Biology subjects

Costantini, V. P.

Publications and source records attributed to Costantini, V. P..

3 recordsLinked to original sources

bioRxiv2023

Heat Inactivation of Aqueous Viable Norovirus and MS2 Bacteriophage

Human norovirus is a leading cause of acute gastroenteritis often associated with contaminated food or water exposure. Many studies have used morphologically similar viruses, such as MS2 bacteriophage, and molecular detection methods to study the environmental fate and inactivation characteristics, given the historical challenges to culture human norovirus. In this study, we used human intestinal enteroids (HIEs) to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50{degrees}C, 60{degrees}C, and 70{degrees}C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50{degrees}C, 60{degrees}C, and 70{degrees}C, respectively. Norovirus and MS2 bacteriophage had significantly different decay rates at all tested temperatures (p = 0.002 - 0.007). No decrease of RNA titers as measured by realtime RT-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation. Overall, our data demonstrate that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. IMPORTANCENorovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide. Treatments to inactivate norovirus are critical to reducing the risk associated with contaminated food and water. The recent developments to replicate human norovirus in human intestinal enteroids (HIE) enables the evaluation of heat inactivation kinetics of viable norovirus. Historically, cultivable surrogate viruses, such as bacteriophage MS2, have been used to measure the environmental fate of human norovirus. Our findings indicate that compared to human norovirus, MS2 bacteriophage is a conservative surrogate to measure the effect of heat inactivation. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with persistence of viable norovirus.

microbiology↗

bioRxiv2023

Glycochenodeoxycholic acid and ceramide suppress the antiviral effect of 25-hydroxycholesterol against human norovirus infection in human intestinal enteroids

The human intestinal enteroid (HIE) cell culture system with the support of glycine-conjugated bile acid glycochenodeoxycholic acid (GCDCA) and ceramide (C2) facilitate successful replication of several norovirus strains. Here we investigate how the presence of GCDCA/C2 impacts gene expression of norovirus-infected HIE and the impact of 25 hydroxycholesterol (25-HC), a key regulator of cholesterol homeostasis and bile acid production on norovirus replication. In absence of GCDCA/C2, 0.01 and 0.1 M 25-HC suppressed virus (GII.4 Sydney[P16]) replication by 1.3 log and 1.1 log respectively (p<0.05). In the presence of GCDCA/C2, 5 M 25-HC was required to achieve a 1 log decrease (p<0.05) in viral titers demonstrating that 25-HC restricts norovirus replication in HIE. RNA sequence analysis showed that during human norovirus infection, 25-HC downregulated expression of genes (CYP3A4, APOB, APOA1, and ABCG1) involved in cholesterol metabolism and transport as well as interferon stimulated genes such as ISG15 and IFIT1. GCDCA/C2 counteracts the suppressive effect of 25-HC expression of some genes related to these pathways including APOA4 and CYP27A1 however, other cholesterol genes such as APOA1 were further suppressed in the presence of GCDCA/C2. ImportanceNorovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and currently, there are no effective therapeutic strategies against this highly contagious pathogen. Our study provides insights into the effect of bile during norovirus infection, highlight the role of the cholesterol/oxysterol pathways during human norovirus replication, and demonstrate the potential utility of oxysterols in developing norovirus therapeutics.

genetics↗

bioRxiv2022

Mucosal and systemic neutralizing antibodies to norovirus and rotavirus by oral immunization with recombinant rotavirus in infant mice

Rotaviruses (RVs) preferentially replicate in the small intestine, frequently cause severe diarrheal disease, and following enteric infection generally induce variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a missed opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued a replication-competent recombinant RRV harboring bicistronic gene segment 7 that encodes both the native RV NSP3 protein and a human norovirus (HuNoV) VP1 protein from the predominant genotype GII.4 (rRRV-HuNoV-VP1). The rRRV-HuNoV-VP1 expressed HuNoV VP1 in infected cells in vitro and importantly, elicited both systemic and local antibody responses to HuNoV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens while providing immunity to RV. Significance statementMucosal immunity is a key component of protection against many pathogens. Robust and effective mucosal immune responses are generally induced following infection with a replication-competent pathogen at a mucosal surface. Several studies have attempted to develop viral vector-based enteric mucosal vaccines; however, the most advanced of these are still in clinical development. Here, we successfully induced systemic and mucosal antibody responses against both rotavirus and norovirus following inoculation of a recombinant rotavirus expressing the human norovirus major capsid protein. These responses are likely to correlate with protective immunity. Live-attenuated rotavirus vaccines have already proven safe and effective worldwide. These findings confirm the potential utility of using rotaviruses as a dual enteric vaccine platform for other important human enteric pathogens.

microbiology↗