Rapid turnaround multiplex sequencing of SARS-CoV-2: comparing tiling amplicon protocol performance

Genome sequencing is pivotal to SARS-CoV-2 surveillance, elucidating the emergence and global dissemination of acquired genetic mutations. Amplicon sequencing has proven very effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated the revision of sequencing protocols in order to maintain performance for emerging virus lineages. We compared the performance of Oxford Nanopore Technologies (ONT) Midnight and ARTIC tiling amplicon protocols using 196 Delta lineage SARS-CoV-2 clinical specimens, and 71 mostly Omicron lineage samples with S gene target failure (SGTF), reflecting circulating lineages in the United Kingdom during December 2021. 96-plexed nanopore sequencing was used. For Delta lineage samples, ARTIC v4 recovered the greatest proportion of >=90% complete genomes (81.1%; 159/193), followed by Midnight (71.5%; 138/193) and ARTIC v3 (34.1%; 14/41). Midnight protocol however yielded higher average genome recovery (mean 98.8%) than ARTIC v4 (98.1%) and ARTIC v3 (75.4%), resulting in less ambiguous final consensus assemblies overall. Explaining these observations were ARTIC v4's superior genome recovery in low viral titre/high cycle threshold (Ct) samples and inferior performance in high titre/low Ct samples, where Midnight excelled. We evaluated Omicron sequencing performance using a revised Midnight primer mix alongside the latest ARTIC v4.1 primers, head-to-head with the existing commercially available Midnight and ARTIC v4 protocols. The revised protocols both improved considerably the recovery of Omicron genomes and exhibited similar overall performance to one another. Revised Midnight protocol recovered >=90% complete genomes for 85.9% (61/71) of Omicron samples vs. 88.7% (63/71) for ARTIC v4.1. Approximate cost per sample for Midnight (12GBP) is lower than ARTIC (16GBP) while hands-on time is considerably lower for Midnight (~7 hours) than ARTIC protocols (~9.5 hours).

Cytokine autoantibodies are stable throughout the haematopoietic stem cell transplantation course and are associated with distinct biomarker and blood cell profiles

Cytokine-specific autoantibodies (c-aAbs) represent an emerging field in endogenous immunodeficiencies, and the immunomodulatory potential of c-aAbs is now well documented. Here, we investigated the hypothesis that c-aAbs affects inflammatory, immunoregulatory and injury-related processes and hence the clinical outcome of haematopoietic stem cell transplantation (HSCT). C-aAbs against IL-1α, IL-6, IL-10, IFNα, IFNγ and GM-CSF were measured in 131 HSCT recipients before and after (days + 7, + 14, + 28) HSCT and tested for associations with 33 different plasma biomarkers, leukocyte subsets, platelets and clinical outcomes, including engraftment, GvHD and infections. We found that c-aAb levels were stable over the course of HSCT, including at high titres, with few individuals seeming to acquire high-titre levels of c-aAbs. Both patients with stable and those with acquired high-titre c-aAb levels displayed significant differences in biomarker concentrations and blood cell counts pre-HSCT and at day 28, and the trajectories of these variables varied over the course of HSCT. No clinical outcomes were associated with high-titre c-aAbs. In this first study of c-aAbs in HSCT patients, we demonstrated that high-titre levels of c-aAb may both persist and emerge in patients over the course of HSCT and may be associated with altered immune biomarkers and cell profiles.

A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilising immunity against SARS-CoV-2 infection

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

Anticoagulation for Stroke Prevention after Restoration of Haemostasis with Emicizumab in Acquired Haemophilia A

Acquired haemophilia A (AHA) is a rare haemorrhagic disorder caused by the development of autoantibodies inhibiting factor VIII function. It predominantly affects the elderly, who are often burdened with a considerable number of comorbidities, and can result in life-threatening bleeding. The management of AHA consists of two aspects: inhibitor eradication with an immunomodulator and bleed control with a bypassing agent. Here we present a case of AHA with a high titre inhibitor in a patient with extensive comorbidities and atrial fibrillation in whom inhibitor eradication could not be achieved within a few weeks using corticosteroids alone. Due to coronavirus disease (COVID)-19 restrictions and complications of care, emicizumab offered an effective and convenient therapy, not only sparing the need for continued and intensified inhibitor eradication, but also allowing anticoagulation for stroke prophylaxis.

Central and peripheral delivery of AAV9-SMN target different pathomechanisms in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy uses a single, high titre intravenous bolus of AAV9-SMN resulting in impressive, yet limited amelioration of the clinical phenotype. However, risks of this treatment include liver toxicity. Intrathecal administration is under clinical trial but was interrupted due to safety concerns in a concomitant animal study. As there is no direct comparison between the different delivery strategies while avoiding high dose toxicity, we injected SMA mice with low dose scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. Here, IV injections restored SMN in peripheral tissues but not CNS, while ICV injections mildly increased SMN in the periphery and the CNS. Consequently, only ICV treatment rescued motor neuron degeneration. Surprisingly, both treatments resulted in an impressive rescue of survival, weight, motor function, and peripheral phenotypes including liver and pancreas pathology. Our work highlights independent contributions of peripheral organs to SMA pathology and suggests that treatments should not be restricted to the motor neuron.

Rapid, dose-dependent and efficient inactivation of surface dried SARS-CoV-2 by 254 nm UV-C irradiation

Background: The COVID-19 pandemic urges for cheap, reliable, and rapid technologies for disinfection and decontamination. One frequently proposed method is ultraviolet (UV)-C irradiation. UV-C doses necessary to achieve inactivation of high-titre SARS-CoV-2 are poorly defined. Aim: We investigated whether short exposure of SARS-CoV-2 to UV-C irradiation sufficiently reduces viral infectivity and doses necessary to achieve an at least 6-log reduction in viral titres. Methods: Using a box and two handheld systems designed to decontaminate objects and surfaces, we evaluated the efficacy of 254 nm UV-C treatment to inactivate surface dried high-titre SARS-CoV-2. Results: Drying for 2 hours did not have a major impact on the infectivity of SARS-CoV-2, indicating that exhaled virus in droplets or aerosols stays infectious on surfaces for at least a certain amount of time. Short exposure of high titre surface dried virus (3–5*10^6 IU/ml) with UV-C light (16 mJ/cm2) resulted in a total inactivation of SARS-CoV-2. Dose-dependency experiments revealed that 3.5 mJ/cm2 were still effective to achieve a > 6-log reduction in viral titres, whereas 1.75 mJ/cm2 lowered infectivity only by one order of magnitude. Conclusions: SARS-CoV-2 is rapidly inactivated by relatively low doses of UV-C irradiation and the relationship between UV-C dose and log-viral titre reduction of surface residing SARS-CoV-2 is nonlinear. Our findings emphasize that it is necessary to assure sufficient and complete exposure of all relevant areas by integrated UV-C doses of at least 3.5 mJ/cm2 at 254 nm. Altogether, UV-C treatment is an effective non-chemical option to decontaminate surfaces from high-titre infectious SARS-CoV-2.

SARS-CoV-2 Infection in Fully Vaccinated Individuals of Old Age Strongly Boosts the Humoral Immune Response

Prophylactic vaccination against SARS-CoV-2 is one of the most important measures to contain the COVID-19 pandemic. Recently, break-through infections following vaccination against this virus have been reported. Here, we describe the humoral immune response of break-through infections in fully vaccinated individuals of old age from an outbreak in a nursing home. In cooperation with the local health authority, blood samples from fully vaccinated and infected as well as fully vaccinated and uninfected residents of the nursing home were collected 4 weeks after the onset of the outbreak. The humoral immune response was determined in a neutralisation assay with replication-competent virus isolates and by a quantitative ELISA. In this outbreak a total of 23 residents and four health care workers were tested positive for SARS-CoV-2. Four residents were unvaccinated, including one with a severe course of disease who later severe disease course who later succumbed to infection. Despite their old age, all vaccinated residents showed no or only mild disease. Comparison of the humoral immune response revealed significantly higher antibody levels in fully vaccinated infected individuals compared to fully vaccinated uninfected individuals (p < 0.001). Notably, although only a minority of the vaccinated uninfected group showed neutralisation capacity against SARS-CoV-2, all vaccinated and infected individuals showed high-titre neutralisation of SARS-CoV-2 including the alpha and beta variant. Large SARS-CoV-2 outbreaks can occur in fully vaccinated populations, but seem to associate with mild disease. SARS-CoV-2 infection in fully vaccinated individuals is a strong booster of the humoral immune response providing enhanced neutralisation capacity against immune evasion variants.

Identification of a novel statovirus in a faecal sample from a calf with enteric disease

A novel clade of RNA viruses was identified in the mammalian gastrointestinal tract by next-generation sequencing. Phylogenetically, these viruses are related to the genera Tombusviridae (plant viruses) and Flaviviridae, which includes mammalian, avian and insect hosts. Named in line with their characterization as stool-associated Tombus-like viruses, it is unclear if statoviruses infect mammals or are dietary in origin. Here, metagenomic sequencing of faecal material collected from a 10-week-old calf with enteric disease found that 20 % of the reads mapped to a de novo-assembled 4 kb contig with homology to statoviruses. Phylogenetic analysis of the statovirus genome found a clear evolutionary relationship with statovirus A, but, with only 47 % similarity, we propose that the statovirus sequence presents a novel species, statovirus F. A TaqMan PCR targeting statovirus F performed on faecal material found a cycle threshold of 11, suggesting a high titre of virus shed from the calf with enteric disease. A collection of 48 samples from bovine enteric disease diagnostic submissions were assayed by PCR to investigate statovirus F prevalence and 6 of 48 (12.5 %) were positive. An ELISA to detect antibodies to the coat protein found that antibodies to statovirus F were almost ubiquitous in bovine serum. Combined, the PCR and ELISA results suggest that statovirus F commonly infects cattle. Further research is needed to elucidate the aetiological significance of statovirus infection.

The frequency of detection of anti-HBc in blood donors from four regions of Russia

Introduction. Occult hepatitis B virus (HBV) revelation in HBV nuclear antigen testing is of particular importance to prevent transfusion infection.Aim — the identification of factors affecting the anti-HBc detection rate in donated transfusable blood components from different regions of Russia.Materials and methods. A cohort screening single-stage epidemiological study was conducted with 2,000 donor blood samples, 500 samples per each of four regions of the country, the Republics of Crimea (Simferopol) and Sakha (Yakutia), the cities of Saransk and Orenburg. Data on 968 blood samples from the National Research Center for Hematology’s donor bank were used as reference. The testing targeted HBV nuclear antigen antibodies. Positive donated blood samples were additionally tested for IgM and virus surface antigen antibodies using Abbott and Vector-Best commercial reagent kits.Results. Donor demographic profiles differed insignificantly across members of the Russian Federation. Males predominated among the donors (69.6 %). Anti-HBc was detected in 219 of 2,000 samples examined (10.9 %). The donor blood sample anti-HBc detection rate ranged from 6.0 to 21.6 %, depending on the region. Anti-HBc-positive proportions in Orenburg, Crimea, Mordovia and Sakha comprised 8.2, 8.0, 6.0 and 21.6 %, respectively (p < 0.01). First-time donors had anti-HBc in 8.06, regular donors — in 11.29 % cases. The anti-HBc detection rate varied with donor’s age, being zero or near 1 % in 20-yo or younger people. Acute HBV antibodies had zero rate in Orenburg at zero or low-titre (< 100 mIU/mL) protective antibodies; 31 total samples, 15 low-titre and 16 negative for protective antibodies. In Simferopol, acute phase antibodies were negative in 7 blood samples containing high-titre protective antibodies (> 100 mIU/mL) and in 5.0 % samples with their low or zero levels. In Yakutian donors, acute phase antibodies were revealed only at protective antibodies negative. In Saransk, this marker was equal-proportion at zero and high-titre protective antibodies (3.3 % each).Conclusion. Transfusion component procurement from younger donors should be prioritised as enhancing haemotransfusion viral safety. Positive occult HBV tests were less common in regions with low HBV incidence.

A case report on COVID-19 associated multisystem inflammatory syndrome in children

As severe acute respiratory syndrome coronavirus 2 continues to spread worldwide, there have been increasing reports from different parts of India during second wave of infection describing children and adolescents with COVID-19-associated multisystem inflammatory conditions. We describe clinical features and management in children with multisystem inflammatory syndrome children (MIS-C) from the Delhi metropolitan area, which had a high incidence of coronavirus disease 2019 (COVID-19). We report a similar case presenting with persisting fever, rashes, bulbar conjunctivitis, abdominal pain, decreased urine output and shock. Initial reports suggestive of high inflammatory markers, neutrophilic leukocytosis, high D-dimer and found to have COVID-19 IgG antibodies positive in high titre. Managed successfully in PICU with bolos fluids, intravenous antibiotics, steroids and anticoagulation.