Plasmodium falciparum OPA3-like protein (PfOPA3) is essential for maintenance of mitochondrial homeostasis and parasite proliferation.

Metabolic pathways and proteins responsible for maintaining mitochondrial dynamics and homeostasis in the Plasmodium parasite, the causative agent of malaria, remain to be elucidated. Here, we identified and functionally characterized a novel OPA3-like domain-containing protein in P. falciparum (PfOPA3). We show that PfOPA3 is expressed in the intraerythrocytic stages of the parasite and localizes to the mitochondria. Inducible knock-down of PfOPA3 using GlmS ribozyme hindered the normal intraerythrocytic cycle of the parasites; specifically, PfOPA3-iKD disrupted parasite development as well as parasite division and segregation at schizont stages, which resulted in a drastic reduction in the number of merozoites progenies. Parasites lacking PfOPA3 show severe defects in the development of functional mitochondria; the mitochondria showed reduced activity of mtETC but not ATP synthesis, as evidenced by reduced activity of complex III of the mtETC, and increased sensitivity for drugs targeting DHODH as well as complex III, but not to the drugs targeting complex V. Further, PfOPA3 downregulation leads to reduction in the level of mitochondrial proton transport uncoupling protein (PfUCP) to compensate reduced activity of complex III and maintain proton efflux across the inner membrane. The reduced activity of DHODH, which is responsible for pyrimidine biosynthesis required for nuclear DNA synthesis, resulted in a significant reduction in parasite nuclear division and generation of progeny. In conclusion, we show that PfOPA3 is essential for the functioning of mtETC and homeostasis required for the development of functional mitochondria as well as for parasite segregation, and thus PfOPA3 is crucial for parasite survival during blood stages.

Fluorescent and Bioluminescent Reporter Mouse-Adapted Ebola Viruses Maintain Pathogenicity and Can Be Visualized in Vivo.

Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA) EBOVs, including 1 based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 73% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using handheld blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models.

Epidemiologic, Immunologic, and Virus Characteristics in Patients With Paired Severe Acute Respiratory Syndrome Coronavirus 2 Serology and Reverse-Transcription Polymerase Chain Reaction Testing.

BACKGROUND: The natural history and clinical progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections can be better understood using combined serological and reverse-transcription polymerase chain reaction (RT-PCR) testing. METHODS: Nasopharyngeal swabs and serum were collected at a single time-point from patients at an urban, public hospital during August-November 2020 and tested for SARS-CoV-2 using RT-PCR, viral culture, and anti-spike pan-immunoglobulin antibody testing. Participant demographics and symptoms were collected through interview. The χ 2 and Fisher exact tests were used to identify associations between RT-PCR and serology results with presence of viable virus and frequency of symptoms. RESULTS: Among 592 participants, 129 (21.8%) had evidence of SARS-CoV-2 infection by RT-PCR or serology. Presence of SARS-CoV-2 antibodies was strongly associated with lack of viable virus (P = .016). COVID-19 symptom frequency was similar for patients testing RT-PCR positive/seronegative and patients testing RT-PCR positive/seropositive. Patients testing RT-PCR positive/seronegative reported headaches, fatigue, diarrhea, and vomiting at rates not statistically significantly different from those testing RT-PCR negative/seropositive. CONCLUSIONS: While patients testing SARS-CoV-2 seropositive were unlikely to test positive for viable virus and were therefore at low risk for forward transmission, coronavirus disease 2019 (COVID-19) symptoms were common. Paired SARS-CoV-2 RT-PCR and antibody testing provides more nuanced understanding of patients' COVID-19 status.

Relationship of SARS-CoV-2 Antigen and Reverse Transcription PCR Positivity for Viral Cultures.

We assessed the relationship between antigen and reverse transcription PCR (RT-PCR) test positivity and successful virus isolation. We found that antigen test results were more predictive of virus recovery than RT-PCR results. However, virus was isolated from some antigen-negative and RT-PCR‒positive paired specimens, providing support for the Centers for Disease Control and Prevention antigen testing algorithm.

Evaluation of Abbott BinaxNOW Rapid Antigen Test for SARS-CoV-2 Infection at Two Community-Based Testing Sites - Pima County, Arizona, November 3-17, 2020.

Rapid antigen tests, such as the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW), offer results more rapidly (approximately 15-30 minutes) and at a lower cost than do highly sensitive nucleic acid amplification tests (NAATs) (1). Rapid antigen tests have received Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use in symptomatic persons (2), but data are lacking on test performance in asymptomatic persons to inform expanded screening testing to rapidly identify and isolate infected persons (3). To evaluate the performance of the BinaxNOW rapid antigen test, it was used along with real-time reverse transcription-polymerase chain reaction (RT-PCR) testing to analyze 3,419 paired specimens collected from persons aged ≥10 years at two community testing sites in Pima County, Arizona, during November 3-17, 2020. Viral culture was performed on 274 of 303 residual real-time RT-PCR specimens with positive results by either test (29 were not available for culture). Compared with real-time RT-PCR testing, the BinaxNOW antigen test had a sensitivity of 64.2% for specimens from symptomatic persons and 35.8% for specimens from asymptomatic persons, with near 100% specificity in specimens from both groups. Virus was cultured from 96 of 274 (35.0%) specimens, including 85 (57.8%) of 147 with concordant antigen and real-time RT-PCR positive results, 11 (8.9%) of 124 with false-negative antigen test results, and none of three with false-positive antigen test results. Among specimens positive for viral culture, sensitivity was 92.6% for symptomatic and 78.6% for asymptomatic individuals. When the pretest probability for receiving positive test results for SARS-CoV-2 is elevated (e.g., in symptomatic persons or in persons with a known COVID-19 exposure), a negative antigen test result should be confirmed by NAAT (1). Despite a lower sensitivity to detect infection, rapid antigen tests can be an important tool for screening because of their quick turnaround time, lower costs and resource needs, high specificity, and high positive predictive value (PPV) in settings of high pretest probability. The faster turnaround time of the antigen test can help limit transmission by more rapidly identifying infectious persons for isolation, particularly when used as a component of serial testing strategies.

Molecular characterization of varicella zoster virus isolated from clinical samples in India.

Background & objectives: Varicella zoster virus (VZV) strains are classified into six different clades based on the sequencing of its genome. Clades 4 and 5 are reported from India based on the single-nucleotide polymorphism (SNP). Till now, multiple clade circulations using partial sequences have been reported from India due to the lack of availability of the full VZV genome sequence. This study conducted a genome sequencing of VZV in India to identify circulating clade. Methods: Four clinical samples obtained from symptomatic patients tested positive for VZV by real-time PCR were used. These four samples were preferred to retrieve the genomic VZV sequence using the next-generation sequencing method. A reference-based assembly method was used to retrieve the genome of VZV, which was further analyzed. Results: At the least, 98 per cent of the whole-genome sequences were recovered from the four samples. The VZV sequences obtained in this study formed a separate monophyletic branch with clade 5, indicating it to be evolved from a distinct ancestor. The nucleotide-based analysis revealed 13 different SNP mutations and one multiple nucleotide variation in the VZV sequences when compared to one of the clade 5 genomes having accession number: DQ457052.1. Interpretation & conclusions: The present study described approximately 98 per cent of the genome sequence of VZV from India. The availability of these genomic sequences will lead to enrichment in the clinical genomic data set from India. The available data would help in the development of diagnostic methods along with evolutionary analysis. We hypothesize the existence of a new sub-clade that belongs to clade 5 and propose further experiments to confirm these results.

Biochar-mediated sequestration of Pb and Cd leads to enhanced productivity in Mentha arvensis.

Immobilization of cadmium (Cd) and lead (Pb) along with the alleviation of their phytotoxicity in Mentha arvensis by biochar was examined in this investigation. A greenhouse experiment was executed to evaluate the effect of biochar (BC) amended Cd and Pb spiked soil on their immobilization and uptake, plant growth, photosynthetic attributes (total chlorophyll, photosynthetic rate, transpiration rate, and stomatal activity) and oxidative enzymes (guaiacol peroxidase: POD; catalase: CAT and superoxide dismutase: SOD). In the present study, the photosynthetic attributes showed that BC significantly improved the total chlorophyll, photosynthetic, transpiration rates, and stomatal activity in the plants. The incorporation of BC in soil increase the Pb and Cd tolerance in M. arvensis vis-à-vis improved the biomass yield and nutrient intake. In addition, biochar has also reduced the POD, CAT, and SOD in the plant as well as improved the soil pH and enzymatic activities. Overall, BC immobilized the Cd and Pb in soil by providing the binding site to the metals and reduced the phytotoxicity in M. arvensis. However, large-scale field trials of BC are required for safe cultivation of M. arvensis which is known for its phytopharmaceuticals importance.

Identification and characterization of novel mosquito-borne (Kammavanpettai virus) and tick-borne (Wad Medani) reoviruses isolated in India.

In 1954, a virus named Wad Medani virus (WMV) was isolated from Hyalomma marginatum ticks from Maharashtra State, India. In 1963, another virus was isolated from Sturnia pagodarum birds in Tamil Nadu, India, and named Kammavanpettai virus (KVPTV) based on the site of its isolation. Originally these virus isolates could not be identified with conventional methods. Here we describe next-generation sequencing studies leading to the determination of their complete genome sequences, and identification of both virus isolates as orbiviruses (family Reoviridae). Sequencing data showed that KVPTV has an AT-rich genome, whereas the genome of WMV is GC-rich. The size of the KVPTV genome is 18 234 nucleotides encoding proteins ranging 238-1290 amino acids (aa) in length. Similarly, the size of the WMV genome is 16 941 nucleotides encoding proteins ranging 214-1305 amino acids in length. Phylogenetic analysis of the VP1 gene, along with the capsid genes VP5 and VP7, revealed that KVPTV is likely a novel mosquito-borne virus and WMV is a tick-borne orbivirus. This study focuses on the phylogenetic comparison of these newly identified orbiviruses with mosquito-, tick- and Culicoides-borne orbiviruses isolated in India and other countries.

Evaluation of therapeutic potential of VB-001, a leave-on formulation, for the treatment of moderate adherent dandruff.

BACKGROUND: Dandruff is a common scalp condition characterized by excessive scaling and itch. Aberrant colonization of the scalp by commensal Malassezia spp. is a major contributor in the multifactorial etiology of dandruff. Literature based understanding of Malassezia linked pathophysiology of dandruff allowed us to comprehend a strategy to potentiate the efficacy of a known antifungal agent used in dandruff therapy. The aim of this study was to determine the efficacy and skin safety of VB-001 antidandruff leave-on formulation in comparison with marketed antidandruff ZPTO shampoo in patients with moderate adherent dandruff of the scalp. METHODS: Healthy males or females aged ≥ 15 years and ≤ 65 with a clinical diagnosis of moderate adherent dandruff of the scalp were recruited for the study to monitor the effects of topical VB-001 versus those of marketed antidandruff ZPTO shampoo. RESULTS: 168 subjects were randomized to the treatment (VB-001, n = 84) and control (ZPTO shampoo, n = 84) groups. The efficacy of each product was evaluated by comparing proportion of subjects who have shown reduction in flaking by ASFS (adherent scalp flaking score) and pruritus by IGA (investigator global assessment) score. VB-001 imparted consistently better reduction in ASFS and enabled early reduction of pruritus in comparison to marketed ZPTO shampoo. CONCLUSION: VB-001, a leave-on formulation with ingredients chosen to selectively disturb the Malassezia niche on dandruff scalp by denying extra nutritional benefits to the microbe, provides unique advantages over existing best in class ZPTO shampoo therapy. It has the potential to emerge as an attractive novel treatment for moderate adherent dandruff. TRIAL REGISTRATION: CTRI Registration number: CTRI/2013/01/003283 . Registered on: 02/01/2013.

Antibiotic-resistant acne: getting under the skin.

Propionibacterium acnes is a key pathogenic factor in the development of acne. Antibiotics are the first choice of treatment for mild-to-moderate, mixed, papular/pustular, and moderate nodular acne, and an alternative choice in severe, nodular/conglobate acne. The emergence of resistance to the currently available antibiotics poses a serious set-back to this algorithm, and the reduced arsenal can diminish efficacy of treatment. This emerging situation should catalyze innovations in dermatology; for example, newer drugs and technologies such as next-generation antibiotics with excellent potency and low propensity to develop resistance, rapid diagnostic platforms to select responders and nonresponders, and delivery technologies that target the bacteria. Such innovations can dramatically expand the arsenal for dermatologists in the management of acne.

Comparative evaluation of marginal leakage of provisional crowns cemented with different temporary luting cements: In vitro study.

BACKGROUND OR STATEMENT OF PROBLEM: As, the longevity of provisional restorations is related to, a perfect adaptation and a strong, long-term union between restoration and teeth structures, therefore, evaluation of marginal leakage of provisional restorative materials luted with cements using the standardized procedures is essential. AIMS AND OBJECTIVES: To compare the marginal leakage of the provisional crowns fabricated from Autopolymerizing acrylic resin crowns and bisphenol A-glycidyl dimethacrylate (BIS-GMA) resin crowns. To compare the marginal leakage of the provisional crowns fabricated from autopolymerizing acrylic resin crowns and BIS-GMA resin crowns cemented with different temporary luting cements. To compare the marginal leakage of the provisional crowns fabricated from autopolymerizing acrylic resin (SC-10) crowns cemented with different temporary luting cements. To compare the marginal leakage of the provisional crowns fabricated from BIS-GMA resin crowns (Protemp 4) cemented with different temporary luting cements. METHODOLOGY: Freshly extracted 60 maxillary premolars of approximately similar dimensions were mounted in dental plaster. Tooth reduction with shoulder margin was planned to use a customized handpiece-holding jig. Provisional crowns were prepared using the wax pattern fabricated from computer aided designing/computer aided manufacturing milling machine following the tooth preparation. Sixty provisional crowns were made, thirty each of SC-10 and Protemp 4 and were then cemented with three different luting cements. Specimens were thermocycled, submerged in a 2% methylene blue solution, then sectioned and observed under a stereomicroscope for the evaluation of marginal microleakage. A five-level scale was used to score dye penetration in the tooth/cement interface and the results of this study was analyzed using the Chi-square test, Mann-Whitney U-test, Kruskal-Wallis H-test and the results were statistically significant P < 0.05 the power of study - 80%. RESULTS: Marginal leakage was significant in both provisional crowns cemented with three different luting cements along the axial walls of teeth (P < 0.05) confidence interval - 95%. CONCLUSION: The temporary cements with eugenol showed more microleakage than those without eugenol. SC-10 crowns showed more microleakage compared to Protemp 4 crowns. SC-10 crowns cemented with Kalzinol showed maximum microleakage and Protemp 4 crowns cemented with HY bond showed least microleakage.

Replicon particle vaccination induces non-neutralizing anti-nucleoprotein antibody-mediated control of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe human disease and is considered a WHO priority pathogen due to the lack of efficacious vaccines and antivirals. A CCHF virus replicon particle (VRP) has previously shown protective efficacy in a lethal Ifnar-/- mouse model when administered as a single dose at least 3 days prior to challenge. Here, we determine that non-specific immune responses are not sufficient to confer short-term protection, since Lassa virus VRP vaccination 3 days prior to CCHFV challenge was not protective. We also investigate how CCHF VRP vaccination confers protective efficacy by examining viral kinetics, histopathology, clinical analytes and immunity early after challenge (3 and 6 days post infection) and compare to unvaccinated controls. We characterize how these effects differ based on vaccination period and correspond to previously reported CCHF VRP-mediated protection. Vaccinating Ifnar-/- mice with CCHF VRP 28, 14, 7, or 3 days prior to challenge, all known to confer complete protection, significantly reduced CCHFV viral load, mucosal shedding, and markers of clinical disease, with greater reductions associated with longer vaccination periods. Interestingly, there were no significant differences in innate immune responses, T cell activation, or antibody titers after challenge between groups of mice vaccinated a week or more before challenge, but higher anti-NP antibody avidity and effector function (ADCD) were positively associated with longer vaccination periods. These findings support the importance of antibody-mediated responses in VRP vaccine-mediated protection against CCHFV infection.

Age-related dysregulation of intestinal epithelium fucosylation is linked to an increased risk of colon cancer.

Colon cancer affects people of all ages. However, its frequency, as well as the related morbidity and mortality, are high among older adults. The complex physiological changes in the aging gut substantially limit the development of cancer therapies. Here, we identify a potentially unique intestinal microenvironment that is linked with an increased risk of colon cancer in older adults. Our findings show that aging markedly influenced persistent fucosylation of the apical surfaces of intestinal epithelial cells, which resulted in a favorable environment for tumor growth. Furthermore, our findings shed light on the importance of the host-commensal interaction, which facilitates the dysregulation of fucosylation and promotes tumor growth as people get older. We analyzed colonic microbial populations at the species level to find changes associated with aging that could contribute to the development of colon cancer. Analysis of single-cell RNA-sequencing data from previous publications identified distinct epithelial cell subtypes involved in dysregulated fucosylation in older adults. Overall, our study provides compelling evidence that excessive fucosylation is associated with the development of colon cancer, that age-related changes increase vulnerability to colon cancer, and that a dysbiosis in microbial diversity and metabolic changes in the homeostasis of older mice dysregulate fucosylation levels with age.

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants.

The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination (∼1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.

Development of reverse genetic tools to study Chapare and Machupo viruses.

Arenaviruses are highly pathogenic viruses that pose a serious public health threat. Chapare virus (CHAV) and Machupo virus (MACV), two New World arenaviruses, cause hemorrhagic fevers with case fatality rates of up to 45%. Research on therapeutic drug targets and vaccines for these viruses is limited because biosafety level 4 containment is required for handling them. In this study, we developed reverse genetics systems, including minigenomes and recombinant viruses, that will facilitate the study of these pathogens. The minigenome system is based on the S segment of CHAV or MACV genomes expressing the fluorescent reporter gene ZsGreen (ZsG). We also generated recombinant CHAV and MACV with and without the ZsG reporter gene. As a proof-of-concept study, we used both minigenomes and recombinant viruses to test the inhibitory effects of previously reported antiviral compounds. The new reverse genetics system described here will facilitate future therapeutic studies for these two life-threatening arenaviruses.

Development of a novel minigenome and recombinant VSV expressing Seoul hantavirus glycoprotein-based assays to identify anti-hantavirus therapeutics.

Seoul virus (SEOV) is an emerging global health threat that can cause hemorrhagic fever with renal syndrome (HFRS), which results in case fatality rates of ∼2%. There are no approved treatments for SEOV infections. We developed a cell-based assay system to identify potential antiviral compounds for SEOV and generated additional assays to characterize the mode of action of any promising antivirals. To test if candidate antivirals targeted SEOV glycoprotein-mediated entry, we developed a recombinant reporter vesicular stomatitis virus expressing SEOV glycoproteins. To facilitate the identification of candidate antiviral compounds targeting viral transcription/replication, we successfully generated the first reported minigenome system for SEOV. This SEOV minigenome (SEOV-MG) screening assay will also serve as a prototype assay for discovery of small molecules inhibiting replication of other hantaviruses, including Andes and Sin Nombre viruses. Ours is a proof-of-concept study in which we tested several compounds previously reported to have activity against other negative-strand RNA viruses using our newly developed hantavirus antiviral screening systems. These systems can be used under lower biocontainment conditions than those needed for infectious viruses, and identified several compounds with robust anti-SEOV activity. Our findings have important implications for the development of anti-hantavirus therapeutics.

Potently neutralizing human mAbs against the zoonotic pararubulavirus Sosuga virus.

Sosuga virus (SOSV) is a recently discovered paramyxovirus with a single known human case of disease. There has been little laboratory research on SOSV pathogenesis or immunity, and no approved therapeutics or vaccines are available. Here, we report the discovery of human mAbs from the circulating memory B cells of the only known human case and survivor of SOSV infection. We isolated 6 mAbs recognizing the functional attachment protein hemagglutinin-neuraminidase (HN) and 18 mAbs against the fusion (F) protein. The anti-HN mAbs all targeted the globular head of the HN protein and could be organized into 4 competition-binding groups that exhibited epitope diversity. The anti-F mAbs can be divided into pre- or postfusion conformation-specific categories and further into 8 competition-binding groups. The only Ab in the panel that did not display neutralization activity was the single postfusion-specific anti-F mAb. Most of the anti-HN mAbs were more potently neutralizing than the anti-F mAbs, with mAbs in 1 of the HN competition-binding groups possessing ultrapotent (<1 ng/mL) half-maximal inhibitory virus neutralization values. These findings provide insight into the molecular basis for human Ab recognition of paramyxovirus surface proteins and the mechanisms of SOSV neutralization.

Development of a neutralization assay using a vesicular stomatitis virus expressing Nipah virus glycoprotein and a fluorescent protein.

Nipah virus (NiV) is a highly pathogenic paramyxovirus with a high case fatality rate. Due to its high pathogenicity, pandemic potential, and lack of therapeutics or approved vaccines, its study requires biosafety level 4 (BSL4) containment. In this report, we developed a novel neutralization assay for use in biosafety level 2 laboratories. The assay uses a recombinant vesicular stomatitis virus expressing NiV glycoprotein and a fluorescent protein. The recombinant virus propagates as a replication-competent virus in a cell line constitutively expressing NiV fusion protein, but it is restricted to a single round of replication in wild-type cells. We used this system to evaluate the neutralization activity of monoclonal and polyclonal antibodies, plasma from NiV-infected hamsters, and serum from human patients. Therefore, this recombinant virus could be used as a surrogate for using pathogenic NiV and may constitute a powerful tool to develop therapeutics in low containment laboratories.

Tissue replication and mucosal swab detection of Sosuga virus in Syrian hamsters in the absence of overt tissue pathology and clinical disease.

Human infection with Sosuga virus (SOSV), a recently discovered pathogenic paramyxovirus, has been reported in one individual to date. No animal models of disease are currently available for SOSV. Here, we describe initial characterization of experimental infection in Syrian hamsters, including kinetics of virus dissemination and replication, and the corresponding clinical parameters, immunological responses, and histopathology. We demonstrate susceptibility of hamsters to infection in the absence of clinical signs or significant histopathologic findings in tissues.