Biology subjects

Sanchez-Tacuba, L.

Publications and source records attributed to Sanchez-Tacuba, L..

3 recordsLinked to original sources

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↗

bioRxiv2022

Mature rotavirus particles contain equivalent amounts of 7meGpppGcap and noncapped viral positive-sense RNAs

Viruses have evolved different strategies to overcome their recognition by the host innate immune system. Addition of cap at their 5RNA ends is an efficient mechanism to ensure escape from detection by the innate immune system, but also to ensure the efficient synthesis of viral proteins. Rotavirus mRNAs contain a type 1 cap structure at their 5end that is added by the viral capping enzyme VP3. This is a multifunctional protein with all the enzymatic activities necessary to add the cap, and also functions as an antagonist of the OAS-RNase L pathway. Here, the relative abundance of capped and noncapped viral RNAs during the replication cycle of rotavirus was determined. We found that both classes of rotaviral +RNAs are encapsidated, and they were present in a 1:1 ratio in the mature infectious particles. The capping of viral +RNAs is dynamic since different ratios of capped and noncapped RNAs were detected at different times post infection. Similarly, when the relative amount of capped and uncapped viral +RNAs produced in an in vitro transcription system was determined, we found that the proportion was very similar to that in the mature viral particles and in infected cells, suggesting that the capping efficiency of VP3 both, in vivo and in vitro, might be close to 50%. Unexpectedly, when the effect of simultaneously knocking down the expression of VP3 and RNase L on the cap status of viral +RNAs was evaluated, we found that even though at late times post infection there was an increased proportion of capped viral RNAs in infected cells, the viral particles isolated from this condition contained an equal ratio of capped and noncapped viral RNA, suggesting that there might be a selective packaging of capped-noncapped RNAs. SIGNIFICANCERotaviruses have a genome composed of eleven segments of double stranded RNA. Whether all 5 ends of the positive sense genomic RNA contained in the mature viral particles are modified by a cap structure is unknown. In this work, using a direct quantitative assay we characterized the relative proportion of capped and noncapped viral RNA in rotavirus infected cells and in viral particles. We found that independently of the relative proportions of cap/noncapped RNA present in rotavirus infected cells, there is a similar proportion of these two kinds of 5-modified positive sense RNAs in the viral particles.

microbiology↗

bioRxiv2022

A recombinant murine rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission

Rotaviruses (RVs) are one of the main causes of severe gastroenteritis, diarrhea, and death in children and young animals. Although suckling mice prove to be highly useful small animal models of RV infection and pathogenesis, direct visualization tools are lacking to track the temporal dynamics of RV replication and transmissibility in vivo. Here, we report the generation of the first recombinant murine RV that encodes a Nano-Luciferase reporter (NLuc) using a newly optimized RV reverse genetics system. The NLuc-expressing RV was replication-competent in cell culture and both infectious and virulent in neonatal mice in vivo. Strong luciferase signals were detected in the proximal and distal small intestines, colon, and mesenteric lymph nodes. We showed, via a noninvasive in vivo imaging system, that RV intestinal replication peaked at day 2 and day 5 post infection. Moreover, we successfully tracked RV transmission to uninoculated littermates as early as 3 days post infection, 1 day prior to clinically apparent diarrhea and 3 days prior to detectable fecal RV shedding in the uninoculated littermates. We also observed significantly increased viral replication in Stat1 knockout mice that lack the host interferon signaling. Our results suggest that the NLuc RV represents a non-lethal powerful tool for the studies of tissue tropism and host and viral factors that regulate RV replication and spread, as providing a new mechanism to facilitate the testing of prophylactic and therapeutic interventions in the future.

microbiology↗