A recombinant murine-like 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. While 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-like 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 days 2 to 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 murine-like 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 well as provides a new tool to facilitate the testing of prophylactic and therapeutic interventions in the future

A recombinant murine-like 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. While 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 transmissibilit

Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces 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 only-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 viv

An Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like Rotaviruses

Copyright © 2020 American Society for Microbiology. An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies

La célula propone y el virus dispone

Publicación trimestral, Enero-Febrero-Marzo, Año 2016, N. 4. Páginas 7 y 8.De manera simplificada podemos definir un virus del latín virus: toxina o veneno como una entidad generalmente conformada por su material genético, que puede ser ácido desoxirribonucleico (ADN) o ácido ribonucleico (ARN) cubierto por una estructura protéica o cápside y que tiene capacidad de infección. Los virus están en todas partes, de hecho vivimos sumergidos en un mar de virus. Los parásitos intracelulares obligados como los virus, sólo pueden replicarse en una célula viva. La búsqueda de soluciones ingeniosas por parte de los virus para tener éxito y lograr replicarse por eso algunos autores consideran a los virus como “maquinas darwinianas perfectas” es un tema central en nuestro grupo de investigación

Appendix S1. Evolution of Zika prevalence in a dengue hyper-endemic municipality in Southern Mexico after the outbreak of 2015 to 2017.

The Zika virus (ZIKV) produced extensive outbreaks in the last decades including a large epidemic across Latin America (2015-2017). Due to Zika is trassmited for the same mosquito-vector of dengue diseases, both diseases coexist in the same areas. During the outbreak, the real magnitude of the epidemic was underestimated due to the high proportion of clinical mild cases and asymptomatic infections, and the similarity of its clinical manifestations with dengue. Another contributing factor was that confirmatory molecular testing (RT-PCR) is only useful during the first 5-7 days after the onset of the symptoms and is not widely available across regions.Population-based serosurveys provide an accurate prevalence estimator to understand the real magnitude of epidemics, however, in dengue-endemic regions, Zika seroprevalence estimations are difficult due to the extensive cross-reactivity of the elicited antibodies in both diseases. Studies of the first Zika epidemics used seroprevalence of IgG against the NS1 Zika protein using enzyme-linked immunosorbent assays (ELISA). The commercial tests demonstrated high diagnostic performance in dengue non-endemic populations. As the Zika epidemic advanced in dengue-hyperendemic areas, most of these ELISA depicted low specificity. Other techniques are also affected, including neutralization tests, which is considered the gold standard test in flavivirus infections. Conversely, the ELISA based on the domain III of E protein (EDIII) reported higher specificity in flavivirus-endemic populations, although there are no commercially available anti-EDIII IgG ELISAs.Commercial ELISAs have important advantages in large sample size serosurveys, such as reproducibility, high throughput and low cost. We used MNT and anti-EDIII IgG ELISA to adjust the cutoff value of the commercial ELISA and correct for possible biases due to cross-reactive antibodies. This study is the supplementary material of the study conducted to describe the extension of the Zika outbreak in a dengue hyper-endemic municipality in southern Mexico</p