Development of recombinant rotavirus carrying herpes simplex virus 2 glycoprotein D gene based on reverse genetics technology.

Vaccine development for herpes simplex virus 2 (HSV-2) has been attempted, but no vaccines are yet available. A plasmid-based reverse genetics system for Rotavirus (RV), which can cause gastroenteritis, allows the generation of recombinant RV containing foreign genes. In this study, we sought to develop simian RV (SA11) as a vector to express HSV-2 glycoprotein D (gD2) and evaluated its immunogenicity in mice. We generated the recombinant SA11-gD2 virus (rSA11-gD2) and confirmed its ability to express gD2 in vitro. The virus was orally inoculated into suckling BALB/c mice and into 8-week-old mice. Serum IgG and IgA titers against RV and gD2 were measured by ELISA. In the 8-week-old mice inoculated with rSA11-gD2, significant increases in not only antibodies against RV but also IgG against gD2 were demonstrated. In the suckling mice, antibodies against RV were induced, but gD2 antibody was not detected. Diarrhea observed after the first inoculation of rSA11-gD2 in suckling mice was similar to that induced by the parent virus. A gD2 expressing simian RV recombinant, which was orally inoculated, induced IgG against gD2. This strategy holds possibility for genital herpes vaccine development.

Synergistic xylan decomposition by a reducing-end xylose-releasing exo-oligoxylanase with other xylanolytic enzymes derived from Paenibacillus xylaniclasticus strain TW1.

Paenibacillus xylaniclasticus strain TW1 is a promising tool for decomposing xylan-containing lignocellulosic biomass, since this strain possesses various genes encoding cellulolytic/hemicellulolytic enzymes. In this study, PxRex8A from the TW1 strain was found to be a reducing-end xylose-releasing exo-oligoxylanase of glycoside hydrolase family 8, which cleaves xylose from xylooligosacchrides of corn core xylan. In synergistic assay, the efficient decomposition of oat spelt xylan (OSX) and beech wood xylan was exemplified in the combination of endo ß-1,4-xylanase (PxXyn11A) and PxRex8A from the TW1 strain in a molar ratio of 4:1. Furthermore, it was found that the addition of ß-d-xylosidase/α-l-arabinofuranosidase (PxXyl43A) from this strain with PxXyn11A and PxRex8A achieved twice the amount of reducing sugars (1.1 mg/mL) against OSX after 24 hours compared to PxXyn11A alone (0.5 mg/mL). These results present that synergy effect of PxRex8A and PxXyl43A with PxXyn11A promotes xylan degradation into xylose.

Establishment of a reverse genetics system for avian rotavirus A strain PO-13.

Avian rotavirus A (RVA) is one of major enteric pathogens that cause diarrhoea in young avian individuals. Importantly, some of the avian RVA strains of G18P[17] genotype are naturally transmitted to and cause clinical diseases in mammalian species, indicating their potential risks to animal health. Although molecular information on the pathogenesis by avian RVA strains will be useful for estimating their risks, the absence of a reverse genetics (RG) system for these strains has hindered the elucidation of their pathogenic mechanisms. In this study, we aimed to establish an RG system for the avian G18P[17] prototype strain PO-13, which was isolated from a pigeon in Japan in 1983 and was experimentally shown to be pathogenic in suckling mice. Transfection with plasmids expressing 11 genomic RNA segments of the strain resulted in rescue of the infectious virus with an artificially introduced genetic marker on its genome, indicating that an RG system for the PO-13 strain was successfully established. The rescued recombinant strain rPO-13 had biological properties almost identical to those of its wild-type strain (wtPO-13). Notably, both rPO-13 and wtPO-13 induced diarrhoea in suckling mice with similar efficiencies. It was thus demonstrated that the RG system will be useful for elucidating the pathogenic mechanisms of the PO-13 strain at the molecular level. This is the first report of the establishment of an RG system for an avian RVA strain.

Rotavirus incapable of NSP6 expression can cause diarrhea in suckling mice.

The group A rotavirus (RVA) genome comprising 11 double-stranded RNAs encodes six structural proteins (VP1-VP4, VP6, and VP7) and six non-structural proteins (NSP1-NSP6). Among these 12 rotaviral proteins, NSP6 has been less studied as to its function. We previously prepared a recombinant NSP6-deficient RVA derived from simian strain SA11-L2 by reverse genetics, and found that the NSP6-deficient virus grew well in cell culture, although its growth was less abundant than that of the parental SA11-L2 strain. In this study, we examined the potency of a recombinant RVA incapable of NSP6 expression to cause diarrhoea in suckling mice. The suckling mice infected with the NSP6-deficient virus apparently experienced diarrhoea, although the symptom was milder and the duration of diarrhoea was shorter than in the mice infected with the authentic SA11-L2 strain. Thus, together with the results obtained for cultured cells in the previous study, it can be concluded that NSP6 is not necessarily required for replication and pathogenicity in vitro and in vivo.

Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes.

With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.

Full genome-based characterization of G4P[6] rotavirus strains from diarrheic patients in Thailand: Evidence for independent porcine-to-human interspecies transmission events.

The exact evolutionary patterns of human G4P[6] rotavirus strains remain to be elucidated. Such strains possess unique and strain-specific genotype constellations, raising the question of whether G4P[6] strains are primarily transmitted via independent interspecies transmission or human-to-human transmission after interspecies transmission. Two G4P[6] rotavirus strains were identified in fecal specimens from hospitalized patients with severe diarrhea in Thailand, namely, DU2014-259 (RVA/Human-wt/THA/DU2014-259/2014/G4P[6]) and PK2015-1-0001 (RVA/Human-wt/THA/PK2015-1-0001/2015/G4P[6]). Here, we analyzed the full genomes of the two human G4P[6] strains, which provided the opportunity to study and confirm their evolutionary origin. On whole genome analysis, both strains exhibited a unique Wa-like genotype constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The NSP1 genotype A8 is commonly found in porcine rotavirus strains. Furthermore, on phylogenetic analysis, each of the 11 genes of strains DU2014-259 and PK2015-1-0001 appeared to be of porcine origin. On the other hand, the two study strains consistently formed distinct clusters for nine of the 11 gene segments (VP4, VP6, VP1-VP3, and NSP2-NSP5), strongly indicating the occurrence of independent porcine-to-human interspecies transmission events. Our observations provide important insights into the origin of zoonotic G4P[6] strains, and into the dynamic interaction between porcine and human rotavirus strains.

The Detection of Rotavirus Antigenemia by Immunochromatographic Kits: a Case Series.

BACKGROUND: Acute gastroenteritis is the most common cause of illness and death in infants and young children worldwide. Rotaviruses (RVs) are the major viruses that cause acute gastroenteritis in young children, especially in developing countries in Asia and Africa. METHODS: The presence of rotavirus antigens in sera of four unvaccinated pediatric patients, aged between 4 and 6 years with severe diarrhea and dehydration, were detected by using three immunochromatographic (IC) kits. In addition, the presence of anti-rotavirus IgG, IgA, and IgM antibodies and their concentrations in patient sera were also determined by enzyme immunoassay (EIA). RESULTS: All three kits could detect rotavirus antigen in patient sera with different intensity of the test lines. When patient sera were pretreated with anti-VP6 rotavirus mouse monoclonal antibody prior to testing, the rotavirus positive test lines disappeared, suggesting that all patient sera contained VP6 protein antigen of rotavirus. Assessment of antibody concentration in these patient sera revealed that all patient sera contained IgG, IgA, and IgM antibodies against rotavirus antigen at different concentrations. CONCLUSIONS: The sensitivity of rotavirus protein detection in the patient sera of one IC kit brand was comparable to those of the EIA, suggesting this IC kit could be an alternative screening method for rapid diagnosis of rotavirus infection.

Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics.

Reassortment is an important mechanism in the evolution of group A rotaviruses (RVAs), yielding viruses with novel genetic and phenotypic traits. The classical methods for generating RVA reassortants with the desired genetic combinations are laborious and time-consuming because of the screening and selection processes required to isolate a desired reassortant. Taking advantage of a recently developed RVA reverse genetics system based on just 11 cloned cDNAs encoding the RVA genome (11 plasmid-only system), we prepared a panel of simian SA11-L2 virus-based single-gene reassortants, each containing 1 segment derived from human KU virus of the G1P[8] genotype. It was shown that there was no gene-specific restriction of the reassortment potential. In addition to these 11 single-gene reassortants, a triple-gene reassortant with KU-derived core-encoding VP1-3 gene segments with the SA11-L2 genetic background, which make up a virion composed of the KU-based core, and SA11-L2-based intermediate and outer layers, could also be prepared with the 11 plasmid-only system. Finally, for possible clinical application of this system, we generated a series of VP7 reassortants representing all the major human RVA G genotypes (G1-4, G9 and G12) efficiently. The preparation of each of these single-gene reassortants was achieved within just 2 weeks. Our results demonstrate that the 11 plasmid-only system allows the rapid and reliable generation of RVA single-gene reassortants, which will be useful for basic research and clinical applications.

Generation of Infectious Recombinant Human Rotaviruses from Just 11 Cloned cDNAs Encoding the Rotavirus Genome.

The generation of recombinant group A rotaviruses (RVAs) entirely from cloned cDNAs has been described only for a single animal RVA strain, simian SA11-L2. We recently developed an optimized RVA reverse genetics system based on only RVA cDNAs (11-plasmid system), in which the concentration of cDNA plasmids containing the NSP2 and NSP5 genes is 3- or 5-fold increased in relation to that of the other plasmids. Based on this approach, we generated a recombinant human RVA (HuRVA)-based monoreassortant virus containing the VP4 gene of the simian SA11-L2 virus using the 11-plasmid system. In addition to this monoreassortant virus, authentic HuRVA (strain KU) was also generated with the 11-plasmid system with some modifications. Our results demonstrate that the 11-plasmid system involving just RVA cDNAs can be used for the generation of recombinant HuRVA and recombinant HuRVA-based reassortant viruses.IMPORTANCE Human group A rotavirus (HuRVA) is a leading pathogen causing severe diarrhea in young children worldwide. In this paper, we describe the generation of recombinant HuRVA (strain KU) from only 11 cloned cDNAs encoding the HuRVA genome by reverse genetics. The growth properties of the recombinant HuRVA were similar to those of the parental RVA, providing a powerful tool for better understanding of HuRVA replication and pathogenesis. Furthermore, the ability to manipulate the genome of HuRVAs "to order" will be useful for next-generation vaccine production for this medically important virus and for the engineering of clinical vectors expressing any foreign genes.

High prevalence of equine-like G3P[8] rotavirus in children and adults with acute gastroenteritis in Thailand.

Group A rotavirus (RVA) is a major cause of acute gastroenteritis in infants and young children worldwide. This study aims to clarify the distribution of G/P types and genetic characteristics of RVAs circulating in Thailand. Between January 2014 and September 2016, 1867 stool specimens were collected from children and adults with acute gastroenteritis in six provinces in Thailand. RVAs were detected in 514/1867 (27.5%) stool specimens. G1P[8] (44.7%) was the most predominant genotype, followed by G3P[8] (33.7%), G2P[4] (11.5%), G8P[8] (7.0%), and G9P[8] (1.3%). Unusual G3P[9] (0.8%), G3P[10] (0.4%), G4P[6] (0.4%), and G10P[14] (0.2%) were also detected at low frequencies. The predominant genotype, G1P[8] (64.4%), in 2014 decreased to 6.1% in 2016. In contrast, the frequency of G3P[8] markedly increased from 5.5% in 2014 to 65.3% in 2015 and 89.8% in 2016. On polyacrylamide gel electrophoresis, most (135/140; 96.4%) of the G3P[8] strains exhibited a short RNA profile. Successful determination of the nucleotide sequences of the VP7 genes of 98 G3P[8] strains with a short RNA profile showed that they are all equine-like G3P[8] strains. On phylogenetic analysis of genome segments of two representative Thai equine-like G3P[8] strains, it was noteworthy that they possessed distinct NSP4 genes, one bovine-like and the other human-like. Thus, we found that characteristic equine-like G3P[8] strains with a short RNA electropherotype are becoming highly prevalent in children and adults in Thailand.

Reverse genetics system for human rotaviruses.

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.

Molecular characterization of rotaviruses obtained from patients with rotavirus-associated encephalitis/encephalopathy.

Group A rotavirus (RVA) rarely causes severe complications such as encephalitis/encephalopathy. However, the pathophysiology of this specific complication remains unclear. Next-generation sequence analysis was used to compare the entire genome sequences of RVAs detected in patients with encephalitis/encephalopathy and gastroenteritis. This study enrolled eight patients with RVA encephalitis/encephalopathy and 10 with RVA gastroenteritis who were treated between February 2013 and July 2014. Viral RNAs were extracted from patients' stool, and whole-genome sequencing analysis was carried out to identify the specific gene mutations in RVA obtained from patients with severe neurological complications. Among the eight encephalitis/encephalopathy cases, six strains were DS-1-like G1P[8] and the remaining two were Wa-like G1P[8] (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1). Meanwhile, eight of the 10 viruses detected in rotavirus gastroenteritis patients were DS-1-like G1P[8], and the remaining two were Wa-like G1P[8]. These strains were further characterized by conducting phylogenetic analysis. No specific clustering was demonstrated in RVAs detected from encephalitis/encephalopathy patients. Although the DS-1-like G1P[8] strain was predominant in both groups, no specific molecular characteristics were detected in RVAs from patients with severe central nervous system complications.

Unusual mono-reassortant of a Wa-like G1P[8] species A rotavirus containing a DS-1-like (genotype 2) NSP4 gene.

Species A rotaviruses are a major cause of acute gastroenteritis in infants and young children worldwide. Reassortment is a common phenomenon due to the segmented nature of the rotavirus genome. The complete coding sequences of a species A rotavirus strain isolated from the feces of a child with acute gastroenteritis in Japan in 2018 were determined using an unbiased viral metagenomics approach. The genetic analysis revealed that the rotavirus strain had an unusual genomic constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), suggesting reassortment of a genotype 1 with a genotype 2 rotavirus, from which the NSP4-encoding gene was acquired.

Entirely plasmid-based reverse genetics system for rotaviruses.

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein-tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIß (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein ß. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.

Generation of Recombinant Rotaviruses Expressing Fluorescent Proteins by Using an Optimized Reverse Genetics System.

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors.IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.

Persistent systemic rotavirus vaccine infection in a child with X-linked severe combined immunodeficiency.

OBJECTIVE: The main aims of the present study were to elucidate the systemic group A rotavirus (RVA) infection and to clarify the genetic changes of persistent virus in the X-linked severe combined immunodeficiency (SCID) patient. METHODS: RotaTeq vaccine (RV5) genotype-specific real-time reverse transcription polymerase chain reaction was used to monitor viral RNA load in serially collected serum and stool samples. Next-generation sequence analysis was used to determine the genotype of the virus by sequencing 11 gene segments. Polyacrylamide gel electrophoresis (PAGE) analysis was used to identify rearrangement of viral genes. The gene rearrangement was examined in NSP5 gene by using Sanger sequence. RESULTS: A 7-month-old boy demonstrated chronic diarrhea following the third administration of RV5 and failure to thrive. He was diagnosed with X-linked SCID and successfully underwent cord blood transplantation. High copy numbers of RV5 genotype G1 RNA were detected in serially collected stool and serum samples and the kinetics of viral RNA loads were correlated with the degree of clinical disease. Next-generation sequence analysis revealed genetic reassortment at least between the strains WI79-9/G1P7[5] and WI79-4/G6P1A[8] in the VP7 gene and the VP4 gene among the vaccine-derived rotavirus strains. In addition, PAGE analysis suggested genetic rearrangements in several genes, and it was confirmed in the NSP5 gene by sequence analysis. CONCLUSIONS: The kinetics of RVA RNA load in serum and stool samples was consistent with the clinical course of the patient. Among five genotypes of RV5 vaccine, G1 genotype replicated well in this patient. Reassortment and rearrangements were demonstrated in persistently infected G1 genotype of RV5.

Rotavirus Vaccination Can Be Performed Without Viral Dissemination in the Neonatal Intensive Care Unit.

Background: This study was conducted to assess the transmissibility of rotavirus vaccine strains after rotavirus vaccination in a neonatal intensive care unit (NICU). Methods: Pentavalent (RV5) or monovalent (RV1) rotavirus vaccine was administered to infants admitted to the NICU. Nineteen vaccinated infants and 49 unvaccinated infants whose beds were located in close proximity to the vaccinated infants were enrolled in this study. Dissemination and fecal shedding of vaccine viruses within the NICU were examined using real-time reverse transcription-polymerase chain reaction. Results: Shedding of the vaccine strain was detected in all 19 vaccinated infants. RV5 virus shedding started 1 day after the first vaccination and persisted for 8 days after the first vaccination, and viral shedding terminated by day 5 after administration of the second RV5 dose. The kinetics of RV1 virus shedding differed among vaccinated infants. The duration of RV1 virus shedding was longer after the first vaccination than after the second vaccination. In contrast to the vaccinated infants, no vaccine virus genomes were detected in any of the stool samples collected from the 49 unvaccinated infants. Conclusions: This study is direct evidence of no transmission of rotavirus vaccine strains between vaccinated infants and unvaccinated infants in close proximity within a NICU.

Monitoring Shedding of Five Genotypes of RotaTeq Vaccine Viruses by Genotype-Specific Real-Time Reverse Transcription-PCR Assays.

RotaTeq (RV5) is a widely used live attenuated pentavalent rotavirus (RV) vaccine. Although fecal shedding of RV vaccine strains persists for long time periods, it is unclear how each vaccine strain replicates in intestinal tissue and is excreted in stool. To examine this issue, we established RV5 genotype-specific real-time reverse transcription-PCR (RT-PCR) assays. Five real-time RT-PCR assays were designed for the VP7 gene in genotypes G1, G2, G3, G4, and G6. All assays exhibited excellent linearity, and the detection limit was 1 infectious unit (IU)/reaction for G2, G4, and G6 and 10 IUs/reaction for G1 and G3. No cross-reactivity was observed among G genotypes. The inter- and intra-assay coefficients of variation were less than 3%. The assays were used to examine 129 stool samples collected from eight infants who received RV5. In cases 1 and 2, who received three rounds of vaccination, RV shedding decreased gradually with the number of vaccinations. G1 and G6 shedding appeared to be predominant in comparison to shedding of the other genotypes. Patterns of fecal shedding of the five genotypes of vaccine viruses differed between the eight vaccine recipients. RV5 genotype-specific real-time RT-PCR assays will be useful to study the molecular biology of RV5 replication in infants and experimental animals.

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth.IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.