Elad Milrot

Background: Botulism is a rare but potentially fatal neuroparalytic illness caused by Clostridium botulinum neurotoxins (BoNTs). While adult cases usually result from foodborne exposure or wound infection, intestinal colonization is exceedingly uncommon. Diagnosis can be delayed by overlap with other neuromuscular syndromes, and confirmation requires specialized assays. Anti-GQ1b antibodies, classically associated with Miller–Fisher syndrome (MFS), have rarely been reported in confirmed botulism, raising questions about shared pathophysiology. Case Presentation: We describe an adult patient with acute dyspnea, xerostomia, and cranial neuropathies. No foodborne source was identified, but intestinal colonization of BoNT/B toxigenic Clostridium botulinum was confirmed by stool enrichment and mouse lethality bioassay. The patient improved promptly following heptavalent antitoxin. Unexpectedly, anti-GQ1b antibodies were detected during recovery, a finding typically linked to MFS rather than botulism. Discussion: This case illustrates the diagnostic challenges of sporadic cases of botulism, especially when respiratory compromise and autonomic dysfunction dominate the initial presentation. The autoantibodies finding raises the possibility of molecular mimicry, whereby toxin–ganglioside interactions expose neuronal epitopes and trigger an immune response. While causality cannot be proven, the overlap between botulism and GQ1b-positive neuropathies merits further investigation. Conclusions: Clinicians should maintain high suspicion for botulism in adults with acute dyspnea, especially when associated with cranial neuropathies, even in the absence of foodborne exposure. Anti-ganglioside antibodies in this context should be interpreted with caution, as they do not exclude botulism but may highlight immunological overlap with autoimmune neuropathies.

Background: Botulism is a rare but potentially fatal neuroparalytic illness caused by Clostridium botulinum neurotoxins (BoNTs). While adult cases usually result from foodborne exposure or wound infection, intestinal colonization is exceedingly uncommon. Diagnosis can be delayed by overlap with other neuromuscular syndromes, and confirmation requires specialized assays. Anti-GQ1b antibodies, classically associated with Miller–Fisher syndrome, have rarely been reported in confirmed botulism, raising questions about shared pathophysiology. Case Presentation: We describe an adult patient with acute dyspnea, xerostomia, and cranial neuropathies. No foodborne source was identified, but intestinal colonization of BoNT/B toxigenic Clostridium botulinum was confirmed by stool enrichment and mouse lethality bioassay. The patient improved promptly following heptavalent antitoxin. Unexpectedly, anti-GQ1b antibodies were detected during recovery, a finding typically linked to MFS rather than botulism. Discussion: This case illustrates the diagnostic challenges of sporadic cases of botulism, especially when respiratory compromise and autonomic dysfunction dominate the initial presentation. The autoantibodies finding raises the possibility of molecular mimicry, whereby toxin–ganglioside interactions expose neuronal epitopes and trigger an immune response. While causality cannot be proven, the overlap between botulism and GQ1bpositive neuropathies merits further investigation. Conclusions: Clinicians should maintain high suspicion for botulism in adults with acute dyspnea especially when associated with cranial neuropathies, even in the absence of foodborne exposure. Anti-ganglioside antibodies in this context should be interpreted with caution, as they do not exclude botulism but may highlight immunological overlap with autoimmune neuropathies.

Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3–4 h post infection (PI), and accumulates as the infection proceeds. By 10–24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis in 2019. Currently, the main method for identification of SARS-CoV-2 is a reverse transcription (RT)-PCR assay designed to detect viral RNA in oropharyngeal (OP) or nasopharyngeal (NP) samples. While the PCR assay is considered highly specific and sensitive, this method cannot determine the infectivity of the sample, which may assist in evaluation of virus transmissibility from patients and breaking transmission chains. Thus, cell-culture-based approaches such as cytopathic effect (CPE) assays are routinely employed for the identification of infectious viruses in NP/OP samples. Despite their high sensitivity, CPE assays take several days and require additional diagnostic tests in order to verify the identity of the pathogen. We have therefore developed a rapid immunofluorescence assay (IFA) for the specific detection of SARS-CoV-2 in NP/OP samples following cell culture infection. Initially, IFA was carried out on Vero E6 cultures infected with SARS-CoV-2 at defined concentrations, and infection was monitored at different time points. This test was able to yield positive signals in cultures infected with 10 pfu/ml at 12 hours postinfection (PI). Increasing the incubation time to 24 hours reduced the detectable infective dose to 1 pfu/ml. These IFA signals occur before the development of CPE. When compared to the CPE test, IFA has the advantages of specificity, rapid detection, and sensitivity, as demonstrated in this work.

Plague, caused by the human pathogen Yersinia pestis, is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca⁺²)- and iron (Fe⁺²)-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca⁺² and Fe⁺². The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an increased sensitivity and without any detectable compromise of the accuracy of the antibiotic susceptibility testing (E-test), critical for subsequent successful therapeutic interventions.

Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561L^D4, with cell wall degrading activity. An A561L^D4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561L^D4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561L^D4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561L^D4 increased the specific infectivity of PBCV-1 from about 25–30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates.

Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

Ricin and abrin are ribosome-inactivating proteins leading to inhibition of protein synthesis and cell death. These toxins are considered some of the most potent and lethal toxins against which there is no available antidote. Digital holographic microscopy (DHM) is a time-lapse, label-free, and noninvasive imaging technique that can provide phase information on morphological features of cells. In this study, we employed DHM to evaluate the morphological changes of cell lines during ricin and abrin intoxication. We showed that the effect of these toxins is characterized by a decrease in cell confluence and changes in morphological parameters such as cell area, perimeter, irregularity, and roughness. In addition, changes in optical parameters such as phase-shift, optical thickness, and effective-calculated volume were observed. These effects were completely inhibited by specific neutralizing antibodies. An enhanced intoxication effect was observed for preadherent compared to adherent cells, as was detected in early morphology changes and confirmed by annexin V/propidium iodide (PI) apoptosis assay. Detection of the dynamic changes in cell morphology at initial stages of cell intoxication by DHM emphasizes the highly sensitive and rapid nature of this method, allowing the early detection of active toxins.