Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses.

Efficacy of Phage-Antibiotic Combinations Against Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates

Background: Increasing antibiotic resistance warrants therapeutic alternatives to eradicate resistant bacteria. Combined phage-antibiotic therapy is a promising approach for eliminating bacterial infections and limiting the evolution of therapy-resistant diseases. Objectives: In the present study, we evaluated the effects of combinations of bacteriophages and antibiotics against multidrug-resistant (MDR) Klebsiella pneumoniae. Methods: Two MDR strains (GenBank no. MF953600 & MF953599) of K. pneumoniae were used. Bacteriophages were isolated from hospital sewage samples by employing a double agar overlay assay and identified by transmission electron microscopy. For further characterization of bacteriophages, the killing assay and host range test were performed. To assess therapeutic efficacy, phages (7.5 × 104 PFU/mL) were used in combination with various antibiotics. Results: The phage-cefepime & tetracycline combinations displayed promising therapeutic effects, restricting the growth of K. pneumoniae isolates, as evidenced by recording OD650nm values. Conclusions: The results of the current study showed that phage-antibiotic combination was a potential therapeutic approach to treat the infections caused by MDR K. pneumoniae.

Study ofLegionellaEffector Domains Revealed Novel and Prevalent Phosphatidylinositol 3-Phosphate Binding Domains

ABSTRACTLegionella pneumophilaand otherLegionellaspecies replicate intracellularly using the Icm/Dot type IV secretion system. InL. pneumophilathis system translocates >300 effectors into host cells and in theLegionellagenus thousands of effectors were identified, the function of most of which is unknown. FourteenL. pneumophilaeffectors were previously shown to specifically bind phosphoinositides (PIs) using dedicated domains. We found that PI-binding domains of effectors are usually not homologous to one another; they are relatively small and located at the effectors' C termini. We used the previously identifiedLegionellaeffector domains (LEDs) with unknown function and the above characteristics of effector PI-binding domains to discover novel PI-binding LEDs. We identified three predicted PI-binding LEDs that are present in 14 L. pneumophilaeffectors and in >200 effectors in theLegionellagenus. Using anin vitroprotein-lipid overlay assay, we found that 11 of theseL. pneumophilaeffectors specifically bind phosphatidylinositol 3-phosphate (PI3P), almost doubling the number ofL. pneumophilaeffectors known to bind PIs. Further, we identified in each of these newly discovered PI3P-binding LEDs conserved, mainly positively charged, amino acids that are essential for PI3P binding. Our results indicate thatLegionellaeffectors harbor unique domains, shared by many effectors, which directly mediate PI3P binding.

Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor

ABSTRACT Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor. IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

Postharvest Reduction of Coliphage MS2 from Romaine Lettuce during Simulated Commercial Processing with and without a Chlorine-Based Sanitizer

ABSTRACT Viral foodborne outbreaks are a serious threat to public health, and fresh produce is becoming increasingly recognized as a transmission vehicle. To limit foodborne disease, ready-to-eat leafy greens are typically washed with a chlorine-based sanitizer during commercial production. This study assessed the efficacy of a chlorine-based sanitizer against coliphage MS2, as a potential surrogate for foodborne viruses, on fresh-cut romaine lettuce during simulated commercial production using a small-scale processing line. Before processing, romaine lettuce was inoculated to contain approximately 105 and 106 PFU/g of MS2 for experiments with and without sanitizer, respectively. Lettuce samples were collected following each stage of processing, which included mechanical shredding, 2 min of flume washing (with or without 25 ppm of free chlorine), shaker table dewatering, and centrifugal drying. In addition, the spent centrifuge water and flume wash water were collected, with the flume water concentrated using hollow-fiber ultrafiltration. MS2 was recovered from lettuce in Tris-glycine buffer and quantified as PFUs in a double-agar overlay assay. The greatest reduction in MS2 occurred between shredding and flume washing, with levels remaining relatively stable following flume washing with or without 25 ppm of free chlorine. Average total reductions of 0.8 and 1.0 log PFU/g were seen after processing with and without the sanitizer, respectively, with no statistical difference observed between the two treatments (P > 0.05). The average MS2 level in the spent centrifugation water started at 4.0 log PFU/ml for experiments with sanitizer and the average MS2 reduction in the flume wash water was 4 log (PFU) for experiments with sanitizer, demonstrating that removals could be achieved in the water itself. These findings suggest that the currently recommended commercial production practices are unable to effectively decrease viruses once they have attached to leafy greens during commercial processing.

Rab11, but not Rab4, facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation to the plasma membrane

Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking. Rab4 and Rab11 are involved in vesicular trafficking to the plasma membrane from early endosomes and recycling endosomes, respectively. Tauroursodeoxycholate (TUDC) and cAMP increase bile formation, in part, by increasing plasma membrane localization of multidrug resistance-associated protein 2 (MRP2). The goal of the present study was to determine the role of these Rab proteins in the trafficking of MRP2 by testing the hypothesis that Rab11 and/or Rab4 facilitate cAMP- and TUDC-induced MRP2 translocation to the plasma membrane. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which constitutively express MRP2. HuH-NTCP cells were transfected with Rab11-WT and GDP-locked dominant inactive Rab11-GDP or with Rab4-GDP to study the role of Rab11 and Rab4. A biotinylation method and a GTP overlay assay were used to determine plasma membrane MRP2 and activation of Rab proteins (Rab11 and Rab4), respectively. Cyclic AMP and TUDC increased plasma membrane MRP2 and stimulated Rab11 activity. Plasma membrane translocation of MRP2 by cAMP and TUDC was increased and inhibited in cells transfected with Rab11-WT and Rab11-GDP, respectively. Cyclic AMP (previous study) and TUDC increased Rab4 activity. However, cAMP- and TUDC-induced increases in MRP2 were not inhibited by Rab4-GDP. Taken together, these results suggest that Rab11 is involved in cAMP- and TUDC-induced MRP2 translocation to the plasma membrane.

Antimicrobial activity and Characterization of Marine bacteria

Marine bacteria were isolated from seawater was collected from different coastal areas of the Tamilnadu Sea. The antimicrobial activities of these bacteria were investigated. Ethyl acetate extracts of marine bacterial fermentation were screened for antimicrobial activities using the method of agar diffusion. The results showed that 25 strains of the isolates have antimicrobial activity. The proportion of active bacteria associated with isolated from seawater. The active marine bacteria were assigned to the genera Alteromonas, Pseudomonas, Bacillus and Marinobacter. The TLC autobiographic overlay assay implied that the antimicrobial metabolites produced by four strains with wide antimicrobial spectrum were different. These marine bacteria were expected to be potential resources of natural antibiotic products. It can be concluded that isolation of Marine bacterial samples can offer a numbers of microbial strains for sources of new biomolecules from Marine sources.