Impact of an autophagy-inducing peptide on immunogenicity and protection efficacy of an adenovirus-vectored SARS-CoV-2 vaccine.

Because of continual generation of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is critical to design the next generation of vaccines to combat the threat posed by SARS-CoV-2 variants. We developed human adenovirus (HAd) vector-based vaccines (HAd-Spike/C5 and HAd-Spike) that express the whole Spike (S) protein of SARS-CoV-2 with or without autophagy-inducing peptide C5 (AIP-C5), respectively. Mice or golden Syrian hamsters immunized intranasally (i.n.) with HAd-Spike/C5 induced similar levels of S-specific humoral immune responses and significantly higher levels of S-specific cell-mediated immune (CMI) responses compared with HAd-Spike vaccinated groups. These results indicated that inclusion of AIP-C5 induced enhanced S-specific CMI responses and similar levels of virus-neutralizing titers against SARS-CoV-2 variants. To investigate the protection efficacy, golden Syrian hamsters immunized i.n. either with HAd-Spike/C5 or HAd-Spike were challenged with SARS-CoV-2. The lungs and nasal turbinates were collected 3, 5, 7, and 14 days post challenge. Significant reductions in morbidity, virus titers, and lung histopathological scores were observed in immunized groups compared with the mock- or empty vector-inoculated groups. Overall, slightly better protection was seen in the HAd-Spike/C5 group compared with the HAd-Spike group.

Inactivation of HCoV-NL63 and SARS-CoV-2 in aqueous solution by 254 nm UV-C.

Ultraviolet germicidal irradiation (UVGI) is a highly effective means of inactivating many bacteria, viruses, and fungi. UVGI is an attractive viral mitigation strategy against coronaviruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-2019 (COVID-19) pandemic. This investigation measures the susceptibility of two human coronaviruses to inactivation by 254 nm UV-C radiation. Human coronavirus NL63 and SARS-CoV-2 were irradiated in a collimated, dual-beam, aqueous UV reactor. By measuring fluence and integrating it in real-time, this reactor accounts for the lamp output transients during UVGI exposures. The inactivation rate constants of a one-stage exponential decay model were determined to be 2.050 cm2/mJ and 2.098 cm2/mJ for the NL63 and SARS-CoV-2 viruses, respectively. The inactivation rate constant for SARS-CoV-2 is within 2% of that of NL63, indicating that in identical inactivation environments, very similar UV 254 nm deactivation susceptibilities for these two coronaviruses would be achieved. Given the inactivation rate constant obtained in this study, doses of 1.1 mJ/cm2, 2.2 mJ/cm2, and 3.3 mJ/cm2 would result in a 90%, 99%, and 99.9% inactivation of the SARS-CoV-2 virus, respectively. The inactivation rate constant obtained in this study is significantly higher than values reported from many 254 nm studies, which suggests greater UV susceptibility to the UV-C than what was believed. Overall, results from this study indicate that 254 nm UV-C is effective for inactivation of human coronaviruses, including SARS-CoV-2.

SARS-CoV-2 Prevalence and Variant Surveillance among Cats in Pittsburgh, Pennsylvania, USA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects many mammals, and SARS-CoV-2 circulation in nonhuman animals may increase the risk of novel variant emergence. Cats are highly susceptible to SARS-CoV-2 infection, and there were cases of virus transmission between cats and humans. The objective of this study was to assess the prevalence of SARS-CoV-2 variant infection of cats in an urban setting. We investigated the prevalence of SARS-CoV-2 variant infections in domestic and community cats in the city of Pittsburgh (n = 272). While no cats tested positive for SARS-CoV-2 viral RNA, 35 cats (12.86%) tested SARS-CoV-2-antibody-positive. Further, we compared a cat-specific experimental lateral flow assay (eLFA) and species-agnostic surrogate virus neutralization assay (sVNT) for SARS-CoV-2 antibody detection in cats (n = 71). The eLFA demonstrated 100% specificity compared to sVNT. The eLFA also showed 100% sensitivity for sera with >90% inhibition and 63.63% sensitivity for sera with 40-89% inhibition in sVNT. Using a variant-specific pseudovirus neutralization assay (pVNT) and antigen cartography, we found the presence of antibodies to pre-Omicron and Omicron SARS-CoV-2 variants. Hence, this approach proves valuable in identifying cat exposure to different SARS-CoV-2 variants. Our results highlight the continued exposure of cats to SARS-CoV-2 and warrant coordinated surveillance efforts.

Little Brown Bats (Myotis lucifugus) Support the Binding of SARS-CoV-2 Spike and Are Likely Susceptible to SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), believed to have originated from a bat species, can infect a wide range of non-human hosts. Bats are known to harbor hundreds of coronaviruses capable of spillover into human populations. Recent studies have shown a significant variation in the susceptibility among bat species to SARS-CoV-2 infection. We show that little brown bats (LBB) express angiotensin-converting enzyme 2 receptor and the transmembrane serine protease 2, which are accessible to and support SARS-CoV-2 binding. All-atom molecular dynamics (MD) simulations revealed that LBB ACE2 formed strong electrostatic interactions with the RBD similar to human and cat ACE2 proteins. In summary, LBBs, a widely distributed North American bat species, could be at risk of SARS-CoV-2 infection and potentially serve as a natural reservoir. Finally, our framework, combining in vitro and in silico methods, is a useful tool to assess the SARS-CoV-2 susceptibility of bats and other animal species.

Optimized conditions for Listeria, Salmonella and Escherichia whole genome sequencing using the Illumina iSeq100 platform with point-and-click bioinformatic analysis.

Whole-genome sequencing (WGS) data have become an integral component of public health investigations and clinical diagnostics. Still, many veterinary diagnostic laboratories cannot afford to implement next generation sequencing (NGS) due to its high cost and the lack of bioinformatic knowledge of the personnel to analyze NGS data. Trying to overcome these problems, and make NGS accessible to every diagnostic laboratory, thirteen veterinary diagnostic laboratories across the United States (US) initiated the assessment of Illumina iSeq100 sequencing platform for whole genome sequencing of important zoonotic foodborne pathogens Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The work presented in this manuscript is a continuation of this multi-laboratory effort. Here, seven AAVLD accredited diagnostic laboratories explored a further reduction in sequencing costs and the usage of user-friendly platforms for genomic data analysis. Our investigation showed that the same genomic library quality could be achieved by using a quarter of the recommended reagent volume and, therefore a fraction of the actual price, and confirmed that Illumina iSeq100 is the most affordable sequencing technology for laboratories with low WGS demand. Furthermore, we prepared step-by-step protocols for genomic data analysis in three popular user-friendly software (BaseSpace, Geneious, and GalaxyTrakr), and we compared the outcomes in terms of genome assembly quality, and species and antimicrobial resistance gene (AMR) identification. No significant differences were found in assembly quality, and the three analysis methods could identify the target bacteria species. However, antimicrobial resistance genes were only identified using BaseSpace and GalaxyTrakr; and GalaxyTrakr was the best tool for this task.

Transmission of SARS-CoV-2 from humans to animals and potential host adaptation.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, can infect a wide range of mammals. Since its spread in humans, secondary host jumps of SARS-CoV-2 from humans to multiple domestic and wild populations of mammals have been documented. Understanding the extent of adaptation to these animal hosts is critical for assessing the threat that the spillback of animal-adapted SARS-CoV-2 into humans poses. We compare the genomic landscapes of SARS-CoV-2 isolated from animal species to that in humans, profiling the mutational biases indicative of potentially different selective pressures in animals. We focus on viral genomes isolated from mink (Neovison vison) and white-tailed deer (Odocoileus virginianus) for which multiple independent outbreaks driven by onward animal-to-animal transmission have been reported. We identify five candidate mutations for animal-specific adaptation in mink (NSP9_G37E, Spike_F486L, Spike_N501T, Spike_Y453F, ORF3a_L219V), and one in deer (NSP3a_L1035F), though they appear to confer a minimal advantage for human-to-human transmission. No considerable changes to the mutation rate or evolutionary trajectory of SARS-CoV-2 has resulted from circulation in mink and deer thus far. Our findings suggest that minimal adaptation was required for onward transmission in mink and deer following human-to-animal spillover, highlighting the 'generalist' nature of SARS-CoV-2 as a mammalian pathogen.

A CNN model for predicting binding affinity changes between SARS-CoV-2 spike RBD variants and ACE2 homologues.

The cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves the association of its receptor binding domain (RBD) with human angiotensin converting enzyme 2 (hACE2) as the first crucial step. Efficient and reliable prediction of RBD-hACE2 binding affinity changes upon amino acid substitutions can be valuable for public health surveillance and monitoring potential spillover and adaptation into non-human species. Here, we introduce a convolutional neural network (CNN) model trained on protein sequence and structural features to predict experimental RBD-hACE2 binding affinities of 8,440 variants upon single and multiple amino acid substitutions in the RBD or ACE2. The model achieves a classification accuracy of 83.28% and a Pearson correlation coefficient of 0.85 between predicted and experimentally calculated binding affinities in five-fold cross-validation tests and predicts improved binding affinity for most circulating variants. We pro-actively used the CNN model to exhaustively screen for novel RBD variants with combinations of up to four single amino acid substitutions and suggested candidates with the highest improvements in RBD-ACE2 binding affinity for human and animal ACE2 receptors. We found that the binding affinity of RBD variants against animal ACE2s follows similar trends as those against human ACE2. White-tailed deer ACE2 binds to RBD almost as tightly as human ACE2 while cattle, pig, and chicken ACE2s bind weakly. The model allows testing whether adaptation of the virus for increased binding with other animals would cause concomitant increases in binding with hACE2 or decreased fitness due to adaptation to other hosts.

Rapid detection of novel coronavirus SARS-CoV-2 by RT-LAMP coupled solid-state nanopores.

The current pandemic of COVID-19 caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concerns. Rapid and accurate testing of SARS-CoV-2 is urgently needed for early detection and control of the disease spread. Here, we present an RT-LAMP coupled glass nanopore digital counting method for rapid detection of SARS-CoV-2. We validated and compared two one-pot RT-LAMP assays targeting nucleocapsid (N) and envelop (E) genes. The nucleocapsid assay was adopted due to its quick time to positive and better copy number sensitivity. For qualitative positive/negative classification of a testing sample, we used the glass nanopore to digitally count the RT-LAMP amplicons and benchmarked the event rate with a threshold. Due to its intrinsic single molecule sensitivity, nanopore sensors could capture the amplification dynamics more rapidly (quick time to positive). We validated our RT-LAMP coupled glass nanopore digital counting method for SARS-CoV-2 detection by using both spiked saliva samples and COVID-19 clinical nasopharyngeal swab samples. The results obtained showed excellent agreement with the gold standard RT-PCR assay. With its integration capability, the electronic nanopore digital counting platform has significant potential to provide a rapid, sensitive, and specific point-of-care assay for SARS-CoV-2.

Transmission of SARS-CoV-2 from humans to a 16-year-old domestic cat with comorbidities in Pennsylvania, USA.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), besides causing human infection, has been shown to naturally infect several susceptible animal species including large cats (tigers, lions, pumas, spotted leopards), dogs, cats, ferrets, gorillas and minks. Cats and minks are continuing to be the most reported species with SARS-CoV-2 infections among animals but it needs to be investigated further. METHODS AND RESULTS: We report the detection of SARS-CoV-2 from a domestic cat that exhibited respiratory disease after being exposed to SARS-CoV-2 virus from humans in the same household. SARS-CoV-2 RNA was detected in two oropharyngeal swabs collected at two time points, 11 days apart; the first, when the cat was reported to be sick and the second, before euthanasia due to poor prognosis. The viral nucleic acid detected at two time points showed no genomic variation and resembled the clade GH circulating in humans in the United States. Clinical and pathological findings noted in this 16-year-old cat were consistent with respiratory and cardiac insufficiency. CONCLUSIONS: SARS-CoV-2 viral infection was likely an incidental clinical finding, as the virus was not detected in fixed lungs, heart, or kidney tissues. Only fresh lung tissue collected at necropsy showed the presence of viral nucleic acid, albeit at a very low level. Further research is needed to clarify the clinical course of SARS-CoV-2 in companion animals of advanced age and underlying cardiac disease.

Computational prediction of the effect of amino acid changes on the binding affinity between SARS-CoV-2 spike RBD and human ACE2.

The association of the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with human angiotensin-converting enzyme 2 (hACE2) represents the first required step for cellular entry. SARS-CoV-2 has continued to evolve with the emergence of several novel variants, and amino acid changes in the RBD have been implicated with increased fitness and potential for immune evasion. Reliably predicting the effect of amino acid changes on the ability of the RBD to interact more strongly with the hACE2 can help assess the implications for public health and the potential for spillover and adaptation into other animals. Here, we introduce a two-step framework that first relies on 48 independent 4-ns molecular dynamics (MD) trajectories of RBD-hACE2 variants to collect binding energy terms decomposed into Coulombic, covalent, van der Waals, lipophilic, generalized Born solvation, hydrogen bonding, π-π packing, and self-contact correction terms. The second step implements a neural network to classify and quantitatively predict binding affinity changes using the decomposed energy terms as descriptors. The computational base achieves a validation accuracy of 82.8% for classifying single-amino acid substitution variants of the RBD as worsening or improving binding affinity for hACE2 and a correlation coefficient of 0.73 between predicted and experimentally calculated changes in binding affinities. Both metrics are calculated using a fivefold cross-validation test. Our method thus sets up a framework for screening binding affinity changes caused by unknown single- and multiple-amino acid changes offering a valuable tool to predict host adaptation of SARS-CoV-2 variants toward tighter hACE2 binding.

Limited window for donation of convalescent plasma with high live-virus neutralizing antibody titers for COVID-19 immunotherapy.

Millions of individuals who have recovered from SARS-CoV-2 infection may be eligible to participate in convalescent plasma donor programs, yet the optimal window for donating high neutralizing titer convalescent plasma for COVID-19 immunotherapy remains unknown. Here we studied the response trajectories of antibodies directed to the SARS-CoV-2 surface spike glycoprotein and in vitro SARS-CoV-2 live virus neutralizing titers (VN) in 175 convalescent donors longitudinally sampled for up to 142 days post onset of symptoms (DPO). We observed robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 that persist, in the aggregate, for at least 100 DPO. However, there is a notable decline in VN titers ≥160 for convalescent plasma therapy, starting 60 DPO. The results also show that individuals 30 years of age or younger have significantly lower VN, IgG and IgM antibody titers than those in the older age groups; and individuals with greater disease severity also have significantly higher IgM and IgG antibody titers. Taken together, these findings define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor.

Trans-Cinnamaldehyde and Eugenol Increase Acinetobacter baumannii Sensitivity to Beta-Lactam Antibiotics.

Multi-drug resistant (MDR) Acinetobacter baumannii is a major nosocomial pathogen causing a wide range of clinical conditions with significant mortality rates. A. baumannii strains are equipped with a multitude of antibiotic resistance mechanisms, rendering them resistant to most of the currently available antibiotics. Thus, there is a critical need to explore novel strategies for controlling antibiotic resistance in A. baumannii. This study investigated the efficacy of two food-grade, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde (TC) and eugenol (EG) in decreasing A. baumannii's resistance to seven ß-lactam antibiotics, including ampicillin, methicillin, meropenem, penicillin, aztreonam, amoxicillin, and piperacillin. Two MDR A. baumannii isolates (ATCC 17978 and AB 251847) were separately cultured in tryptic soy broth (∼6 log CFU/ml) containing the minimum inhibitory concentration (MIC) of TC or EG with or without the MIC of each antibiotic at 37°C for 18 h. A. baumannii strains not exposed to the PDAs or antibiotics served as controls. Following incubation, A. baumannii counts were determined by broth dilution assay. In addition, the effect of PDAs on the permeability of outer membrane and efflux pumps in A. baumannii was measured. Further, the effect of TC and EG on the expression of A. baumannii genes encoding resistance to ß-lactam antibiotics (blaP), efflux pumps (adeABC), and multi-drug resistant protein (mdrp) was studied using real-time quantitative PCR (RT-qPCR). The experiment was replicated three times with duplicate samples of each treatment and control. The results from broth dilution assay indicated that both TC and EG in combination with antibiotics increased the sensitivity of A. baumannii to all the tested antibiotics (P < 0.05). The two PDAs inhibited the function of A. baumannii efflux pump, (AdeABC), but did not increase the permeability of its outer membrane. Moreover, RT-qPCR data revealed that TC and EG down-regulated the expression of majority of the genes associated with ß-lactam antibiotic resistance, especially blaP and adeABC (P < 0.05). The results suggest that TC and EG could potentially be used along with ß-lactam antibiotics for controlling MDR A. baumannii infections; however, their clinical significance needs to be determined using in vivo studies.

Inhibition and Inactivation of Escherichia coli O157:H7 Biofilms by Selenium.

The present study investigated the efficacy of selenium (Se) in reduction of enterohemorrhagic Escherichia coli (EHEC) exopolysaccharide (EPS) synthesis, inhibition of biofilm formation at 25 and 4°C on polystyrene surface, and inactivation of mature EHEC biofilms in combination with hot water. Sterile 96-well polystyrene plates inoculated with EHEC (∼6.0 log CFU per well) were treated with a subinhibitory concentration (SIC) of Se, and biofilms were allowed to mature at 4 and 25°C for 96 h. Biofilm-associated bacterial population was determined by scraping and plating, whereas the extent of EPS production was determined using ruthenium red staining assay. Solid surface assay was used to study the effect of Se on early attachment of EHEC cells to polystyrene. The efficacy of Se in rapid inactivation of preformed, mature EHEC biofilm was investigated by treating biofilms on polystyrene plates with the MBC of Se in combination with hot water at 80°C with a contact time of 0 min, 30 s, 2 min, and 5 min. Furthermore, the effect of Se on EHEC biofilm architecture was visualized using confocal microscopy, whereas the effect of Se on EHEC biofilm genes was determined using real-time quantitative PCR (RT-qPCR). Finally, the potential feasibility of coating stainless steel surfaces with Se nanoparticles to inhibit EHEC biofilm formation was studied. Se reduced early attachment of planktonic cells, biofilm formation, and EPS synthesis in EHEC ( P < 0.05). Se in combination with hot water reduced biofilm-associated bacterial counts by 3 to 4 log CFU/mL at 5 min of exposure compared with the control ( P < 0.05). However, hot water treatment alone decreased biofilm-associated bacterial counts by only 1.0 log CFU/mL. RT-qPCR results revealed that Se down-regulated the transcription of critical genes associated with biofilm synthesis in EHEC ( P < 0.05). The results collectively suggest that Se could potentially be used to control EHEC biofilms in food processing environments, but appropriate applications need to be validated.

Inactivation of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 on cantaloupes by octenidine dihydrochloride.

The efficacy of a new generation disinfectant, octenidine dihydrochloride (OH), as wash and coating treatments for reducing Listeria monocytogenes (LM), Salmonella spp. (SAL), and Escherichia coli O157:H7 (EC) on cantaloupe was investigated. Cantaloupe rind plugs inoculated separately with the three bacterial species (∼8 log CFU/cm(2)) were washed for 1, 3, 5 min at 25 °C in water, or chlorine (200 ppm), ethanol (1%), OH (0.01, 0.05, 0.1%) and surviving populations were measured after treatment. Additionally, inoculated cantaloupe rind plugs were coated with 2% chitosan or chitosan containing OH (0.01, 0.05, 0.1%) and sampled for surviving pathogens. Subsequently, the antimicrobial efficacy of OH wash and coating (0.1, 0.2%) on whole cantaloupes was determined. All OH wash reduced LM, SAL, and EC on cantaloupe rinds by > 5 log CFU/cm(2) by 2 min, and reduced populations to undetectable levels (below 2 log CFU/cm(2)) by 5 min (P < 0.05). Similarly, OH coating on cantaloupe rinds reduced the pathogens by 3-5 log /cm(2) (P < 0.05). Washing and coating whole cantaloupes with OH reduced the three pathogens by at least 5 log and 2 log CFU/cm(2), respectively (P < 0.05). Results suggest that OH could be used as antimicrobial wash and coating to reduce LM, SAL, and EC on cantaloupes.

Efficacy of Plant-Derived Antimicrobials in Controlling Enterohemorrhagic Escherichia coli Virulence In Vitro.

Escherichia coli O157:H7 is a major foodborne pathogen that can cause serious human illness characterized by hemorrhagic diarrhea and kidney failure. The pathology of enterohemorrhagic E. coli O157:H7 (EHEC) infection is primarily mediated by verotoxins, which bind to the globotriaosylceramide receptor on host cells. Antibiotics are contraindicated for treating EHEC infection because they lead to increased verotoxin release, thereby increasing the risk of renal failure and death in patients. Thus, alternative strategies are needed for controlling EHEC infections in humans. This study investigated the effect of subinhibitory concentrations of five plant-derived antimicrobial agents (PDAs) that are generally considered as safe, i.e., trans-cinnamaldehyde, eugenol, carvacrol, thymol, and ß-resorcylic acid, on EHEC motility, adhesion to human intestinal epithelial cells, verotoxin production, and virulence gene expression. All tested PDAs reduced EHEC motility and attachment to human intestinal epithelial cells (P < 0.05) and decreased verotoxin synthesis by EHEC. The reverse transcription real-time PCR data revealed that PDAs decreased the expression of critical virulence genes in EHEC (P < 0.05). The results collectively suggest that these PDAs could be used to reduce EHEC virulence, but follow-up studies in animal models are necessary to validate these findings.

Inhibiting Microbial Toxins Using Plant-Derived Compounds and Plant Extracts.

Many pathogenic bacteria and fungi produce potentially lethal toxins that cause cytotoxicity or impaired cellular function either at the site of colonization or other locations in the body through receptor-mediated interactions. Various factors, including biotic and abiotic environments, competing microbes, and chemical cues affect toxin expression in these pathogens. Recent work suggests that several natural compounds can modulate toxin production in pathogenic microbes. However, studies explaining the mechanistic basis for their effect are scanty. This review discusses the potential of various plant-derived compounds for reducing toxin production in foodborne and other microbes. In addition, studies highlighting their anti-toxigenic mechanism(s) are discussed.