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

Characterization of Homologous and Heterologous Rotavirus-Specific T-Cell Responses in Infant and Adult Mice

During primary rotavirus (RV) infection, CD8(+) T cells play an important role in viral clearance as well as providing partial protection against reinfection. CD4(+) T cells are essential for maximal development of RV-specific intestinal immunoglobulin A. In this study, we took advantage of the cytokine flow cytometry technique to obtain a detailed map of H-2(b)- and H-2(d)-restricted CD8(+) and CD4(+) T-cell epitopes from the RV proteins VP6 and VP7. Three new CD8(+) T-cell epitopes (H-2(d) and H-2(b) restricted) and one new CD4(+) T-cell epitope (H-2(d) and H-2(b) restricted) were identified. Using these newly identified targets, we characterized the development and specificity of cellular immune responses in C57BL/6 and BALB/c mice during acute infection of infants and adults. We found that both the CD4(+) and CD8(+) responses peaked on days 5 to 7 after infection and then declined rapidly. Interestingly, both the response kinetics and tissue distributions were different when epitopes on VP6 and VP7 were compared. VP6 elicited a response which predominated in the intestine, while the response to VP7 was more systemic. Additionally, the T-cell responses elicited after homologous versus heterologous infection differed substantially. We found that during homologous infection, there was a greater response toward VP6 than that toward VP7, especially in the intestine, while after heterologous infection, this was not the case. Finally, in suckling mice, we found two peaks in the CD8 response on days 7 and 14 postinfection, which differed from the single peak found in adults and likely mimics the biphasic pattern of rotavirus shedding in infant mice

Qualitative and Quantitative Characteristics of Rotavirus-Specific CD8 T Cells Vary Depending on the Route of Infection▿

CD8 T-cell response provides an important defense against rotavirus, which infects a variety of systemic locations in addition to the gut. Here we investigated the distribution, phenotype, and function of rotavirus-specific CD8 T cells in multiple organs after rotavirus infection initiated via the intranasal, oral, or intramuscular route. The highest level of virus-specific CD8 T cells was observed in the Peyer's patches of orally infected mice and in the lungs of intranasally infected animals. Very low levels of virus-specific CD8 T cells were detected in peripheral blood or spleen irrespective of the route of infection. Rotavirus-specific CD8 T cells from Peyer's patches of orally infected mice expressed high levels of CCR9, while CXCR6 and LFA-1 expression was associated with virus-specific CD8 T cells in lungs of intranasally infected mice. Oral infection induced the highest proportion of gamma interferon− CD107a/b+ CD8 T cells in Peyer's patches. When equal numbers of rotavirus-specific CD8 T cells were transferred into Rag-1 knockout mice chronically infected with rotavirus, the donor cells derived from Peyer's patches of orally infected mice were more efficient than those derived from lungs of intranasally infected animals in clearing intestinal infection. These results suggest that different routes of infection induce virus-specific CD8 T cells with distinct homing phenotypes and effector functions as well as variable abilities to clear infection

Roles of VP4 and NSP1 in Determining the Distinctive Replication Capacities of Simian Rotavirus RRV and Bovine Rotavirus UK in the Mouse Biliary Tract▿

Rotavirus replication and virulence are strongly influenced by virus strain and host species. The rotavirus proteins VP3, VP4, VP7, NSP1, and NSP4 have all been implicated in strain and species restriction of replication; however, the mechanisms have not been fully determined. Simian (RRV) and bovine (UK) rotaviruses have distinctive replication capacities in mouse extraintestinal organs such as the biliary tract. Using reassortants between UK and RRV, we previously demonstrated that the differential replication of these viruses in mouse embryonic fibroblasts is determined by the respective NSP1 proteins, which differ substantially in their abilities to degrade interferon (IFN) regulatory factor 3 (IRF3) and suppress the type I IFN response. In this study, we used an in vivo model of rotavirus infection of mouse gallbladder with UK × RRV reassortants to study the genetic and mechanistic basis of systemic rotavirus replication. We found that the low-replication phenotype of UK in biliary tissues was conferred by UK VP4 and that the high-replication phenotype of RRV was conferred by RRV VP4 and NSP1. Viruses with RRV VP4 entered cultured mouse cholangiocytes more efficiently than did those with UK VP4. Reassortants with RRV VP4 and UK NSP1 genes induced high levels of expression of IRF3-dependent p54 in biliary tissues, and their replication was increased 3-fold in IFN-α/β and -γ receptor or STAT1 knockout (KO) mice compared to wild-type mice. Our data indicate that systemic rotavirus strain-specific replication in the murine biliary tract is determined by both viral entry mediated by VP4 and viral antagonism of the host innate immune response mediated by NSP1

IRF3 Inhibition by Rotavirus NSP1 Is Host Cell and Virus Strain Dependent but Independent of NSP1 Proteasomal Degradation▿

Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1's ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation

Keratin 20 Helps Maintain Intermediate Filament Organization in Intestinal Epithelia

Of the >20 epithelial keratins, keratin 20 (K20) has an unusual distribution and is poorly studied. We began to address K20 function, by expressing human wild-type and Arg80→His (R80H) genomic (18 kb) and cDNA K20 in cells and mice. Arg80 of K20 is conserved in most keratins, and its mutation in epidermal keratins causes several skin diseases. R80H but not wild-type K20 generates disrupted keratin filaments in transfected cells. Transgenic mice that overexpress K20 R80H have collapsed filaments in small intestinal villus regions, when expressed at moderate levels, whereas wild-type K20-overexpressing mice have normal keratin networks. Overexpressed K20 maintains its normal distribution in several tissues, but not in the pancreas and stomach, without causing any tissue abnormalities. Hence, K20 pancreatic and gastric expression is regulated outside the 18-kb region. Cross-breeding of wild-type or R80H K20 mice with mice that overexpress wild-type K18 or K18 that is mutated at the conserved K20 Arg80-equivalent residue show that K20 plays an additive and compensatory role with K18 in maintaining keratin filament organization in the intestine. Our data suggest the presence of unique regulatory domains for pancreatic and gastric K20 expression and support a significant role for K20 in maintaining keratin filaments in intestinal epithelia