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A 7-year-old boy presented to the emergency department with fever, cough, congestion, abdominal pain, myalgias, and morbilliform rash. Several aspects of the patient’s history, including recent travel, living on a farm, exposure to sick contacts, and new medications, resulted in a wide differential diagnosis. Initial laboratory testing revealed leukocytosis with neutrophilia and elevated atypical lymphocytes, but did not reveal any infectious causes of illness. He was discharged from the hospital, but then represented to the emergency department a day later with worsening rash, continued fever, abdominal pain, and poor intake. He was then admitted. A more comprehensive laboratory evaluation was initiated. During this hospital course, the patient’s physical examination changed when he developed head and neck edema, and certain laboratory trends became clearer. With the assistance of several specialists, the team was able to reach a more definitive diagnosis and initiate treatment to appropriately manage his condition.

Many mechanisms responsible for COVID-19 pathogenesis are well-established, but COVID-19 includes features with unclear pathogenesis, such as autonomic dysregulation, coagulopathies, and high levels of inflammation. The receptor for SARS-CoV-2 spike protein’s receptor binding domain (RBD) is angiotensin converting enzyme 2 (ACE2). We hypothesized that some COVID-19 patients may develop antibodies that have negative molecular image of RBD sufficiently similar to ACE2 to yield ACE2-like catalytic activity – ACE2-like abzymes. To explore this hypothesis, we studied patients hospitalized with COVID-19 who had plasma samples available obtained about 7 days after admission. ACE2 is a metalloprotease that requires Zn2+for activity. However, we found that the plasma from some patients studied could cleave a synthetic ACE2 peptide substrate, even though the plasma samples were collected using disodium ethylenediaminetetraacetate (EDTA) anticoagulant. When we spiked plasma with synthetic ACE2, no ACE2 substrate cleavage activity was observed unless Zn2+was added or the plasma was diluted to decrease EDTA concentration. After processing samples by 100 kDa size exclusion columns and protein A/G adsorption, which depleted immunoglobulin by >99.99%, the plasma samples did not cleave the ACE2 substrate peptide. The data suggest that some patients with COVID-19 develop antibodies with abzyme-like activity capable of cleaving synthetic ACE2 substrate. Since abzymes can exhibit promiscuous substrate specificities compared to the enzyme whose active site image they resemble, and since proteolytic cascades regulate many physiologic processes, anti-RBD abzymes may contribute to some otherwise obscure COVID-19 pathogenesis.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to-volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

Beyond their pulmonary disease, many COVID-19 patients experience a complex constellation of characteristics, including hyperinflammatory responses, autoimmune disorders, and coagulopathies. However, the pathogenesis of these aspects of COVID-19 is obscure. More than 90% of people are latently infected with the lymphotropic herpesviruses Epstein–Barr Virus (EBV) and/or Human Herpesvirus-6 (HHV-6). Some of the inflammatory features of COVID-19 resemble clinical syndromes seen during EBV and HHV-6 infection, and these latent viruses can be reactivated by inflammatory mediators. We hypothesized that EBV and HHV-6 reactivation might be a common feature of early COVID-19, particularly in patients with more inflammation. We tested for EBV and HHV-6 reactivation in 67 patients acutely hospitalized with COVID-19 using previously validated quantitative PCR assays on the plasma. In our cohort, we found that 15/67 (22.4%) patients had detectable EBV and 3/67 (4.5%) had detectable HHV-6. This frequency of activation is somewhat more than the frequency reported for some healthy cohorts, such as blood donors and other healthy control cohorts. There was no association between EBV or HHV-6 and markers indicative of more inflammatory disease. We conclude that EBV and HHV-6 activation at about day 7 of hospitalization occurred in a modest fraction of our cohort of COVID-19 patients and was not associated with high levels of inflammation. In the modest fraction of patients, EBV and HHV-6 reactivation could contribute to some features of acute disease and pre-disposition to post-acute sequelae in a subset of patients.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated

* Abbreviations: COVID-19 — : coronavirus disease 2019 nAb — : neutralizing antibody SARS-CoV-2 — : severe acute respiratory syndrome coronavirus 2 After having coronavirus disease 2019 (COVID-19), patients develop a humoral immune response thought to protect against reinfection, but antibody levels can decline over time. Understanding how long antibody levels remain high enough to prevent infection is important in understanding, absent vaccination, whether children may be vulnerable to COVID-19 and in modeling how COVID-19 spreads through the population. The levels of antibodies capable of neutralizing the virus (neutralizing antibodies [nAbs]) provide a direct marker of a protective humoral immune response. The nAbs can be measured directly by determining how well a patient’s serum inactivates virus when a known amount of virus is placed on cells in tissue culture, which is known as a plaque reduction neutralization test. The remarkably safe and effective approved COVID-19 vaccines elicit potent antibody responses against portions of the virus mediating attachment to host cells and viral entry, the spike protein and its receptor-binding domain. The concentration of antibodies a patient has against spike … Address correspondence to: Andrea T. Cruz, Department of Pediatrics, Baylor College of Medicine, 6621 Fannin St, Suite A2210, Houston, TX 77030. E-mail: acruz{at}bcm.edu

Multisystem inflammatory syndrome in children (MIS-C) is a serious, sometimes life-threatening late complication of coronavirus disease 2019 (COVID-19) with multiorgan involvement and evidence of immune activation. The pathogenesis of MIS-C is not known, nor is the pathogenesis of the severe organ damage that is the hallmark of MIS-C. Human herpesvirus 6 (HHV-6), the virus responsible for roseola, is a ubiquitous herpesvirus that causes close to universal infection by the age of 3 years. HHV-6 remains latent for life and can be activated during inflammatory states, by other viruses, and by host cell apoptosis. HHV-6 has been associated with end-organ diseases, including hepatitis, carditis, and encephalitis. In addition, ∼1% of people have inherited chromosomally integrated human herpesvirus 6 (iciHHV-6), which is HHV-6 that has been integrated into chromosomal telomeric regions and is transmitted through the germ line. iciHHV-6 can be reactivated and has been associated with altered immune responses. We report here a case of MIS-C in which an initial high HHV-6 DNA polymerase chain reaction viral load assay prompted testing for iciHHV-6, which yielded a positive result. Additional research may be warranted to determine if iciHHV-6 is commonly observed in patients with MIS-C and, if so, whether it may play a part in MIS-C pathogenesis.

Significance We report a vaccine platform to express vaccine antigens on the surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrate the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface of Escherichia coli to produce killed whole-cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates interferon-γ responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly protective coronavirus vaccine since a betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against alphacoronavirus porcine epidemic diarrhea virus. When using a vaccine-appropriate bacteria vector, this inexpensive vaccine platform offers the potential for use in developing countries., As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible Gram-negative autotransporter to express vaccine antigens on surface of genome-reduced bacteria to enhance interaction of vaccine antigen with immune system. As a proof of principle, we utilized genome-reducedE. colito express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as a killed whole cell vaccine. The FP sequence is highly conserved across coronaviruses; the 6 FP core amino acid residues along with the 4 adjacent residues upstream and the 3 residues downstream the core are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated IFN-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly-protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.Significance StatementWe report a new vaccine platform to express vaccine antigens on surface of genome-reduced bacteria to enhance vaccine immunogenicity. We demonstrated the utility of this vaccine platform by expressing the highly conserved fusion peptide (FP) of SARS-CoV-2 and porcine epidemic diarrhea virus on the surface ofE.colito produce killed whole cell bacterial vaccines. The vaccine primes a potent anamnestic response, potentiates IFN-γ responses, and provides significant protection in pigs against disease following virus challenge. The FP could be a target for a broadly-protective coronavirus vaccine since a Betacoronavirus SARS-CoV-2 FP vaccine provided cross-protection against Alphacoronavirus PEDV. When using a vaccine appropriate bacteria vector, this inexpensive new vaccine platform offers the potential for use in developing countries.

* Abbreviations: COVID-19 — : coronavirus disease 2019 MIS-C — : multisystem inflammatory syndrome in children RBD — : receptor-binding domain S — : severe acute respiratory syndrome coronavirus 2 spike protein SARS-Co-V-2 — : severe acute respiratory syndrome coronavirus 2 Coronavirus disease 2019 (COVID-19) is arguably the most socially and economically disruptive pandemic since the 1918 influenza pandemic. Although pediatric COVID-19 shares features with the adult disease, there are several differences. Children produce virus in amounts at least equal to adults, if not higher,1 and can transmit the virus, just as adults can.2 School-aged children are generally less-severely affected than infants or adults, but some children without significant underlying disease become ill or die in a disease process analogous to the one most commonly seen in adults: severe pulmonary disease and respiratory failure.3–6 Children and adults appear to have different humoral immune responses to COVID-19.7 A small fraction of children with COVID-19 experience a hyperinflammatory process,8–13 termed multisystem inflammatory syndrome in children (MIS-C) in the United States, with features distinct from Kawasaki disease.14 This case definition includes ≥2 of these symptoms: rash, conjunctivitis, or mucocutaneous inflammation; hypotension; cardiac disease; coagulopathy; or acute gastrointestinal problems.15 MIS-C, which is not correlated with viral load levels, typically appears some time after initial infection.13 MIS-C’s incidence is difficult to determine, given the high rate of asymptomatic infection.12 Adults with COVID-19 also can experience inflammatory disorders: coagulopathies, vasculitis, cardiomyositis, and neuroinflammatory processes.16–18 That the most effective therapy to reduce mortality in adults with severe COVID-19 yet established is an immune suppressant, dexamethasone,19 reveals the importance of inflammatory and immune-mediated pathologies. In this issue of … Address correspondence to Steven Zeichner, MD, PhD, Department of Pediatrics, University of Virginia, Box 801349, Charlottesville, VA 22908. E-mail: zeichner{at}virginia.edu

* Abbreviation: COVID-19 — : coronavirus disease of 2019 The impact of the coronavirus disease of 2019 (COVID-19), which is caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2,1 has been widespread, with >500 000 cases diagnosed in >170 countries since the virus was identified in January 2020.2 The preliminary data have been focused on severe respiratory manifestations, which are seen predominantly in adults, with scant initial data on the burden of COVID-19 in children.3 We, therefore, read with interest the findings of Dong et al,4 who reported in this issue of Pediatrics a series of >2000 children with suspected or confirmed COVID-19. The authors found that 4% of virologically confirmed cases had asymptomatic infection, and this rate almost certainly understates the true rate of asymptomatic infection because many children who are asymptomatic are unlikely to be tested. Among children who were symptomatic, 5% had dyspnea or hypoxemia (a substantially lower percentage than what has been reported for adults3), and 0.6% progressed to acute respiratory distress syndrome or multiorgan system dysfunction (a rate that is also lower than that seen in adults). Preschool-aged children and infants were more likely than older children to have severe clinical manifestations. There are several salient points … Address correspondence to Andrea T. Cruz, MD, MPH, Department of Pediatrics, Baylor College of Medicine, 6621 Fannin St, Suite A2210, Houston, TX 77030. E-mail: acruz{at}bcm.edu

A 16-year-old girl presented to the emergency department with intermittent fevers and worsening abdominal pain of 5 weeks duration. She had a history of travel to a less developed country and exposure to possible infectious diseases. Abdominal imaging and blood tests revealed diffuse mesenteric lymphadenopathy, elevated transaminases, and elevation of inflammatory markers. Gastroesophageal and colon endoscopies revealed gastric ulcers, and the patient was discharged with a presumptive diagnosis of systemic juvenile idiopathic arthritis given the lymphadenopathy seen on imaging, serositis, sacroiliac joint stiffness noted on physical examination, and pain relief with celecoxib. She presented again 4 days later with worsening abdominal tenderness, elevated transaminases, and new-onset abdominal distention. Tissue biopsy yielded the diagnosis and directed appropriate treatment.

Background/objectives Atopic dermatitis (AD) is a common chronic inflammatory skin condition associated with high transepidermal water loss, high skin pH, and Staphylococcus aureus skin colonization. The treatment of AD with bath additives remains highly debated. Recent evidence suggests that dilute apple cider vinegar (ACV) may improve skin barrier integrity in AD, but its safety and efficacy are not well studied. This pilot split-arm study analyzed the effect of dilute apple cider vinegar soaks on skin barrier integrity in patients with atopic dermatitis as measured by skin transepidermal water loss and skin pH. Methods A total of 22 subjects (11 AD and 11 healthy controls) were enrolled. Subjects soaked both of their forearms for 14 days, with one arm in dilute ACV (0.5% acetic acid) and the other in water 10 minutes daily. Transepidermal water loss and pH were measured pre- and post-treatment. Results In both groups, transepidermal water loss increased and pH decreased at 0 minutes post-ACV treatment, but these effects were not sustained at 60 minutes. In total, 72.7% (16/22) of subjects reported mild side effects from ACV with improvement after discontinuing the soaks. Conclusions Dilute ACV soaks have no significant effect on skin barrier integrity but caused skin irritation in a majority of subjects. Study limitations include analysis of a single brand, dilution, and application of ACV. Future studies are needed to explore whether lower concentrations of ACV soaks or other applications such as a leave-on acidic ointment could improve skin barrier integrity in a safe, nonirritating way.

No effective therapy to eliminate the HIV latently infected cell reservoir has been developed. One approach, “shock and kill”, employs agents that activate HIV, subsequently killing the activated infected cells and/or virus. Shock and kill requires agents that safely and effectively activate HIV. One class of activation agents works through classical NF-κB pathways, but global NF-κB activators are non-specific and toxic. There exist two major IκBs: IκBα, and IκBε, which hold activating NF-κB subunits in the cytoplasm, releasing them for nuclear transit upon cell stimulation. IκBα was considered the main IκB responsible for gene expression regulation, including HIV activation. IκBε is expressed in cells constituting much of the latent HIV reservoir, and IκBε knockout mice have a minimal phenotype, suggesting that IκBε could be a valuable target for HIV activation and reservoir depletion. We previously showed that targeting IκBε yields substantial increases in HIV expression. Here, we show that IκBε holds c-Rel and p65 activating NF-κB subunits in the cytoplasm, and that targeting IκBε with siRNA produces a strong increase in HIV expression associated with enhanced c-Rel and p65 transit to the nucleus and binding to the HIV LTR of the activating NF-κBs, demonstrating a mechanism through which targeting IκBε increases HIV expression. The findings suggest that it may be helpful to develop HIV activation approaches, acting specifically to target IκBε and its interactions with the NF-κBs.

Short bowel syndrome (SBS) presents an increasing problem in pediatrics. SBS often results from surgical resection of necrotic bowel following necrotizing enterocolitis or treatment of anatomic gastrointestinal defects. SBS is associated with significant morbidity and mortality, and creates substantial burdens for patients, families, and the health system. Recent reports have demonstrated that the fecal microbiome of children with SBS is significantly different from healthy control and severe intestinal microbial imbalances is associated with poor growth. We hypothesized that children with SBS and adverse clinical features such as PN dependent, shorter bowel length and lack of ileocecal valve would demonstrate more gut dysbiosis compare with the SBS non-PN dependent. An improved understanding of SBS pathogenesis would enhance management and potentially suggest new interventions. We studied microbial communities of SBS and control non-SBS patients from the jejunum, obtained endoscopically or by ostomy aspiration, and stool. We enrolled SBS patients who did and did not require parenteral nutrition (PN), as a surrogate marker for the seriousness of their disease. We studied the microbiota using high-throughput DNA sequencing of 16S rRNA genes and statistical analyses. We found that microbial diversity was significantly greater in jejunal aspirate than in stool samples in SBS patients, unlike non-SBS patients; that SBS patients receiving enteral feeds had greater diversity, and that SBS patients on PN and enteral feeds had lower differences in diversity in jejunal vs. stool samples. We found a trend toward increased diversity in patients with an intact ileocecal valve, and found that certain taxa were more abundant in the certain sample types, and in SBS patients vs. non-SBS patients. SBS patients have lower microbial diversity, especially patients with more severe disease, patients requiring PN, and those lacking an ileocecal valve. SBS patients, particularly those with more complex characteristics, exhibit differences in their intestinal microbiota. Particular individual taxa were over- and under-represented in patients with more unfavorable disease. While diminished diversity and alterations in microbiota composition are likely consequences of SBS, future efforts aimed at increasing microbial diversity and interventions targeting specific microbiota characteristics might constitute a testable approach to ameliorate some clinical SBS clinical consequences.

Introduction Atopic dermatitis is a common skin disease characterized by altered cutaneous immunity in which patients often exhibit lower skin microbiota diversity compared to healthy skin and are prone to colonization by Staphylococcus aureus. Apple cider vinegar has been shown to have antibacterial effects; however, its effects on the skin microbiome have not previously been well-described. Objectives We aimed to examine the effects of topical dilute apple cider vinegar soaks on Staphylococcus aureus abundance, skin bacterial microbiome composition, and skin bacterial microbiome diversity in atopic dermatitis participants compared to healthy skin. Methods Eleven subjects with atopic dermatitis and 11 healthy controls were enrolled in this randomized, non-blinded, single-institution, split-arm pilot study. Subjects soaked one forearm in dilute apple cider vinegar (0.5% acetic acid) and the other forearm in tap water for 10 minutes daily. Skin bacteria samples were collected from subjects’ volar forearms before and after 14 days of treatment. 16S sequencing was used to analyze Staphylococcus aureus abundance and skin bacterial microbiome composition, and alpha diversity of microbiota were determined using Shannon diversity index. Results There was no difference in skin bacterial microbiome in atopic dermatitis subjects after 2 weeks of daily water or apple cider vinegar treatments (p = 0.056 and p = 0.22, respectively), or in mean abundance of S. aureus on apple cider vinegar-treated forearms (p = 0.60). At 2 weeks, the skin bacterial microbiomes of healthy control subjects were not significantly different from the skin bacterial microbiome of atopic dermatitis subjects (p = 0.14, 0.21, 0.12, and 0.05). Conclusions Our results suggest that daily soaks in 0.5% apple cider vinegar are not an effective method of altering the skin bacterial microbiome in atopic dermatitis. Further studies are needed to explore the effects of different concentrations of apple cider vinegar on skin microflora and disease severity. Trial number UVA IRB-HSR #19906.

Regulation of expression of HTLV-1 gene products from integrated proviruses plays an important role in HTLV-1-associated disease pathogenesis. Previous studies have shown that T cell receptor (TCR)- and phorbol ester (PMA) stimulation of chronically infected CD4 T cells increases the expression of integrated HTLV-1 proviruses in latently infected cells, however the mechanism remains unknown. Analysis of HTLV-1 RNA and protein species following PMA treatment of the latently HTLV-1-infected, FS and SP T cell lines demonstrated rapid induction of tax/rex mRNA. This rapid increase in tax/rex mRNA was associated with markedly enhanced tax/rex mRNA stability while the stability of unspliced or singly spliced HTLV-1 RNAs did not increase. Tax/rex mRNA in the HTLV-1 constitutively expressing cell lines exhibited high basal stability even without PMA treatment. Our data support a model whereby T cell activation leads to increased HTLV-1 gene expression at least in part through increased tax/rex mRNA stability.

Background Despite much work, safe and effective approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1 remain an elusive goal. Patients infected with HIV-1 treated with cytotoxic agents or bone marrow transplantation can experience decreases in the reservoir of HIV-1 latently infected cells. Other viruses capable of long-term latency, such as herpesviruses, can sense host cell apoptosis and respond by initiating replication. These observations suggest that other viruses capable of long-term latency, like HIV-1, might also sense when its host cell is about to undergo apoptosis and respond by initiating replication. Results Pro-monocytic (U1) and lymphoid (ACH-2) HIV-1 persistently infected cell lines were treated with cytotoxic drugs – doxorubicin, etoposide, fludarabine phosphate, or vincristine – and activation of latent HIV-1 was evaluated using assays for HIV-1 RNA and p24 production. Both cell lines showed dose-dependent increases in apoptosis and associated HIV-1 activation following exposure to the cytotoxic agents. Pretreatment of the cells with the pan-caspase inhibitor Z-VAD-FMK prior to exposure to the cytotoxic agents inhibited apoptosis and viral activation. Direct exposure of the latently infected cell lines to activated caspases also induced viral replication. HIV-1 virions produced in association with host cell apoptosis were infectious. Conclusions The results indicate that latent HIV-1 can sense when its host cell is undergoing apoptosis and responds by completing its replication cycle. The results may help explain why patients treated with cytotoxic regimens for bone marrow transplantation showed reductions in the reservoir of latently infected cells. The results also suggest that the mechanisms that HIV-1 uses to sense and respond to host cell apoptosis signals may represent helpful new targets for approaches to attack and deplete the long-lived reservoir of cells latently infected with HIV-1.

The oral microbial community (microbiota) plays a critical role in human health and disease. Alterations in the oral microbiota may be associated with disorders such as gingivitis, periodontitis, childhood caries, alveolar osteitis, oral candidiasis and endodontic infections. In the immunosuppressed population, the spectrum of potential oral disease is even broader, encompassing candidiasis, necrotizing gingivitis, parotid gland enlargement, Kaposi’s sarcoma, oral warts and other diseases. Here, we used 454 pyrosequencing of bacterial 16S rRNA genes to examine the oral microbiome of saliva, mucosal and tooth samples from HIV-positive and negative children. Patient demographics and clinical characteristics were collected from a cross-section of patients undergoing routine dental care. Multiple specimens from different sampling sites in the mouth were collected for each patient. The goal of the study was to observe the potential diversity of the oral microbiota among individual patients, sample locations, HIV status and various dental characteristics. We found that there were significant differences in the microbiome among the enrolled patients, and between sampling locations. The analysis was complicated by uneven enrollment in the patient cohorts, with only five HIV-negative patients enrolled in the study and by the rapid improvement in the health of HIV-infected children between the time the study was conceived and completed. The generally good oral health of the HIV-negative patients limited the number of dental plaque samples that could be collected. We did not identify significant differences between well-controlled HIV-positive patients and HIV-negative controls, suggesting that well-controlled HIV-positive patients essentially harbor similar oral flora compared to patients without HIV. Nor were significant differences in the oral microbiota identified between different teeth or with different dental characteristics. Additional studies are needed to better characterize the oral microbiome in children and those with poorly-controlled HIV infections.

A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.