Adeno-associated virus type 2 in US children with acute severe hepatitis.

As of August 2022, clusters of acute severe hepatitis of unknown aetiology in children have been reported from 35 countries, including the USA1,2. Previous studies have found human adenoviruses (HAdVs) in the blood from patients in Europe and the USA3-7, although it is unclear whether this virus is causative. Here we used PCR testing, viral enrichment-based sequencing and agnostic metagenomic sequencing to analyse samples from 16 HAdV-positive cases from 1 October 2021 to 22 May 2022, in parallel with 113 controls. In blood from 14 cases, adeno-associated virus type 2 (AAV2) sequences were detected in 93% (13 of 14), compared to 4 (3.5%) of 113 controls (P < 0.001) and to 0 of 30 patients with hepatitis of defined aetiology (P < 0.001). In controls, HAdV type 41 was detected in blood from 9 (39.1%) of the 23 patients with acute gastroenteritis (without hepatitis), including 8 of 9 patients with positive stool HAdV testing, but co-infection with AAV2 was observed in only 3 (13.0%) of these 23 patients versus 93% of cases (P < 0.001). Co-infections by Epstein-Barr virus, human herpesvirus 6 and/or enterovirus A71 were also detected in 12 (85.7%) of 14 cases, with higher herpesvirus detection in cases versus controls (P < 0.001). Our findings suggest that the severity of the disease is related to co-infections involving AAV2 and one or more helper viruses.

Four superoxide dismutases contribute to Bacillus anthracis virulence and provide spores with redundant protection from oxidative stress.

The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (Delta sod15 Delta sodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the Delta sod15 Delta sodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (Delta sod15 Delta sodA1 Delta sodC Delta sodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophage-mediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.

Recovery efficiencies of anthrax spores and ricin from nonporous or nonabsorbent and porous or absorbent surfaces by a variety of sampling methods*.

The 2001 anthrax letter cases brought into focus the need to establish the most effective environmental sampling procedures. Results are presented from two studies aimed at establishing the best procedures for everyday surfaces likely to be contaminated after the release of environmentally stable bioaggressive agents, as exemplified by anthrax spores and ricin. With anthrax spores, contact plates, with mean retrieval rates of 28-54%, performed better than other methods by a wide margin for flat nonporous, nonabsorbent surfaces. They also proved best on flat porous, absorbent materials, although recoveries were low (<7%). For both agents, dry devices (swabs, wipes, Trace Evidence Collection Filters) had universally poor retrieval efficiencies with no significant differences between them. Among moistened devices (wipes, swabs, and Sample Collection and Recovery Devices), wipes were generally best, albeit with considerable cross-over among individual readings (highest mean recoveries for anthrax spores and ricin 5.5% and 2.5%, respectively, off plastic).

Recombinant Bacillus anthracis spore proteins enhance protection of mice primed with suboptimal amounts of protective antigen.

Inactivated Bacillus anthracis spores given with protective antigen (PA) contribute to immunity against anthrax in several animal models. Antiserum raised against whole irradiated B. anthracis spores has been shown to have anti-germination and opsonic activities in vitro. Based on these observations, we hypothesized that surface-exposed spore proteins might serve as supplemental components of a PA-based anthrax vaccine. The protective anti-spore serum was tested for reactivity with recombinant forms of 30 proteins known, or believed to be, present within the B. anthracis exosporium. Eleven of those proteins were reactive with this antiserum, and, subsequently a subset of this group was used to generate rabbit polyclonal antibodies. These sera were evaluated for recognition of the immunogens on intact spores generated from Sterne strain, as well as from an isogenic mutant lacking the spore surface protein Bacillus collagen-like antigen (BclA). The data were consistent with the notion that the antigens in question were located beneath BclA on the basal surface of the exosporium. A/J mice immunized with either the here-to-for hypothetical protein p5303 or the structural protein BxpB, each in combination with subprotective levels of PA, showed enhanced protection against subcutaneous spore challenge. While neither anti-BxpB or anti-p5303 antibodies reduced the rate of spore germination in vitro, both caused increased uptake and lead to a higher rate of destruction by phagocytic cells. We conclude that by facilitating more efficient phagocytic clearance of spores, antibodies against individual exosporium components can contribute to protection against B. anthracis infection.

Recombinant exosporium protein BclA of Bacillus anthracis is effective as a booster for mice primed with suboptimal amounts of protective antigen.

Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis. We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protection to only 1 of 10 animals. However, all mice that received suboptimal amounts of recombinant PA and that then received rBclA 2 weeks later survived spore challenge. Additionally, anti-rBclA IgG, compared to anti-PA IgG, promoted a sevenfold-greater uptake of opsonized spores by mouse macrophages and markedly decreased intramacrophage spore germination. Since BclA has some sequence similarity to human collagen, we also tested the extent of binding of anti-rBclA antibodies to human collagen types I, III, and V and found no discernible cross-reactivity. Taken together, these results support the concept of rBclA as being a safe and effective boost for a PA-primed individual against anthrax and further suggest that such rBclA-enhanced protection occurs by the induction of spore-opsonizing and germination-inhibiting antibodies.

The US capitol bioterrorism anthrax exposures: clinical epidemiological and immunological characteristics.

BACKGROUND: Bioterrorism-related anthrax exposures occurred at the US Capitol in 2001. Exposed individuals received antibiotics and anthrax vaccine adsorbed immunization. METHODS: A prospective longitudinal study of 124 subjects--stratified on the basis of spore exposure, nasopharyngeal culture results, and immunization status from inside and outside an epidemiologically defined exposure zone--was performed to describe clinical outcome and immune responses after Bacillus anthracis exposure. Antibody and cell-mediated immune (CMI) responses to protective antigen (PA) and lethal factor were assayed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting. RESULTS: Antibody and CMI dose-exposure responses, albeit generally of low magnitude, were seen for unimmunized subjects from inside, within the perimeter, and outside the exposure zone and in nonexposed control subjects. Anti-PA antibody and CMI responses were detected in 94% and 86% of immunized subjects. No associations were seen between symptoms and exposure levels or immune responses. CONCLUSIONS: Anthrax spores primed cellular and possibly antibody immune responses in a dose-dependent manner and may have enhanced vaccine boost and recall responses. Immune responses were detected inside the perimeter and outside the exposure zone, which implies more-extensive spore exposure than was predicted. Despite postexposure prophylaxis with antibiotics, inhalation of B. anthracis spores resulted in stimulation of the immune system and possibly subclinical infection, and the greater the exposure, the more complete the immune response. The significance of low-level exposure should not be underestimated.

Heat activation/shock temperatures for Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores.

Assessing true numbers of viable anthrax spores is complex. Optimal heat activation conditions vary with species, media and germinants. Published time/temperature combinations for Bacillus anthracis spores range from 60 degrees C for 1, post-heating counts were less than their pre-heating counterparts on between 71% and 88% of occasions. A high probability was found of viable spore counts differing significantly from counts determined microscopically, with differences of almost 1 log possible. Viable counts were lower than microscopic counts in 15 of 18 tests.

Simultaneous identification and verification of Bacillus anthracis.

Specific identification of Bacillus anthracis (B. anthracis) is vital for the accurate treatment of afflicted personnel during biological warfare situations and civilian terrorist attacks. In order to accomplish this, we have subjected the lysates from B. anthracis to affinity purification using monoclonal antibodies for the selected antigenic protein present in the bacteria. The bound antigenic protein was identified by multi-dimensional protein identification technology (MudPIT) to be a surface layer protein EA1. The same antigen was identified from the lysates from a few strains of B. anthracis demonstrating the observation to be common for B. anthracis strains. Hence, this presents an effective pathway for the identification of the bacteria present in unknown samples of various origins. Generation of a database containing the EA1 protein has been found to be useful in the database search of unknown samples.

Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis.

The suitability and sensitivity of two in vitro lateral-flow assays for detecting Clostridium botulinum neurotoxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid, solid, and high-fat-content foods were developed. The lateral-flow assays, one developed by the Naval Medical Research Center (Silver Spring, MD) and the other by Alexeter Technologies (Gaithersburg, MD), are based on the immunodetection of BoNT types A, B, and E. The assays were found to be rapid and easy to perform with minimum requirements for laboratory equipment or skills. They can readily detect 10 ng/ml of BoNT types A and B and 20 ng/ml of BoNT type E. Compared to other in vitro detection methods, these assays are less sensitive, and the assessment of a result is strictly qualitative. However, the assay was found to be simple to use and to require minimal training. The assays successfully detected BoNT types A, B, and E in a wide variety of foods, suggesting their potential usefulness as a preliminary screening system for triaging food samples with elevated BoNT levels in the event of a C. botulinum contamination event.