Comparison of SARS-CoV-2 variants of concern in primary human nasal cultures demonstrates Delta as most cytopathic and Omicron as fastest replicating.

The SARS-CoV-2 pandemic was marked with emerging viral variants, some of which were designated as variants of concern (VOCs) due to selection and rapid circulation in the human population. Here, we elucidate functional features of each VOC linked to variations in replication rate. Patient-derived primary nasal cultures grown at air-liquid interface were used to model upper respiratory infection and compared to cell lines derived from human lung epithelia. All VOCs replicated to higher titers than the ancestral virus, suggesting a selection for replication efficiency. In primary nasal cultures, Omicron replicated to the highest titers at early time points, followed by Delta, paralleling comparative studies of population sampling. All SARS-CoV-2 viruses entered the cell primarily via a transmembrane serine protease 2 (TMPRSS2)-dependent pathway, and Omicron was more likely to use an endosomal route of entry. All VOCs activated and overcame dsRNA-induced cellular responses, including interferon (IFN) signaling, oligoadenylate ribonuclease L degradation, and protein kinase R activation. Among the VOCs, Omicron infection induced expression of the most IFN and IFN-stimulated genes. Infections in nasal cultures resulted in cellular damage, including a compromise of cell barrier integrity and loss of nasal cilia and ciliary beating function, especially during Delta infection. Overall, Omicron was optimized for replication in the upper respiratory tract and least favorable in the lower respiratory cell line, and Delta was the most cytopathic for both upper and lower respiratory cells. Our findings highlight the functional differences among VOCs at the cellular level and imply distinct mechanisms of pathogenesis in infected individuals. IMPORTANCE: Comparative analysis of infections by SARS-CoV-2 ancestral virus and variants of concern, including Alpha, Beta, Delta, and Omicron, indicated that variants were selected for efficiency in replication. In infections of patient-derived primary nasal cultures grown at air-liquid interface to model upper respiratory infection, Omicron reached the highest titers at early time points, a finding that was confirmed by parallel population sampling studies. While all infections overcame dsRNA-mediated host responses, infections with Omicron induced the strongest interferon and interferon-stimulated gene response. In both primary nasal cultures and lower respiratory cell line, infections by Delta were most damaging to the cells as indicated by syncytia formation, loss of cell barrier integrity, and nasal ciliary function.

The HIV-1 Capsid-Targeted Inhibitor GSK878 Alters Selection of Target Sites for HIV DNA Integration.

Decades of effort have yielded highly effective antiviral agents to treat HIV, but viral strains have evolved resistance to each inhibitor type, focusing attention on the importance of developing new inhibitor classes. A particularly promising new target is the HIV capsid, the function of which can be disrupted by highly potent inhibitors that persist long term in treated subjects. Studies with such inhibitors have contributed to an evolving picture of the role of capsid itself-the inhibitors, like certain capsid protein (CA) amino acid substitutions, can disrupt intracellular trafficking to alter the selection of target sites for HIV DNA integration in cellular chromosomes. In this study, we compare effects on HIV integration targeting for two potent inhibitors-a new molecule targeting CA, GSK878, and the previously studied lenacapavir (LEN, formerly known as GS-6207). We find that both inhibitors reduce integration in active transcription units and near epigenetic marks associated with active transcription. A careful study of integration near repeated sequences indicated frequencies were also altered for integration within multiple repeat classes. One notable finding was increased integration in centromeric satellite repeats in the presence of LEN and GSK878, which is of interest because proviruses integrated in centromeric repeats have been associated with transcriptional repression, inducibility, and latency. These data add to the picture that CA protein remains associated with preintegration complexes through the point in infection during which target sites for integration are selected, and specify new aspects of the consequences of disrupting this mechanism.

Virome in immunodeficiency: what we know currently.

ABSTRACT: Over the past few years, the human virome and its complex interactions with microbial communities and the immune system have gained recognition as a crucial factor in human health. Individuals with compromised immune function encounter distinctive challenges due to their heightened vulnerability to a diverse range of infectious diseases. This review aims to comprehensively explore and analyze the growing evidence regarding the role of the virome in immunocompromised disease status. By surveying the latest literature, we present a detailed overview of virome alterations observed in various immunodeficiency conditions. We then delve into the influence and mechanisms of these virome changes on the pathogenesis of specific diseases in immunocompromised individuals. Furthermore, this review explores the clinical relevance of virome studies in the context of immunodeficiency, highlighting the potential diagnostic and therapeutic gains from a better understanding of virome contributions to disease manifestations.

Tracking gut microbiome and bloodstream infection in critically ill adults.

BACKGROUND: The gut microbiome is believed to contribute to bloodstream infection (BSI) via translocation of dominant gut bacteria in vulnerable patient populations. However, conclusively linking gut and blood organisms requires stringent approaches to establish strain-level identity. METHODS: We enrolled a convenience cohort of critically ill patients and investigated 86 bloodstream infection episodes that occurred in 57 patients. Shotgun metagenomic sequencing was used to define constituents of their gut microbiomes, and whole genome sequencing and assembly was done on 23 unique bloodstream isolates that were available from 21 patients. Whole genome sequences were downloaded from public databases and used to establish sequence-identity distribution and define thresholds for unrelated genomes of BSI species. Gut microbiome reads were then aligned to whole genome sequences of the cognate bloodstream isolate and unrelated database isolates to assess identity. RESULTS: Gut microbiome constituents matching the bloodstream infection species were present in half of BSI episodes, and represented >30% relative abundance of gut sequences in 10% of episodes. Among the 23 unique bloodstream organisms that were available for whole genome sequencing, 14 were present in gut at the species level. Sequence alignment applying defined thresholds for identity revealed that 6 met criteria for identical strains in blood and gut, but 8 did not. Sequence identity between BSI isolates and gut microbiome reads was more likely when the species was present at higher relative abundance in gut. CONCLUSION: In assessing potential gut source for BSI, stringent sequence-based approaches are essential to determine if organisms responsible for BSI are identical to those in gut: of 14 evaluable patients in which the same species was present in both sites, they were identical in 6/14, but were non-identical in 8/14 and thus inconsistent with gut source. This report demonstrates application of sequencing as a key tool to investigate infection tracking within patients.

Comparison of SARS-CoV-2 variants of concern in primary human nasal cultures demonstrates Delta as most cytopathic and Omicron as fastest replicating.

The SARS-CoV-2 pandemic was marked with emerging viral variants, some of which were designated as variants of concern (VOCs) due to selection and rapid circulation in the human population. Here we elucidate functional features of each VOC linked to variations in replication rate. Patient-derived primary nasal cultures grown at air-liquid-interface (ALI) were used to model upper-respiratory infection and human lung epithelial cell lines used to model lower-respiratory infection. All VOCs replicated to higher titers than the ancestral virus, suggesting a selection for replication efficiency. In primary nasal cultures, Omicron replicated to the highest titers at early time points, followed by Delta, paralleling comparative studies of population sampling. All SARS-CoV-2 viruses entered the cell primarily via a transmembrane serine protease 2 (TMPRSS2)-dependent pathway, and Omicron was more likely to use an endosomal route of entry. All VOCs activated and overcame dsRNA-induced cellular responses including interferon (IFN) signaling, oligoadenylate ribonuclease L degradation and protein kinase R activation. Among the VOCs, Omicron infection induced expression of the most IFN and IFN stimulated genes. Infections in nasal cultures resulted in cellular damage, including a compromise of cell-barrier integrity and loss of nasal cilia and ciliary beating function, especially during Delta infection. Overall, Omicron was optimized for replication in the upper-respiratory system and least-favorable in the lower-respiratory cell line; and Delta was the most cytopathic for both upper and lower respiratory cells. Our findings highlight the functional differences among VOCs at the cellular level and imply distinct mechanisms of pathogenesis in infected individuals.

Exposure of progressive immune dysfunction by SARS-CoV-2 mRNA vaccination in patients with chronic lymphocytic leukemia: A prospective cohort study.

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) have reduced seroconversion rates and lower binding antibody (Ab) and neutralizing antibody (NAb) titers than healthy individuals following Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) mRNA vaccination. Here, we dissected vaccine-mediated humoral and cellular responses to understand the mechanisms underlying CLL-induced immune dysfunction. METHODS AND FINDINGS: We performed a prospective observational study in SARS-CoV-2 infection-naïve CLL patients (n = 95) and healthy controls (n = 30) who were vaccinated between December 2020 and June 2021. Sixty-one CLL patients and 27 healthy controls received 2 doses of the Pfizer-BioNTech BNT162b2 vaccine, while 34 CLL patients and 3 healthy controls received 2 doses of the Moderna mRNA-1273 vaccine. The median time to analysis was 38 days (IQR, 27 to 83) for CLL patients and 36 days (IQR, 28 to 57) for healthy controls. Testing plasma samples for SARS-CoV-2 anti-spike and receptor-binding domain Abs by enzyme-linked immunosorbent assay (ELISA), we found that all healthy controls seroconverted to both antigens, while CLL patients had lower response rates (68% and 54%) as well as lower median titers (23-fold and 30-fold; both p < 0.001). Similarly, NAb responses against the then prevalent D614G and Delta SARS-CoV-2 variants were detected in 97% and 93% of controls, respectively, but in only 42% and 38% of CLL patients, who also exhibited >23-fold and >17-fold lower median NAb titers (both p < 0.001). Interestingly, 26% of CLL patients failed to develop NAbs but had high-titer binding Abs that preferentially reacted with the S2 subunit of the SARS-CoV-2 spike. Since these patients were also seropositive for endemic human coronaviruses (HCoVs), these responses likely reflect cross-reactive HCoV Abs rather than vaccine-induced de novo responses. CLL disease status, advanced Rai stage (III-IV), elevated serum beta-2 microglobulin levels (ß2m >2.4 mg/L), prior therapy, anti-CD20 immunotherapy (<12 months), and intravenous immunoglobulin (IVIg) prophylaxis were all predictive of an inability to mount SARS-CoV-2 NAbs (all p ≤ 0.03). T cell response rates determined for a subset of participants were 2.8-fold lower for CLL patients compared to healthy controls (0.05, 95% CI 0.01 to 0.27, p < 0.001), with reduced intracellular IFNγ staining (p = 0.03) and effector polyfunctionality (p < 0.001) observed in CD4+ but not in CD8+ T cells. Surprisingly, in treatment-naïve CLL patients, BNT162b2 vaccination was identified as an independent negative risk factor for NAb generation (5.8, 95% CI 1.6 to 27, p = 0.006). CLL patients who received mRNA-1273 had 12-fold higher (p < 0.001) NAb titers and 1.7-fold higher (6.5, 95% CI 1.3 to 32, p = 0.02) response rates than BNT162b2 vaccinees despite similar disease characteristics. The absence of detectable NAbs in CLL patients was associated with reduced naïve CD4+ T cells (p = 0.03) and increased CD8+ effector memory T cells (p = 0.006). Limitations of the study were that not all participants were subjected to the same immune analyses and that pre-vaccination samples were not available. CONCLUSIONS: CLL pathogenesis is characterized by a progressive loss of adaptive immune functions, including in most treatment-naïve patients, with preexisting memory being preserved longer than the capacity to mount responses to new antigens. In addition, higher NAb titers and response rates identify mRNA-1273 as a superior vaccine for CLL patients.

A Germline-Targeting Chimpanzee SIV Envelope Glycoprotein Elicits a New Class of V2-Apex Directed Cross-Neutralizing Antibodies.

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germ line-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these animals identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, nonneutralizing antibodies revealed that SCIV-expressed, but not wild-type SIVcpz Envs, as well as a subset of primary HIV-1 Envs, preferentially adopted a more open trimeric state. These results reveal the existence of a cryptic V2 epitope that is exposed in occluded-open SIVcpz and HIV-1 Env trimers and elicits cross-neutralizing responses of limited breadth and potency. IMPORTANCE An effective HIV-1 vaccination strategy will need to stimulate rare precursor B cells of multiple bNAb lineages and affinity mature them along desired pathways. Here, we searched for V2-apex germ line-targeting Envs among a large set of diverse primate lentiviruses and identified minimally modified versions of one chimpanzee SIV Env that bound several human V2-apex bNAb precursors and stimulated one of these in a V2-apex bNAb precursor-expressing knock-in mouse. We also generated chimeric simian-chimpanzee immunodeficiency viruses and showed that they elicit low-titer V2-directed heterologous plasma breadth in six of seven infected rhesus macaques. Characterization of this antibody response identified a new class of weakly cross-reactive neutralizing antibodies that target the V2-apex, but only in occluded-open Env trimers. The existence of this cryptic epitope, which in some Env backgrounds is immunodominant, needs to be considered in immunogen design.

Multiple Introductions of SARS-CoV-2 Alpha and Delta Variants into White-Tailed Deer in Pennsylvania.

The SARS-CoV-2 pandemic began by viral spillover from animals to humans; today multiple animal species are known to be susceptible to infection. White-tailed deer, Odocoileus virginianus, are infected in North America at substantial levels, and genomic data suggests that a variant in deer may have spilled back to humans. Here, we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole genome sequences were obtained, together with six more partial spike gene sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer. IMPORTANCE Coronaviruses have been documented to replicate in numerous species of vertebrates, and multiple spillovers of coronaviruses from animals into humans have founded human epidemics. The COVID-19 epidemic likely derived from a spillover of SARS-CoV-2 from bats into humans, possibly via an intermediate host. There are now several examples of SARS-CoV-2 jumping from humans into other mammals, including mink and deer, creating the potential for new animal reservoirs from which spillback into humans could occur. For this reason, data on formation of new animal reservoirs is of great importance for understanding possible sources of future infection. Here, we identify extensive infection in white-tailed deer in Pennsylvania, including what appear to be multiple independent transmissions. Data further suggests possible transmission among deer. These data thus help identify a potential new animal reservoir and provide background information relevant to its management.

SARS-CoV-2 mRNA vaccination exposes progressive adaptive immune dysfunction in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) patients have lower seroconversion rates and antibody titers following SARS-CoV-2 vaccination, but the reasons for this diminished response are poorly understood. Here, we studied humoral and cellular responses in 95 CLL patients and 30 healthy controls after two BNT162b2 or mRNA-2173 mRNA immunizations. We found that 42% of CLL vaccinees developed SARS-CoV-2-specific binding and neutralizing antibodies (NAbs), while 32% had no response. Interestingly, 26% were seropositive, but had no detectable NAbs, suggesting the maintenance of pre-existing endemic human coronavirus-specific antibodies that cross-react with the S2 domain of the SARS-CoV-2 spike. These individuals had more advanced disease. In treatment-naïve CLL patients, mRNA-2173 induced 12-fold higher NAb titers and 1.7-fold higher response rates than BNT162b2. These data reveal a graded loss of immune function, with pre-existing memory being preserved longer than the capacity to respond to new antigens, and identify mRNA-2173 as a superior vaccine for CLL patients.

A Summary of the Sixth International Workshop on Microbiome in HIV Pathogenesis, Prevention, and Treatment.

In October of 2020, researchers from around the world met online for the sixth annual International Workshop on Microbiome in HIV Pathogenesis, Prevention, and Treatment. New research was presented on the roles of the microbiome on immune response and HIV transmission and pathogenesis and the potential for alterations in the microbiome to decrease transmission and affect comorbidities. This article presents a summary of the findings reported.

DNA binding to TLR9 expressed by red blood cells promotes innate immune activation and anemia.

Red blood cells (RBCs) are essential for aerobic respiration through delivery of oxygen to distant tissues. However, RBCs are currently considered immunologically inert, and few, if any, secondary functions of RBCs have been identified. Here, we showed that RBCs serve as critical immune sensors through surface expression of the nucleic acid­sensing Toll-like receptor 9 (TLR9). Mammalian RBCs expressed TLR9 on their surface and bound CpG-containing DNA derived from bacteria, plasmodia, and mitochondria. RBC-bound mitochondrial DNA was increased during human and murine sepsis and pneumonia. In vivo, CpG-carrying RBCs drove accelerated erythrophagocytosis and innate immune activation characterized by increased interferon signaling. Erythroid-specific deletion of TLR9 abrogated erythrophagocytosis and decreased local and systemic cytokine production during CpG-induced inflammation and polymicrobial sepsis. Thus, detection and capture of nucleic acid by TLR9-expressing RBCs regulated red cell clearance and inflammatory cytokine production, demonstrating that RBCs function as immune sentinels during pathologic states. Consistent with these findings, RBC-bound mitochondrial DNA was elevated in individuals with viral pneumonia and sepsis secondary to coronavirus disease 2019 (COVID-19) and associated with anemia and severity of disease. These findings uncover a previously unappreciated role of RBCs as critical players in inflammation distinct from their function in gas transport.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

Predictors of Nonseroconversion after SARS-CoV-2 Infection.

Not all persons recovering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection develop SARS-CoV-2-specific antibodies. We show that nonseroconversion is associated with younger age and higher reverse transcription PCR cycle threshold values and identify SARS-CoV-2 viral loads in the nasopharynx as a major correlate of the systemic antibody response.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses.

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4+ T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNß that reduced viral replication in vitro by 50% (IC50) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNß resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients.IMPORTANCE Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.

Molecular Beacons Allow Specific RT-LAMP Detection of B.1.1.7 Variant SARS-CoV-2.

Over the course of the coronavirus disease 2019 (COVID-19) pandemic, several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants are important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive quantitative PCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons combined with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants that contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific and affordable and allows multiplexing with other SARS-CoV-2 loop-mediated amplification primer sets.

SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

A cone-beam computed tomographic evaluation of alveolar bone dimensional changes and the periodontal limits of mandibular incisor advancement in skeletal Class II patients.

OBJECTIVES: To evaluate the presence of dehiscences and changes in alveolar bone height and width in the area of the mandibular central incisors pre- and post-orthodontic treatment. MATERIALS AND METHODS: In 60 skeletal Class II patients, cone-beam computed tomographic (CBCT) images were obtained and the patients were divided into four groups based on the presence of dehiscences at pre- and post-orthodontic treatment. The alveolar bone height and width were measured on CBCT in cross section along the long axis of the teeth. Lateral cephalograms were analyzed. RESULTS: The changes in L1-NB and IMPA appeared to be correlated with vertical bone loss and dehiscence. Alveolar bone height appeared to follow a segmented relationship with these two variables, with changes below a threshold (L1-NB = 0.71 mm, IMPA = 3.02°) having relatively minimal or no effect on bone loss but with changes beyond the threshold correlated with extensive bone loss. Similarly, increases in L1-NB or IMPA correlated with decreases in alveolar bone width (L1-NB: -0.25 mm/mm, IMPA: -0.07 mm/°) and increased the probability of developing dehiscences, with an estimated 50% probability of vertical bone loss at a L1-NB change of 2.00 mm or, equivalently, an IMPA change of 8.02° was estimated. CONCLUSIONS: When treating skeletal Class II patients, the limits of incisor proclination/protraction are less than previously thought. To prevent undesired periodontal outcomes, careful three-dimensional diagnosis is advisable. Furthermore, when excessive protrusion and/or proclination is planned, additional treatment modalities, including orthognathic surgery, tooth extraction, and corticotomy with bone graft, should be considered.