Production of OSU G5P[7] Porcine Rotavirus Expressing a Fluorescent Reporter via Reverse Genetics.

Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in infants and the young of many economically important animals. Although vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal. Thus, there is a need for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents a Rotavirus A species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined via Nanopore sequencing, we developed a robust reverse genetics system enabling the recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers (~107 plaque-forming units/mL), its growth kinetics were modestly decreased in comparison to the laboratory-adapted OSU virus. The reverse genetics system was used to generate the rOSU rotavirus, which served as an expression vector for a foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced a genetically stable rOSU virus that expressed the fluorescent UnaG protein as a functional separate product. Together, these findings raise the possibility of producing improved live oral porcine rotavirus vaccines through reverse-genetics-based modification or combination porcine rotavirus vaccines that can express neutralizing antigens for other porcine enteric diseases.

Recovery of Recombinant Rotaviruses by Reverse Genetics.

Rotaviruses are the primary cause of severe gastroenteritis in infants and young children throughout the world. To combat rotavirus illness, several live oral vaccines have been developed, or are under development, that are formulated from attenuated human or human-animal reassortant strains of rotavirus. While the effectiveness of these vaccines is generally high in developed countries, the same vaccines are significantly less effective in many developing countries, where the need for rotavirus vaccines is greatest. Recently, reverse genetics systems have been developed that allow modification of the segmented double-stranded (ds)RNA genome of rotavirus, including reprogramming the genome to allow expression of additional proteins that may stimulate expanded neutralizing antibody responses in vaccinated children. The use of reverse genetics systems may not only lead to the development of more potent classes of vaccines but can be used to better explore the intricacies of rotavirus molecular biology and pathogenesis. In this article, we share protocols that can be used to generate recombinant rotaviruses, including modified strains that express foreign proteins.

Rotavirus capping enzyme VP3 inhibits interferon expression by inducing MAVS degradation during viral replication.

The binding of viral RNA to RIG-I-like receptors triggers the formation of mitochondrial antiviral signaling (MAVS) protein aggregates critical for interferon (IFN) expression. Several rotavirus strains have been shown to suppress IFN expression by inducing MAVS degradation. Relying on transient expression assays, previous studies reached different conclusions regarding the identity of the rotavirus protein responsible for MAVS degradation, suggesting it was an activity of the rotavirus capping enzyme VP3 or the interferon antagonist NSP1. Here, we have used recombinant SA11 rotaviruses to identify the endogenous viral protein responsible for MAVS degradation and to analyze how the attack on MAVS impacts IFN expression. The recombinant viruses included those expressing modified VP3 or NSP1 proteins deficient in the ability to induce the degradation of MAVS or interferon regulatory factor-3 (IRF3), or both. With these viruses, we determined that VP3 directs the proteasomal degradation of MAVS but plays no role in IRF3 degradation. Moreover, NSP1 was determined to induce IRF3 degradation but to have no impact on MAVS degradation. Analysis of rotavirus-infected cells indicated that IRF3 degradation was more efficient than MAVS degradation and that NSP1 was primarily responsible for suppressing IFN expression in infected cells. However, VP3-mediated MAVS degradation contributed to IFN suppression in cells that failed to produce functional NSP1, pointing to a subsidiary role for VP3 in the IFN antagonist activity of NSP1. Thus, VP3 is a multifunctional protein with several activities that counter anti-rotavirus innate immune responses, including capping of viral (+)RNAs, hydrolysis of the RNase L 2-5A (2'-5' oligoadenylate) signaling molecule, and proteasomal degradation of MAVS. IMPORTANCE Rotavirus is an enteric RNA virus that causes severe dehydrating gastroenteritis in infants and young children through infection of enterocytes in the small intestine. Timely clearance of the virus demands a robust innate immune response by cells associated with the small intestine, including the expression of interferon (IFN). Previous studies have shown that some rotavirus strains suppress the production of interferon, by inducing the degradation of mitochondrial antiviral signaling (MAVS) protein and interferon regulatory factor-3 (IRF3). In this study, we have used reverse genetics to generate recombinant rotaviruses expressing compromised forms of VP3 or NSP1, or both, to explore the function of these viral proteins in the degradation of MAVS and IRF3. Our results demonstrate that VP3 is responsible for MAVS depletion in rotavirus-infected cells, and through this activity, helps to suppress IFN production. Thus, VP3 functions to support the activity of rotavirus NSP1, the major interferon antagonist of the virus.

T7 expression plasmids for producing a recombinant human G1P[8] rotavirus comprising RIX4414 sequences of the RV1 (Rotarix , GSK) vaccine strain.

The live oral rotavirus RV1 (Rotarix) vaccine is formulated from the human G1P[8] RIX4414 virus. Based on RIX4414 sequences, T7 expression plasmids were constructed that supported recovery of recombinant RIX4414-like viruses by reverse genetics. These plasmids will advance the study of the RV1 vaccine, possibly allowing improvements to its efficacy.

Recombinant rotavirus expressing the glycosylated S1 protein of SARS-CoV-2.

Reverse genetic systems have been used to introduce heterologous sequences into the rotavirus segmented double-stranded (ds)RNA genome, enabling the generation of recombinant viruses that express foreign proteins and possibly serve as vaccine vectors. Notably, insertion of SARS-CoV-2 sequences into the segment seven (NSP3) RNA of simian SA11 rotavirus was previously shown to result in the production of recombinant viruses that efficiently expressed the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the S1 region of the SARS-CoV-2 spike protein. However, efforts to generate a similar recombinant (r) SA11 virus that efficiently expressed full-length S1 were less successful. In this study, we describe modifications to the S1-coding cassette inserted in the segment seven RNA that allowed recovery of second-generation rSA11 viruses that efficiently expressed the ~120-kDa S1 protein. The ~120-kDa S1 products were shown to be glycosylated, based on treatment with endoglycosidase H, which reduced the protein to a size of ~80 kDa. Co-pulldown assays demonstrated that the ~120-kDa S1 proteins had affinity for the human ACE2 receptor. Although all the second-generation rSA11 viruses expressed glycosylated S1 with affinity for the ACE receptor, only the S1 product of one virus (rSA11/S1f) was appropriately recognized by anti-S1 antibodies, suggesting the rSA11/S1f virus expressed an authentic form of S1. Compared to the other second-generation rSA11 viruses, the design of the rSA11/S1f was unique, encoding an S1 product that did not include an N-terminal FLAG tag. Probably due to the impact of FLAG tags upstream of the S1 signal peptides, the S1 products of the other viruses (rSA11/3fS1 and rSA11/3fS1-His) may have undergone defective glycosylation, impeding antibody binding. In summary, these results indicate that recombinant rotaviruses can serve as expression vectors of foreign glycosylated proteins, raising the possibility of generating rotavirus-based vaccines that can induce protective immune responses against enteric and mucosal viruses with glycosylated capsid components, including SARS-CoV-2.

Human Rotavirus Replicates in Salivary Glands and Primes Immune Responses in Facial and Intestinal Lymphoid Tissues of Gnotobiotic Pigs.

Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs.

Rotavirus NSP1 Subverts the Antiviral Oligoadenylate Synthetase-RNase L Pathway by Inducing RNase L Degradation.

The interferon (IFN)-inducible 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway plays a critical role in antiviral immunity. Group A rotaviruses, including the simian SA11 strain, inhibit this pathway through two activities: an E3-ligase related activity of NSP1 that degrades proteins necessary for IFN signaling, and a phosphodiesterase (PDE) activity of VP3 that hydrolyzes the RNase L-activator 2',5'-oligoadenylate. Unexpectedly, we found that a recombinant (r) SA11 double mutant virus deficient in both activities (rSA11-VP3H797R-NSP1ΔC17) retained the ability to prevent RNase L activation. Mass spectrometry led to the discovery that NSP1 interacts with RNase L in rSA11-infected HT29 cells. This interaction was confirmed through copulldown assay of cells transiently expressing NSP1 and RNase L. Immunoblot analysis showed that infection with wild-type rSA11 virus, rSA11-VP3H797R-NSP1ΔC17 double mutant virus, or single mutant forms of the latter virus all resulted in the depletion of endogenous RNase L. The loss of RNase L was reversed by addition of the neddylation inhibitor MLN4924, but not the proteasome inhibitor MG132. Analysis of additional mutant forms of rSA11 showed that RNase L degradation no longer occurred when either the N-terminal RING domain of NSP1 was mutated or the C-terminal 98 amino acids of NSP1 were deleted. The C-terminal RNase L degradation domain is positioned upstream and is functionally independent of the NSP1 domain necessary for inhibiting IFN expression. Our studies reveal a new role for NSP1 and its E3-ligase related activity as an antagonist of RNase L and uncover a novel virus-mediated strategy of inhibiting the OAS-RNase L pathway. IMPORTANCE For productive infection, rotavirus and other RNA viruses must suppress interferon (IFN) signaling and the expression of IFN-stimulated antiviral gene products. Particularly important is inhibiting the interferon (IFN)-inducible 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway, as activated RNase L can direct the nonspecific degradation of viral and cellular RNAs, thereby blocking viral replication and triggering cell death pathways. In this study, we have discovered that the simian SA11 strain of rotavirus employs a novel strategy of inhibiting the OAS-RNase L pathway. This strategy is mediated by SA11 NSP1, a nonstructural protein that hijacks E3 cullin-RING ligases, causing the ubiquitination and degradation of host proteins essential for IFN induction. Our analysis shows that SA11 NSP1 also recognizes and causes the ubiquitination of RNase L, an activity resulting in depletion of endogenous RNase L. These data raise the possibility of using therapeutics targeting cellular E3 ligases to control rotavirus infections.

ICTV Virus Taxonomy Profile: Spinareoviridae 2022.

Spinareoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (9-12 linear segments) dsRNA genomes of 23-29 kbp. Spinareovirids have a broad host range, infecting animals, fungi and plants. Some have important pathogenic potential for humans (e.g. Colorado tick fever virus), livestock (e.g. avian orthoreoviruses), fish (e.g. aquareoviruses) and plants (e.g. rice ragged stunt virus and rice black streaked dwarf virus). This is a summary of the ICTV Report on the family Spinareoviridae, which is available at ictv.global/report/spinareoviridae.

ICTV Virus Taxonomy Profile: Sedoreoviridae 2022.

Sedoreoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (10-12 linear segments) dsRNA genomes of 18-26 kbp. Sedoreovirids have a broad host range, infecting mammals, birds, crustaceans, arthropods, algae and plants. Some of them have important pathogenic potential for humans (e.g. rotavirus A), livestock (e.g. bluetongue virus) and plants (e.g. rice dwarf virus). This is a summary of the ICTV Report on the family Sedoreoviridae, which is available at ictv.global/report/sedoreoviridae.

Generation of Recombinant Rotaviruses Expressing Human Norovirus Capsid Proteins.

Rotavirus, a segmented double-stranded RNA virus of the Reoviridae family, is a primary cause of acute gastroenteritis in young children. In countries where rotavirus vaccines are widely used, norovirus (NoV) has emerged as the major cause of acute gastroenteritis. Towards the goal of creating a combined rotavirus-NoV vaccine, we explored the possibility of generating recombinant rotaviruses (rRVs) expressing all or portions of the NoV GII.4 VP1 capsid protein. This was accomplished by replacing the segment 7 NSP3 open reading frame with a cassette encoding, sequentially, NSP3, a 2A stop-restart translation element, and all or portions (P, P2) of NoV VP1. In addition to successfully recovering rRVs with modified SA11 segment 7 RNAs encoding NoV capsid proteins, analogous rRVs were recovered through modification of the segment 7 RNA of the RIX4414 vaccine strain. An immunoblot assay confirmed that rRVs expressed NoV capsid proteins as independent products. Moreover, VP1 expressed by rRVs underwent dimerization and was recognized by conformational-dependent anti-VP1 antibodies. Serially passaged rRVs that expressed the NoV P and P2 were genetically stable, retaining additional sequences of up to 1.1 kbp without change. However, serially passaged rRVs containing the longer 1.6-kb VP1 sequence were less stable and gave rise to virus populations with segment 7 RNAs lacking VP1 coding sequences. Together, these studies suggest that it may be possible to develop combined rotavirus-NoV vaccines using modified segment 7 RNA to express NoV P or P2. In contrast, development of potential rotavirus-NoV vaccines expressing NoV VP1 will need additional efforts to improve genetic stability. IMPORTANCE Rotavirus (RV) and norovirus (NoV) are the two most important causes of acute viral gastroenteritis (AGE) in infants and young children. While the incidence of RV AGE has been brought under control in many countries through the introduction of universal mass vaccination with live attenuated RV vaccines, similar highly effective NoV vaccines are not available. To pursue the development of a combined RV-NoV vaccine, we examined the potential of using RV as an expression vector of all or portions of the NoV capsid protein VP1. Our results showed that by replacing the NSP3 open reading frame in RV genome segment 7 RNA with a coding cassette for NSP3, a 2A stop-restart translation element, and VP1, recombinant RVs can be generated that express NoV capsid proteins. These findings raise the possibility of developing new generations of RV-based combination vaccines that provide protection against a second enteric pathogen, such as NoV.

Type III and Not Type I Interferons Efficiently Prevent the Spread of Rotavirus in Human Intestinal Epithelial Cells.

Rotavirus infects intestinal epithelial cells and is the leading cause of gastroenteritis in infants worldwide. Upon viral infection, intestinal cells produce type I and type III interferons (IFNs) to alert the tissue and promote an antiviral state. These two types of IFN bind to different receptors but induce similar pathways that stimulate the activation of interferon-stimulated genes (ISGs) to combat viral infection. In this work, we studied the spread of a fluorescent wild-type (WT) SA11 rotavirus in human colorectal cancer cells lacking specific interferon receptors and compared it to that of an NSP1 mutant rotavirus that cannot interfere with the host intrinsic innate immune response. We could show that the WT rotavirus efficiently blocks the production of type I IFNs but that type III IFNs are still produced, whereas the NSP1 mutant rotavirus allows the production of both. Interestingly, while both exogenously added type I and type III IFNs could efficiently protect cells against rotavirus infection, endogenous type III IFNs were found to be key to limit infection of human intestinal cells by rotavirus. By using a fluorescent reporter cell line to highlight the cells mounting an antiviral program, we could show that paracrine signaling driven by type III IFNs efficiently controls the spread of both WT and NSP1 mutant rotavirus. Our results strongly suggest that NSP1 efficiently blocks the type I IFN-mediated antiviral response; however, its restriction of the type III IFN-mediated one is not sufficient to prevent type III IFNs from partially inhibiting viral spread in intestinal epithelial cells. Additionally, our findings further highlight the importance of type III IFNs in controlling rotavirus infection, which could be exploited as antiviral therapeutic measures. IMPORTANCE Rotavirus is one of the most common causes of gastroenteritis worldwide. In developing countries, rotavirus infections lead to more than 200,000 deaths in infants and children. The intestinal epithelial cells lining the gastrointestinal tract combat rotavirus infection by two key antiviral compounds known as type I and III interferons. However, rotavirus has developed countermeasures to block the antiviral actions of the interferons. In this work, we evaluated the arms race between rotavirus and type I and III interferons. We determined that although rotavirus could block the induction of type I interferons, it was unable to block type III interferons. The ability of infected cells to produce and release type III interferons leads to the protection of the noninfected neighboring cells and the clearance of rotavirus infection from the epithelium. This suggests that type III interferons are key antiviral agents and could be used to help control rotavirus infections in children.

Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein.

Rotavirus, a segmented double-stranded RNA virus, is a major cause of acute gastroenteritis in young children. The introduction of live oral rotavirus vaccines has reduced the incidence of rotavirus disease in many countries. To explore the possibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding sequences for NSP3 and FLAG-tagged portions of the SARS-CoV-2 spike (S) protein. A 2A translational element was used to drive separate expression of NSP3 and the S product. rSA11 viruses were recovered that encoded the S-protein S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended receptor-binding domain (ExRBD), and S2 core (CR) domain (rSA11/NSP3-fS1, -fNTD, -fRBD, -fExRBD, and -fCR, respectively). Generation of rSA11/fS1 required a foreign-sequence insertion of 2.2-kbp, the largest such insertion yet made into the rotavirus genome. Based on isopycnic centrifugation, rSA11 containing S sequences were denser than wildtype virus, confirming the capacity of the rotavirus to accommodate larger genomes. Immunoblotting showed that rSA11/-fNTD, -fRBD, -fExRBD, and -fCR viruses expressed S products of expected size, with fExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, rSA11/NSP3-fS1 failed to express its expected 80-kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of portions of the SARS-CoV-2 S protein (e.g., NTD, RBD, ExRBD, and CR) with sizes smaller than the S1 fragment.

Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element.

The segmented 18.5-kbp dsRNA genome of rotavirus expresses 6 structural and 6 nonstructural proteins. We investigated the possibility of using the recently developed plasmid-based rotavirus reverse genetics (RG) system to generate recombinant viruses that express a separate heterologous protein in addition to the 12 viral proteins. To address this, we replaced the NSP3 open reading frame (ORF) of the segment 7 (pT7/NSP3) transcription vector used in the RG system with an ORF encoding NSP3 fused to a fluorescent reporter protein (i.e., UnaG, mRuby, mKate, or TagBFP). Inserted at the fusion junction was a teschovirus translational 2A stop-restart element designed to direct the separate expression of NSP3 and the fluorescent protein. Recombinant rotaviruses made with the modified pT7/NSP3 vectors were well growing and generally genetically stable, and they expressed NSP3 and a separate fluorescent protein detectable by live cell imaging. NSP3 made by the recombinant viruses was functional, inducing nuclear accumulation of cellular poly(A)-binding protein. Further modification of the NSP3 ORF showed that it was possible to generate recombinant viruses encoding 2 heterologous proteins (mRuby and UnaG) in addition to NSP3. Our results demonstrate that, through modification of segment 7, the rotavirus genome can be increased in size to at least 19.8 kbp and can be used to produce recombinant rotaviruses expressing a full complement of viral proteins and multiple heterologous proteins. The generation of recombinant rotaviruses expressing fluorescent proteins will be valuable for the study of rotavirus replication and pathogenesis by live cell imagining and suggest that rotaviruses will prove useful as expression vectors.IMPORTANCE Rotaviruses are a major cause of severe gastroenteritis in infants and young children. Recently, a highly efficient reverse genetics system was developed that allows genetic manipulation of the rotavirus segmented double-stranded RNA genome. Using the reverse genetics system, we show that it is possible to modify one of the rotavirus genome segments (segment 7) such that virus gains the capacity to express a separate heterologous protein in addition to the full complement of viral proteins. Through this approach, we have generated wild-type-like rotaviruses that express various fluorescent reporter proteins, including UnaG (green), mRuby (far red), mKate (red), and TagBFP (blue). Such strains will be of value in probing rotavirus biology and pathogenesis by live cell imagining techniques. Notably, our work indicates that the rotavirus genome is remarkably flexible and able to accommodate significant amounts of heterologous RNA sequence, raising the possibility of using the virus as a vaccine expression vector.

Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins.

Rotaviruses are a large and evolving population of segmented double-stranded RNA viruses that cause severe gastroenteritis in the young of many mammalian and avian host species, including humans. With the recent advent of rotavirus reverse genetics systems, it has become possible to use directed mutagenesis to explore rotavirus biology, modify and optimize existing rotavirus vaccines, and develop rotavirus multitarget vaccine vectors. In this report, we describe a simplified reverse genetics system that allows the efficient and reliable recovery of recombinant rotaviruses. The system is based on co-transfection of T7 transcription vectors expressing full-length rotavirus (+)RNAs and a CMV vector encoding an RNA capping enzyme into BHK cells constitutively producing T7 RNA polymerase (BHK-T7). Recombinant rotaviruses are amplified by overseeding the transfected BHK-T7 cells with MA104 cells, a monkey kidney cell line that is highly permissive for virus growth. In this report, we also describe an approach for generating recombinant rotaviruses that express a separate fluorescent reporter protein through the introduction of a 2A translational stop-restart element into genome segment 7 (NSP3). This approach avoids deleting or modifying any of the viral open reading frames, thus allowing the production of recombinant rotaviruses that retain fully functional viral proteins while expressing a fluorescent protein.

Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System.

Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein.

Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

Collection of Recombinant Rotaviruses Expressing Fluorescent Reporter Proteins.

A collection of recombinant rotaviruses that express the fluorescent markers UnaG, mKate, mRuby, TagBFP, CFP, or YFP as separate proteins was generated. Genes for the fluorescent proteins were inserted into genome segment 7 without compromising expression of the protein NSP3. These recombinant rotaviruses are valuable for analyzing rotavirus biology by fluorescence-based live-cell imaging.

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum.

Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca2+]cyt) and decreasing endoplasmic reticulum (ER) stores. While an overall, monophasic increase in [Ca2+]cyt during RV infection has been shown, the nature of the RV-induced aberrant calcium signals and how they manifest over time at the single-cell level have not been characterized. Thus, we generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted genetically-encoded calcium indicators to characterize calcium signaling throughout RV infection by time-lapse imaging. We found that RV induces highly dynamic [Ca2+]cyt signaling that manifest as hundreds of discrete [Ca2+]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca2+]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca2+]cyt spikes were primarily from ER calcium release and were attenuated by inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs also exhibited prominent [Ca2+]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca2+]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca2+]cyt by dynamic calcium signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced calcium signaling.

Multiple Introductions and Antigenic Mismatch with Vaccines May Contribute to Increased Predominance of G12P[8] Rotaviruses in the United States.

Rotavirus is the leading global cause of diarrheal mortality for unvaccinated children under 5 years of age. The outer capsid of rotavirus virions consists of VP7 and VP4 proteins, which determine viral G and P types, respectively, and are primary targets of neutralizing antibodies. Successful vaccination depends upon generating broadly protective immune responses following exposure to rotaviruses presenting a limited number of G- and P-type antigens. Vaccine introduction resulted in decreased rotavirus disease burden but also coincided with the emergence of uncommon G and P genotypes, including G12. To gain insight into the recent predominance of G12P[8] rotaviruses in the United States, we evaluated 142 complete rotavirus genome sequences and metadata from 151 clinical specimens collected in Nashville, TN, from 2011 to 2013 through the New Vaccine Surveillance Network. Circulating G12P[8] strains were found to share many segments with other locally circulating strains but to have distinct constellations. Phylogenetic analyses of G12 sequences and their geographic sources provided evidence for multiple separate introductions of G12 segments into Nashville, TN. Antigenic epitopes of VP7 proteins of G12P[8] strains circulating in Nashville, TN, differ markedly from those of vaccine strains. Fully vaccinated children were found to be infected with G12P[8] strains more frequently than with other rotavirus genotypes. Multiple introductions and significant antigenic mismatch may in part explain the recent predominance of G12P[8] strains in the United States and emphasize the need for continued monitoring of rotavirus vaccine efficacy against emerging rotavirus genotypes.IMPORTANCE Rotavirus is an important cause of childhood diarrheal disease worldwide. Two immunodominant proteins of rotavirus, VP7 and VP4, determine G and P genotypes, respectively. Recently, G12P[8] rotaviruses have become increasingly predominant. By analyzing rotavirus genome sequences from stool specimens obtained in Nashville, TN, from 2011 to 2013 and globally circulating rotaviruses, we found evidence of multiple introductions of G12 genes into the area. Based on sequence polymorphisms, VP7 proteins of these viruses are predicted to present themselves to the immune system very differently than those of vaccine strains. Many of the sick children with G12P[8] rotavirus in their diarrheal stools also were fully vaccinated. Our findings emphasize the need for continued monitoring of circulating rotaviruses and the effectiveness of the vaccines against strains with emerging G and P genotypes.