestine and spreads systemically, whereas strain type 3 Dearing (T3D) does not. These differences segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. In this study, we define genetic determinants that regulate reovirus-induced pathology following intranasal inoculation and respiratory infection. We report that two laboratory isolates of T3D, T3D(C) and T3D(F), differ in the capacity to replicate in the respiratory tract and spread systemically; the T3D(C) isolate replicates to higher titers in the lungs and disseminates, while T3D(F) does not. Two nucleotide polymorphisms in the S1 gene influence these differences, and both S1 gene products are involved. T3D(C) amino acid polymorphisms in the tail and head domains of σ1 protein influence the sensitivity of virions to protease-mediated loss of infectivity. The T3D(C) polymorphism at nucleotide 77, which leads to coding changes in both S1 gene products, promotes systemic dissemination from the respiratory tract. A σ1s-null virus produces lower titers in the lung after intranasal inoculation and disseminates less efficiently to sites of secondary replication. These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung.

Comparative analysis of Reoviridae reverse genetics methods.

Effective methods to engineer the segmented, double-stranded RNA genomes of Reoviridae viruses have only recently been developed. Mammalian orthoreoviruses (MRV) and bluetongue virus (BTV) can be recovered from entirely recombinant reagents, significantly improving the capacity to study the replication, pathogenesis, and transmission of these viruses. Conversely, rotaviruses (RVs), which are the major etiological agent of severe gastroenteritis in infants and children, have thus far only been modified using single-segment replacement methods. Reoviridae reverse genetics techniques universally rely on site-specific initiation of transcription by T7 RNA polymerase to generate the authentic 5' end of recombinant RNA segments, but they vary in how the RNAs are introduced into cells: recombinant BTV is recovered by transfection of in vitro transcribed RNAs, whereas recombinant MRV and RV RNAs are transcribed intracellularly from transfected plasmid cDNAs. Additionally, several parameters have been identified in each system that are essential for recombinant virus recovery. Generating recombinant BTV requires the use of 5' capped RNAs and is enhanced by multiple rounds of RNA transfection, suggesting that translation of viral proteins is likely the rate-limiting step. For RV, the efficiency of recovery is almost entirely dependent on the strength of the selection mechanism used to isolate the single-segment recombinant RV from the unmodified helper virus. The reverse genetics methods for BTV and RV are presented and compared to the previously described MRV methods. Analysis and comparison of each method suggest several key lines of research that might lead to a reverse genetics system for RV, analogous to those used for MRV and BTV.

Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus.

Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms. Recovery is rapid and simple; after two rounds of selective passage the recombinant virus reaches titers of ≥10(4) pfu/mL. We used this reverse genetics method to generate a panel of viruses with chimeric NSP2 genes. For one of the chimeric viruses, the introduced NSP2 sequence was obtained from a pathogenic, noncultivated human RV isolate, demonstrating that this reverse genetics system can be used to study the molecular biology of circulating RVs. Combining characterized RV ts mutants and validated siRNA targets should permit the extension of this "two-hit" reverse genetics methodology to other RV genes. Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery.

A longitudinal study of SARS-CoV-2 antibody seroprevalence and mitigation behaviors among college students at an Arkansas University.

Objective: Assess university students' SARS-CoV-2 antibody seroprevalence and mitigation behaviors over time. Participants: Randomly selected college students (N = 344) in a predominantly rural Southern state. Methods: Participants provided blood samples and completed self-administered questionnaires at three timepoints over the academic year. Adjusted odds ratios and 95% confidence intervals were estimated from logistic regression analyses. Results: SARS-CoV-2 antibody seroprevalence was 18.2% in September 2020, 13.1% in December, and 45.5% in March 2021 (21% for those with no vaccination history). SARS-CoV-2 antibody seroprevalence was associated with large social gatherings, staying local during the summer break, symptoms of fatigue or rhinitis, Greek affiliation, attending Greek events, employment, and using social media as the primary COVID-19 information source. In March 2021, seroprevalence was associated with receiving at least one dose of a COVID-19 vaccination. Conclusion: SARS-CoV-2 seroprevalence was higher in this population of college students than previous studies. Results can assist leaders in making informed decisions as new variants threaten college campuses.

The reovirus sigma1s protein is a determinant of hematogenous but not neural virus dissemination in mice.

Nonstructural protein σ1s is a critical determinant of hematogenous dissemination by type 1 reoviruses, which reach the central nervous system (CNS) by a strictly blood-borne route. However, it is not known whether σ1s contributes to neuropathogenesis of type 3 reoviruses, which disseminate by both vascular and neural pathways. Using isogenic type 3 viruses that vary only in σ1s expression, we observed that mice survived at a higher frequency following hind-limb inoculation with σ1s-null virus than when inoculated with wild-type virus. This finding suggests that σ1s is essential for reovirus virulence when inoculated at a site that requires systemic spread to cause disease. Wild-type and σ1s-null viruses produced comparable titers in the spinal cord, suggesting that σ1s is dispensable for invasion of the CNS. Although the two viruses ultimately achieved similar peak titers in the brain, loads of wild-type virus were substantially greater than those of the σ1s-null mutant at early times after inoculation. In contrast, wild-type virus produced substantially higher titers than the σ1s-null virus in peripheral organs to which reovirus spreads via the blood, including the heart, intestine, liver, and spleen. Concordantly, viral titers in the blood were higher following infection with wild-type virus than following infection with the σ1s-null mutant. These results suggest that differences in viral brain titers at early time points postinfection are due to limited virus delivery to the brain by hematogenous pathways. Transection of the sciatic nerve prior to hind-limb inoculation diminished viral spread to the spinal cord. However, wild-type virus retained the capacity to disseminate to the brain following sciatic nerve transection, indicating that wild-type reovirus can spread to the brain by the blood. Together, these results indicate that σ1s is not required for reovirus spread by neural mechanisms. Instead, σ1s mediates hematogenous dissemination within the infected host, which is required for full reovirus neurovirulence.

Reverse genetics for mammalian reovirus.

Mammalian orthoreoviruses (reoviruses) are highly tractable models for studies of viral replication and pathogenesis. The versatility of reovirus as an experimental model has been enhanced by development of a plasmid-based reverse genetics system. Infectious reovirus can be recovered from cells transfected with plasmids encoding cDNAs of each reovirus gene segment using a strategy that does not require helper virus and is independent of selection. In this system, transcription of each gene segment is driven by bacteriophage T7 RNA polymerase, which can be supplied transiently by recombinant vaccinia virus (rDIs-T7pol) or by cells that constitutively express the enzyme. Reverse genetics systems have been developed for two prototype reovirus strains, type 1 Lang (T1L) and type 3 Dearing (T3D). Each reovirus cDNA was encoded on an independent plasmid for the first-generation rescue system. The efficiency of virus recovery was enhanced in a second-generation system by combining the cDNAs for multiple reovirus gene segments onto single plasmids to reduce the number of plasmids from 10 to 4. The reduction in plasmid number and the use of baby hamster kidney cells that express T7 RNA polymerase increased the efficiency of viral rescue, reduced the incubation time required to recover infectious virus, and eliminated potential biosafety concerns associated with the use of recombinant vaccinia virus. Reovirus reverse genetics has been used to introduce mutations into viral capsid and nonstructural components to study viral protein-structure activity relationships and can be exploited to engineer recombinant reoviruses for vaccine and oncolytic applications.

Reovirus nonstructural protein sigma1s is required for establishment of viremia and systemic dissemination.

Serotype-specific patterns of reovirus disease in the CNS of newborn mice segregate with the viral S1 gene segment, which encodes attachment protein sigma1 and nonstructural protein sigma1s. The importance of receptor recognition in target cell selection by reovirus implicates the sigma1 protein as the primary effector of disease outcome. However, the contribution of sigma1s to reovirus disease is unknown. To define the function of sigma1s in reovirus pathogenesis, we generated a sigma1s-deficient virus by altering a single nucleotide to disrupt the sigma1s translational start site. Viruses were recovered that contain nine gene segments from strain type 3 Dearing and either the wild-type or sigma1s-null S1 gene segment from strain type 1 Lang. Following peroral inoculation of newborn mice, both viruses replicated in the intestine, although the wild-type virus achieved higher yields than the sigma1s-null virus. However, unlike the wild-type virus, the sigma1s-deficient virus failed to disseminate to sites of secondary viral replication, including the brain, heart, and liver. Within the small intestine, both viruses were detected in Peyer's patches, but only the wild-type virus reached the mesenteric lymph node. Concordantly, wild-type virus, but not sigma1s-deficient virus, was detected in the blood of infected animals. Wild-type and sigma1s-null viruses produced equivalent titers following intracranial inoculation, indicating that sigma1s is dispensable for viral growth in the murine CNS. These results suggest a key role for sigma1s in virus spread from intestinal lymphatics to the bloodstream, thereby allowing the establishment of viremia and dissemination to sites of secondary replication within the infected host.

Seroprevalence of SARS-CoV-2 antibodies in front-line pediatric health care workers.

Objective: The goal of this study was to determine the prevalence of SARS-CoV-2 infections in pediatric front-line health care workers (HCWs) using SARS-CoV-2 serum antibodies as an indicator of infection. Methods: In this cross-sectional study, we collected blood samples and survey responses from HCWs in a 38-bed pediatric emergency department. Serum antibodies to SARS-CoV-2 (IgM and/or IgG) were measured using a 2-step enzyme-linked immunosorbent assay (ELISA) to detect antibodies against the Spike protein receptor binding domain (RBD), the ectodomain of Spike (S), and the nucleoprotein (N). Results: We collected survey responses and serum samples from 54 pediatric front-line HCWs from October 2020 through April 2021. Among the 29 unvaccinated HCWs, 4 (13.7%) had antibodies to SARS-CoV-2. For the 25 vaccinated HCWs, 10 (40%) were seropositive; 3 were <10 days from the first vaccine dose and 7 were ≥10 days after the first dose. Two of the 10 seropositive vaccines had a prior positive reverse transcription polymerase chain reaction test. Individuals ≥10 days from receiving the first vaccine dose were 37.5 (95% CI: 3.5-399.3) times more likely to have SARS-CoV-2 antibodies than unvaccinated individuals or those <10 days from first vaccine dose. Conclusions: Evidence of widespread SARS-CoV-2 infections was not found in unvaccinated front-line HCWs from a pediatric ED as of April 2021. Future work will be required to determine the reasons underlying the lower SARS-CoV-2 antibody prevalence compared to adult HCWs.

State-wide random seroprevalence survey of SARS-CoV-2 past infection in a southern US State, 2020.

The purpose of this cross-sectional study was to estimate the proportion of Arkansas residents who were infected with the SARS-CoV-2 virus between May and December 2020 and to assess the determinants of infection. To estimate seroprevalence, a state-wide population-based random-digit dial sample of non-institutionalized adults in Arkansas was surveyed. Exposures were age, sex, race/ethnicity, education, occupation, contact with infected persons, comorbidities, height, and weight. The outcome was past COVID-19 infection measured by serum antibody test. We found a prevalence of 15.1% (95% CI: 11.1%, 20.2%) by December 2020. Seropositivity was significantly elevated among participants who were non-Hispanic Black, Hispanic (prevalence ratio [PRs]:1.4 [95% CI: 0.8, 2.4] and 2.3 [95% CI: 1.3, 4.0], respectively), worked in high-demand essential services (PR: 2.5 [95% CI: 1.5, 4.1]), did not have a college degree (PR: 1.6 [95% CI: 1.0, 2.4]), had an infected household or extra-household contact (PRs: 4.7 [95% CI: 2.1, 10.1] and 2.6 [95% CI: 1.2, 5.7], respectively), and were contacted in November or December (PR: 3.6 [95% CI: 1.9, 6.9]). Our results indicate that by December 2020, one out six persons in Arkansas had a past SARS-CoV-2 infection.

Pediatric SARS-CoV-2 Seroprevalence in Arkansas Over the First Year of the COVID-19 Pandemic.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) seroprevalence studies largely focus on adults, but little is known about spread in children. We determined SARS-CoV-2 seroprevalence in children and adolescents from Arkansas over the first year of the coronavirus disease of 2019 (COVID-19) pandemic. METHODS: We tested remnant serum samples from children ages 1-18 years who visited Arkansas hospitals or clinics for non-COVID-19-related reasons from April 2020 through April 2021 for SARS-CoV-2 antibodies. We used univariable and multivariable regression models to determine the association between seropositivity and participant characteristics. RESULTS: Among 2357 participants, seroprevalence rose from 7.9% in April/May 2020 (95% CI, 4.9-10.9) to 25.0% in April 2021 (95% CI, 21.5-28.5). Hispanic and black children had a higher association with antibody positivity than non-Hispanic and white children, respectively, in multiple sampling periods. CONCLUSIONS: By spring 2021, most children in Arkansas were not infected with SARS-CoV-2. With the emergence of SARS-CoV-2 variants, recognition of long-term effects of COVID-19, and the lack of an authorized pediatric SARS-CoV-2 vaccine at the time, these results highlight the importance of including children in SARS-CoV-2 public health, clinical care, and research strategies.

Temporal Variations in Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus 2 Infections by Race and Ethnicity in Arkansas.

Background: The aim of this study was to estimate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates in the small rural state of Arkansas, using SARS-CoV-2 antibody prevalence as an indicator of infection. Methods: We collected residual serum samples from adult outpatients seen at hospitals or clinics in Arkansas for non-coronavirus disease 2019 (COVID-19)-related reasons. A total of 5804 samples were identified over 3 time periods: 15 August-5 September 2020 (time period 1), 12 September-24 October 2020 (time period 2), and 7 November-19 December 2020 (time period 3). Results: The age-, sex-, race-, and ethnicity-standardized SARS-CoV-2 seroprevalence during each period, from 2.6% in time period 1 to 4.1% in time period 2 and 7.4% in time period 3. No statistically significant difference in seroprevalence was found based on age, sex, or residence (urban vs rural). However, we found higher seroprevalence rates in each time period for Hispanics (17.6%, 20.6%, and 23.4%, respectively) and non-Hispanic Blacks (4.8%, 5.4%, and 8.9%, respectively) relative to non-Hispanic Whites (1.1%, 2.6%, and 5.5%, respectively). Conclusions: Our data imply that the number of Arkansas residents infected with SARS-CoV-2 rose steadily from 2.6% in August to 7.4% in December 2020. There was no statistical difference in seroprevalence between rural and urban locales. Hispanics and Blacks had higher rates of SARS-CoV-2 antibodies than Whites, indicating that SARS-CoV-2 spread disproportionately in racial and ethnic minorities during the first year of the COVID-19 pandemic.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.