A Novel Virus Discovered in the Yeast Pichia membranifaciens.

Mycoviruses are widely distributed across fungi, including yeasts of the Saccharomycotina subphylum. It was recently discovered that the yeast species Pichia membranifaciens contained double stranded RNAs (dsRNAs) that were predicted to be of viral origin. The fully sequenced dsRNA is 4,578 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses of the family Totiviridae. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is related to other totiviruses previously described within the Saccharomycotina yeasts. PmV-L-A is part of a monophyletic subgroup within the I-A totiviruses, implying a common ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the previously solved structure of the Saccharomyces cerevisiae virus L-A (ScV-L-A) Gag protein. The predicted tertiary structure of the PmV-L-A Pol and its homologs provide details of the potential mechanism of totivirus RNA-dependent RNA polymerases (RdRps) because of structural similarities to the RdRps of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts is important because of their parallels with mammalian viruses and the emerging role of totiviruses in animal disease.

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which—Partitiviridae and Mitoviridae—were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin—two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

High-Resolution Structure of the Nuclease Domain of the Human Parvovirus B19 Main Replication Protein NS1

Two new structures of the N-terminal domain of the main replication protein, NS1, of Human Parvovirus B19 (B19V) are presented. This domain (NS1-nuc) plays an important role in the “rolling hairpin” replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e. nicking) single-stranded DNA at a nearby site known as the trs. One structure of NS1-nuc is solved to 2.4 Å and shows the positions of two bound phosphate ions. A second structure shows the position of a single divalent cation in the DNA nicking active site. The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain, however shown here at significantly higher resolution. Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented which predict residues important for DNA cleavage and for sequence specific recognition at the viral origin of replication.

Buckeye rot of tomato in India: Present status, challenges, and future research perspectives

Tomato in India is commonly exposed to various diseases of fungal, bacterial, and viral origin, resulting in substantial yield losses (≥50%). Buckeye rot (caused by Phytophthora nicotianae var. parasitica) is among the major constraints in the successful cultivation of tomato crops in various parts of the world including the Solan district of Himachal Pradesh state, India. The fruit rot becomes more devastating under high humidity and wet soils. Symptoms generally appear on green fruit as alternate dark and light brown concentric rings. The genome size of P. nicotianae var. parasitica is 82 Mb with >23,000 predicted genes. High humidity (> 60%) and optimal temperatures (20-25 ºC), along with rainfall (≥ 10 mm), help to disperse the pathogen as the inoculum reaches the fruit through splashing rains. Sporangia germinate indirectly by producing zoospores at 20-25o C, or directly via germ tubes at >25ºC. In the absence of suitable resistant varieties, no single management practice is sufficient to keep the disease below the economic threshold level; therefore, integration of cultural and chemical methods is preferable. This paper aims to focus on the etiology, and management challenges of buckeye rot. We recommend innovative disease management strategies like identification and deployment of resistant cultivars as well as spraying of synthetic chemical fungicides, biocontrol agents, and use of abiotic chemicals that induce resistance, for developing sustainable crop production practices.

IOT BASED MOLECULAR MARKERS FOR DISEASE RESISTANCE STUDIES IN RICE - STRATEGY AND CHALLENGES

Rice production is constrained by diseases of fungal, bacterial and viral origin. The Internet of Things (IoT) – network of interconnected devices - is an application for disease related uses, collection of data, processing for testing and monitoring. This review article aims about how IoT can track and allows disease resistance studies in in Oryza species. Among them Xanthomonas oryzae, Magnaporthe grisea, RYMV (Rice yellow mottle virus), and brown planthopper causes the high yield losses. Disease resistance genes are identified and they are screened by the SSR (simple sequence repeats), RAPD (Randomly Amplified Polymorphic DNA) and RFLP (restriction fragment length polymorphisms) analysis.

Acute Respiratory Distress Syndrome: Focus on Viral Origin and Role of Pulmonary Lymphatics

Acute respiratory distress syndrome (ARDS) is a serious affection of the lung caused by a variety of pathologies. Great interest is currently focused on ARDS induced by viruses (pandemic influenza and corona viruses). The review describes pulmonary changes in ARDS and specific effects of the pandemic viruses in ARDS, and summarizes treatment options. Because the known pathogenic mechanisms cannot explain all aspects of the syndrome, the contribution of pulmonary lymphatics to the pathology is discussed. Organization and function of lymphatics in a healthy lung and in resorption of pulmonary edema are described. A future clinical trial may provide more insight into the role of hyaluronan in ARDS but the development of promising pharmacological treatments is unlikely because drugs play no important role in lymphedema therapy.

A tripartite structure, the complex nuclear receptor element (cNRE), is a cis-regulatory module of viral origin required for atrial chamber preferential gene expression

Optimal cardiac function requires appropriate contractile proteins in each heart chamber. Atria require slow myosins to act as variable reservoirs, while ventricles demand fast myosin for swift pumping functions. Hence, myosin is under chamber-biased cis-regulatory control to achieve this functional distribution. Failure in proper regulation of myosin genes can lead to severe congenital heart dysfunction. The precise regulatory input leading to cardiac chamber-biased expression remains uncharted. To address this, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives specific gene expression to the atria to uncover the regulatory information leading to chamber expression and understand their evolutionary origins. We show that SMyHC III gene states are autonomously orchestrated by a complex nuclear receptor cis-regulatory element (cNRE), a 32-bp sequence with hexanucleotide binding repeats. Using in vivo transgenic assays in zebrafish and mouse models, we demonstrate that preferential atrial expression is achieved by the combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Through comparative genomics, we provide evidence that the cNRE emerged from an endogenous viral element, most likely through infection of an ancestral host germline. Our study reveals an evolutionary pathway to cardiac chamber-specific expression.

Recent Development of Ruminant Vaccine Against Viral Diseases

Pathogens of viral origin produce a large variety of infectious diseases in livestock. It is essential to establish the best practices in animal care and an efficient way to stop and prevent infectious diseases that impact animal husbandry. So far, the greatest way to combat the disease is to adopt a vaccine policy. In the fight against infectious diseases, vaccines are very popular. Vaccination's fundamental concept is to utilize particular antigens, either endogenous or exogenous to induce immunity against the antigens or cells. In light of how past emerging and reemerging infectious diseases and pandemics were handled, examining the vaccination methods and technological platforms utilized for the animals may provide some useful insights. New vaccine manufacturing methods have evolved because of developments in technology and medicine and our broad knowledge of immunology, molecular biology, microbiology, and biochemistry, among other basic science disciplines. Genetic engineering, proteomics, and other advanced technologies have aided in implementing novel vaccine theories, resulting in the discovery of new ruminant vaccines and the improvement of existing ones. Subunit vaccines, recombinant vaccines, DNA vaccines, and vectored vaccines are increasingly gaining scientific and public attention as the next generation of vaccines and are being seen as viable replacements to conventional vaccines. The current review looks at the effects and implications of recent ruminant vaccine advances in terms of evolving microbiology, immunology, and molecular biology.

349. Diagnostic Testing and Antibiotic Utilization in Patients with COVID-19

Background Patients with COVID-19 receive high rates of antibiotic therapy, despite viral origin of infection. Reports of bacterial coinfection range from 3.5 to 8% in the early phase of infection. This study aimed to evaluate the relationship between diagnostic tests and antibiotic utilization in patients admitted with COVID-19 at the University of Maryland Medical Center to better inform future prescribing practices. Methods Retrospective cohort study of adult patients with a positive SARS-CoV-2 PCR on admission from March 2020 through June 2020. Associations between diagnostic tests employed and antibiotic initiation and duration were explored using bivariate analysis (SPSS®). Results Baseline characteristics of 224 included patients are reported in Table 1. Excluding SARS-CoV-2 PCRs, most frequently performed diagnostic tests included blood cultures (65.6%), MRSA nasal surveillance (45.1%), respiratory cultures (36.2%), respiratory viral panel (RVP) (33.0%), and Legionella (28.6%) and pneumococcal (26.3%) urine antigens. Positivity of RVP, Legionella, pneumococcus, blood, and respiratory tests were low (1.3%, 0.4%, 0.9%, 1.8%, and 6.7%, respectively). A total of 62% of patients were initiated on antibacterial therapy with a median cumulative antibiotic duration of 77.9 hours (IQR 41.4, 111.8). History of chronic respiratory disease (76% vs. 58.6%; P=0.025), any degree of oxygen requirement on admission (72% vs. 42.6%; P=0.006), and performance of blood cultures (70.7% vs. 46.8%, P< 0.0001) were associated with antibiotic initiation. Positive bacterial diagnostic respiratory culture (median duration 72.8h [IQR 46.7, 96.6] vs. 97.3h [IQR 79.8, 194.1]; P=0.027) and positive blood culture (median duration 80.1h [IQR 42.1, 111.7] vs. 97.5h [IQR 71.8, 164.8]; P=0.046) were associated with longer antibiotic duration. Patients who did not have respiratory cultures performed had similar antibiotic durations as those with negative respiratory cultures. Table 1. Baseline Characteristics Conclusion Despite low coinfection rates, negative diagnostic tests did not result in shorter empiric antibacterial duration. These findings highlight the ongoing need for both diagnostic and antimicrobial stewardship in COVID-19. Disclosures Emily Heil, PharmD, MS, BCIDP, Nothing to disclose Kimberly C. Claeys, PharmD, GenMark (Speaker’s Bureau)

284. Antibiotic Prescribing Trends in Hospitalized Influenza Versus COVID-19 Patients at a Community-Based Health System

Background The 2019 coronavirus SARS-CoV-2 continues to affect global population health. Patients with severe disease that require hospitalization due to COVID-19 pneumonia remain at further risk of bacterial co-infections. There is limited evidence suggesting up to 3.5% bacterial co-infection upon admission and up to 13.5% of secondary infections after hospitalization for pneumonia yet antibacterial therapy usage remain as high, or even higher, than data seen for viral pneumonia, such as influenza. Unnecessary use of antimicrobial therapy may lead to further resistance and requires stewardship attention. Methods A single-center retrospective chart review was conducted in a community health system on all inpatient influenza admissions between October 1st 2019 to March 31st 2020 and all COVID-19 admissions during the same 6-month period one year later. Patients were excluded if age < 18, observation or emergency visit. The study aims to determine the percentage of patients that were prescribed antibacterial therapy during influenza season compared to during the COVID-19 pandemic. Results A total of 175 patients were included in the influenza group while 1411 patients were included in the COVID-19 group (Table 1). The percent of inpatients with positive bacterial respiratory cultures were 12% in both influenza and COVID-19 groups. Positive bacterial respiratory cultures collected within 48 hours of admission were 3.4% in the influenza group compared to 1.2% in the COVID -19 group. Seventy-three percent of patients in the influenza group received antibiotics during admission compared to 78% in the COVID -19 group. Azithromycin and/or ceftriaxone was most commonly prescribed (58% vs. 60%) (Figure 1). The median length of stay was 3 days in the influenza group compared to 5 days in the COVID-19 group. In hospital mortality was higher in the COVID-19 group (1.7% vs. 9%). Conclusion Despite the viral origin of influenza and COVID-19 and low incidence of bacterial infection, antibacterials were frequently prescribed in both indications but it appears to trend more so in the COVID-19 group. There is an opportunity to enhance antimicrobial stewardship for the treatment of COVID-19 in acute care settings. Disclosures All Authors: No reported disclosures