Evaluation of different genomic regions of rotavirus B and rotavirus C for development of real-time RT‒PCR assays.

BACKGROUND: The causative agents of diarrhea, rotavirus B (RVB) and rotavirus C (RVC) are common in adults and patients of all age groups, respectively. Due to the Rotavirus A (RVA) vaccination program, a significant decrease in the number of gastroenteritis cases has been observed globally. The replacement of RVA infections with RVB, RVC, or other related serogroups is suspected due to the possibility of reducing natural selective constraints due to RVA infections. The data available on RVB and RVC incidence are scant due to the lack of cheap and rapid commercial diagnostic assays and the focus on RVA infections. The present study aimed to develop real-time RT‒PCR assays using the data from all genomic RNA segments of human RVB and RVC strains available in the Gene Bank. RESULTS: Among the 11 gene segments, NSP3 and NSP5 of RVB and the VP6 gene of RVC were found to be suitable for real-time RT‒PCR (qRT‒PCR) assays. Fecal specimens collected from diarrheal patients were tested simultaneously for the presence of RVB (n = 192) and RVC (n = 188) using the respective conventional RT‒PCR and newly developed qRT‒PCR assays. All RVB- and RVC-positive specimens were reactive in their respective qRT‒PCR assays and had Ct values ranging between 23.69 and 41.97 and 11.49 and 36.05, respectively. All known positive and negative specimens for other viral agents were nonreactive, and comparative analysis showed 100% concordance with conventional RT‒PCR assays. CONCLUSIONS: The suitability of the NSP5 gene of RVB and the VP6 gene of RVC was verified via qRT‒PCR assays, which showed 100% sensitivity and specificity. The rapid qRT‒PCR assays developed will be useful diagnostic tools, especially during diarrheal outbreaks for testing non-RVA rotaviral agents and reducing the unnecessary use of antibiotics.

The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Human transmission and outbreaks of feline-like G6 rotavirus revealed with whole-genome analysis of G6P[9] feline rotavirus.

Group A rotaviruses (RVAs) are generally highly species-specific; however, some strains infect across species. Feline RVAs sporadically infect humans, causing gastroenteritis. In 2012 and 2013, rectal swab samples were collected from 61 asymptomatic shelter cats at a public health center in Mie Prefecture, Japan, to investigate the presence of RVA and any association with human infections. The analysis identified G6P[9] strains in three cats and G3P[9] strains in two cats, although no feline RVA sequence data were available for the former. A whole-genome analysis of these G6P[9] strains identified the genotype constellation G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. The nucleotide identity among these G6P[9] strains exceeded 99.5% across all 11 gene segments, indicating the circulation of this G6P[9] strain among cats. Notably, strain RVA/Human-wt/JPN/KF17/2010/G6P[9], previously detected in a 3-year-old child with gastroenteritis, shares high nucleotide identity (>98%) with Mie20120017f, the representative G6P[9] strain in this study, across all 11 gene segments, confirming feline RVA infection and symptomatic presentation in this child. The VP7 gene of strain Mie20120017f also shares high nucleotide identity with other sporadically reported G6 RVA strains in humans. This suggests that feline-origin G6 strains as the probable source of these sporadic G6 RVA strains causing gastroenteritis in humans globally. Moreover, a feline-like human G6P[8] strain circulating in Brazil in 2022 was identified, emphasizing the importance of ongoing surveillance to monitor potential global human outbreaks of RVA.

Impact of Limosilactobacillus fermentum probiotic treatment on gut microbiota composition in sahiwal calves with rotavirus diarrhea: A 16S metagenomic analysis study".

BACKGROUND: Diarrhea poses a major threat to bovine calves leading to mortality and economic losses. Among the causes of calf diarrhea, bovine rotavirus is a major etiological agent and may result in dysbiosis of gut microbiota. The current study was designed to investigate the effect of probiotic Limosilactobacillus fermentum (Accession No.OR504458) on the microbial composition of rotavirus-infected calves using 16S metagenomic analysis technique. Screening of rotavirus infection in calves below one month of age was done through clinical signs and Reverse Transcriptase PCR. The healthy calves (n = 10) were taken as control while the infected calves (n = 10) before treatment was designated as diarrheal group were treated with Probiotic for 5 days. All the calves were screened for the presence of rotavirus infection on each day and fecal scoring was done to assess the fecal consistency. Infected calves after treatment were designated as recovered group. Fecal samples from healthy, recovered and diarrheal (infected calves before sampling) were processed for DNA extraction while four samples from each group were processed for 16S metagenomic analysis using Illumina sequencing technique and analyzed via QIIME 2. RESULTS: The results show that Firmicutes were more abundant in the healthy and recovered group than in the diarrheal group. At the same time Proteobacteria was higher in abundance in the diarrheal group. Order Oscillospirales dominated healthy and recovered calves and Enterobacterials dominated the diarrheal group. Alpha diversity indices show that diversity indices based on richness were higher in the healthy group and lower in the diarrheal group while a mixed pattern of clustering between diarrheal and recovered groups samples in PCA plots based on beta diversity indices was observed. CONCLUSION: It is concluded that probiotic Limosilactobacillus Fermentum N-30 ameliorate the dysbiosis caused by rotavirus diarrhea and may be used to prevent diarrhea in pre-weaned calves after further exploration.

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Introduction: Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. Methods: In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. Results: The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. Discussion: The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

Epidemiological Surveillance: Genetic Diversity of Rotavirus Group A in the Pearl River Delta, Guangdong, China in 2019.

Objective: This study aimed to understand the epidemic status and phylogenetic relationships of rotavirus group A (RVA) in the Pearl River Delta region of Guangdong Province, China. Methods: This study included individuals aged 28 days-85 years. A total of 706 stool samples from patients with acute gastroenteritis collected between January 2019 and January 2020 were analyzed for 17 causative pathogens, including RVA, using a Gastrointestinal Pathogen Panel, followed by genotyping, virus isolation, and complete sequencing to assess the genetic diversity of RVA. Results: The overall RVA infection rate was 14.59% (103/706), with an irregular epidemiological pattern. The proportion of co-infection with RVA and other pathogens was 39.81% (41/103). Acute gastroenteritis is highly prevalent in young children aged 0-1 year, and RVA is the key pathogen circulating in patients 6-10 months of age with diarrhea. G9P[8] (58.25%, 60/103) was found to be the predominant genotype in the RVA strains, and the 41 RVA-positive strains that were successfully sequenced belonged to three different RVA genotypes in the phylogenetic analysis. Recombination analysis showed that gene reassortment events, selection pressure, codon usage bias, gene polymorphism, and post-translational modifications (PTMs) occurred in the G9P[8] and G3P[8] strains. Conclusion: This study provides molecular evidence of RVA prevalence in the Pearl River Delta region of China, further enriching the existing information on its genetics and evolutionary characteristics and suggesting the emergence of genetic diversity. Strengthening the surveillance of genotypic changes and gene reassortment in RVA strains is essential for further research and a better understanding of strain variations for further vaccine development.

Recent Progress in Human Milk Oligosaccharides and Its Antiviral Efficacy.

Gastrointestinal (GI)-associated viruses, including rotavirus (RV), norovirus (NV), and enterovirus, usually invade host cells, transmit, and mutate their genetic information, resulting in influenza-like symptoms, acute gastroenteritis, encephalitis, or even death. The unique structures of human milk oligosaccharides (HMOs) enable them to shape the gut microbial diversity and endogenous immune system of human infants. Growing evidence suggests that HMOs can enhance host resistance to GI-associated viruses but without a systematic summary to review the mechanism. The present review examines the lactose- and neutral-core HMOs and their antiviral effects in the host. The potential negative impacts of enterovirus 71 (EV-A71) and other GI viruses on children are extensive and include neurological sequelae, neurodevelopmental retardation, and cognitive decline. However, the differences in the binding affinity of HMOs for GI viruses are vast. Hence, elucidating the mechanisms and positive effects of HMOs against different viruses may facilitate the development of novel HMO derived oligosaccharides.

Epidemiology of pathogens causing acute diarrhea in patients presenting to the emergency departments in 4 hospitals in Lebanon.

Acute diarrhea is a prevalent disease worldwide and a cause of mortality in low and middle-income countries. In previous studies, the causative enteropathogen is found in 38% to 58% of patients, leaving a considerable diagnostic gap. In this study, we intend to determine the pathogenic agents responsible for acute diarrhea in patients presenting to the Emergency Departments of several Lebanese hospitals. A total of 100 stool samples were collected between June 2022 and June 2023 from patients presenting with acute diarrhea to the Emergency Departments. Pathogens were detected by multiplex polymerase chain reaction. The average age for the patients was 53 years old. All patients presented with diarrhea, 15 of them had fever associated. In total, 46 patients were admitted to the hospital (70%), among them 80% received Intravenous antibiotics. Campylobacter was the most common agent detected in 36%, followed by Rotavirus 19%, and Noroviruses 15%. The rest was detected at lower percentages. Bacteria accounted for 49% of cases, viruses for 39%, and parasitic infection 6%. Acute diarrhea epidemiology is understudied in Lebanon. This study is the first Lebanese data about acute diarrhea pathogens. Avoiding overuse of antibiotics in bacterial versus viral infections can be achieved while prevention campaigns can raise awareness about food and water safety at the community level.

Rates and determinants of Rotavirus vaccine uptake among children in Italy: a cross-sectional study within the 2022 OBVIOUS* project.

INTRODUCTION: The World Health Organization defines rotavirus as among the most severe causes of viral gastroenteritis affecting children under 5 year old. Italy and other European countries do not release disaggregated data on rotavirus vaccination coverage. This study aimed to assess the uptake and drivers of rotavirus vaccination in Italy. METHODS: We administered a survey to 10,000 Italian citizens recruited via an online panel and proportionate to key demographic strata. We examined rotavirus vaccine uptake among parents whose youngest child was aged 6 weeks to 4 years, their sociodemographic characteristics, their beliefs about vaccine administration, and who recommended the rotavirus vaccination. RESULTS: A total of 711 respondents met the inclusion criteria for the rotavirus vaccine questionnaire. The uptake was estimated at 60.3% nationwide (66.4% among mothers and 50.2% among fathers). Being a mother and living in cities/suburbs was significantly associated with a higher likelihood of vaccine uptake, while fathers were more likely to be uncertain of their children's vaccine status. Living in Central Italy and having friends/relatives opposed to vaccination were found to be significantly associated with a lower likelihood of vaccine uptake, while parents' education level and children's demographics were not found to correlate with any outcomes. In 90.3% of cases, the rotavirus vaccination was recalled as being recommended by a paediatrician. CONCLUSIONS: Consistent collection of behavioural preferences and socioeconomic characteristics of recipients of rotavirus vaccine campaigns, their epidemiological information, cost-benefit, and national policy data are crucial for designing effective vaccination strategies in Italy and other European countries with similar social profiles to reach the target uptake.

Lipidomics reveals the significance and mechanism of the cellular ceramide metabolism for rotavirus replication.

As one of the most important causative agents of severe gastroenteritis in children, piglets, and other young animals, species A rotaviruses have adversely impacted both human health and the global swine industry. Vaccines against rotaviruses (RVs) are insufficiently effective, and no specific treatment is available. To understand the relationships between porcine RV (PoRV) infection and enterocytes in terms of the cellular lipid metabolism, we performed an untargeted liquid chromatography mass spectrometry (LC-MS) lipidomics analysis of PoRV-infected IPEC-J2 cells. Herein, a total of 451 lipids (263 upregulated lipids and 188 downregulated lipids), spanning sphingolipid, glycerolipid, and glycerophospholipids, were significantly altered compared with the mock-infected group. Interestingly, almost all the ceramides among these lipids were upregulated during PoRV infection. LC-MS analysis was used to validated the lipidomics data and demonstrated that PoRV replication increased the levels of long-chain ceramides (C16-ceramide, C18-ceramide, and C24-ceramide) in cells. Furthermore, we found that these long-chain ceramides markedly inhibited PoRV infection and that their antiviral actions were exerted in the replication stage of PoRV infection. Moreover, downregulation of endogenous ceramides with the ceramide metabolic inhibitors enhanced PoRV propagation. Increasing the levels of ceramides by the addition of C6-ceramide strikingly suppressed the replication of diverse RV strains. We further found that the treatment with an apoptotic inhibitor could reverse the antiviral activity of ceramide against PoRV replication, demonstrating that ceramide restricted RV infection by inducing apoptosis. Altogether, this study revealed that ceramides played an antiviral role against RV infection, providing potential approaches for the development of antiviral therapies.IMPORTANCERotaviruses (RVs) are among the most important zoonosis viruses, which mainly infected enterocytes of the intestinal epithelium causing diarrhea in children and the young of many mammalian and avian species. Lipids play an essential role in viral infection. A comprehensive understanding of the interaction between RV and lipid metabolism in the enterocytes will be helpful to control RV infection. Here, we mapped changes in enterocyte lipids following porcine RV (PoRV) infection using an untargeted lipidomics approach. We found that PoRV infection altered the metabolism of various lipid species, especially ceramides (derivatives of the sphingosine). We further demonstrated that PoRV infection increased the accumulation of ceramides and that ceramides exerted antiviral effects on RV replication by inducing apoptosis. Our findings fill a gap in understanding the alterations of lipid metabolism in RV-infected enterocytes and highlight the antiviral effects of ceramides on RV infection, suggesting potential approaches to control RV infection.

Assessment of Gastroenteric Viruses in Marketed Bivalve Mollusks in the Tourist Cities of Rio de Janeiro, Brazil, 2022.

This study investigated the prevalence and genetic diversity of gastroenteric viruses in mussels and oysters in Rio de Janeiro, Brazil. One hundred and thirty-four marketed bivalve samples were obtained between January and December 2022. The viral analysis was performed according to ISO/TS 15216, and the screening revealed the detection of norovirus GII/GI (40.3%), sapovirus (SaV; 12.7%), human mastadenovirus (7.5%), and rotavirus A (RVA; 5.9%). In total, 44.8% (60) of shellfish samples tested positive for one or more viruses, 46.7% (28/60) of the positive samples tested positive for a single viral agent, 26.7% (16) tested positive for two viral agents, 8.3% (5) for three viral agents, and 13.3% (8) for four viral agents. Additionally, three mussel samples were contaminated with the five investigated viruses (5%, 3/60). Norovirus GII showed the highest mean viral load (3.4 × 105 GC/g), followed by SaV (1.4 × 104 GC/g), RVA (1.1 × 104 GC/g), human mastadenovirus (3.9 × 103 GC/g), and norovirus GI (6.7 × 102 GC/g). Molecular characterization revealed that the recovered norovirus strains belonged to genotypes GII.2, GII.6, GII.9, GII.17, and GII.27; SaV belonged to genotypes GI.1 and GIV.1; RVA to genotypes G6, G8, P[8]-III, and human mastadenovirus to types F40 and F41. The GII.27 norovirus characterized in this study is the only strain of this genotype reported in Brazil. This study highlights the dissemination and diversity of gastroenteric viruses present in commercialized bivalves in a touristic area, indicating the potential risk to human health and the contribution of bivalves in the propagation of emerging pathogens.

An isothermal nucleic acid amplification-based enzymatic recombinase amplification method for dual detection of porcine epidemic diarrhea virus and porcine rotavirus A.

Viral diarrhea is the predominant digestive tract sickness in piglings, resulting in substantial profit losses in the porcine industry. Porcine rotavirus A (PoRVA) and porcine epidemic diarrhea virus (PEDV) are the main causes of grave gastroenteritis and massive dysentery, especially in piglets. PoRVA and PEDV have high transmissibility, exhibit similar clinical symptoms, and frequently co-occur. Therefore, to avoid financial losses, a quick, highly efficient, objective diagnostic test for the prevention and detection of these diseases is required. Enzymatic recombinase amplification (ERA) is a novel technology based on isothermal nucleic acid amplification. It demonstrates high sensitivity and excellent specificity, with a short processing time and easy operability, compared with other in vitro nucleic acid amplification technologies. In this study, a dual ERA method to detect and distinguish between PEDV and PoRVA nucleic acids was established. The method shows high sensitivity, as the detection limits were 101 copies/µL for both viruses. To test the usefulness of this method in clinical settings, we tested 64 swine clinical samples. Our results were 100% matched with those acquired using a commercially available kit. Therefore, we have successfully developed a dual diagnostic ERA nucleic acids method for detecting and distinguishing between PEDV and PoRVA.

Emergence and dissemination of equine-like G3P[8] rotavirus A in Brazil between 2015 and 2021.

Rotavirus A (RVA) is a major cause of acute gastroenteritis globally that is classically genotyped by its two immunodominant outer capsid proteins, VP7 (G-) and VP4 (P-). Recent evidence suggests that the reassortant equine-like G3P[8] strain played a substantial role in RVA transmission in Brazil since 2015. To understand its global emergence and dissemination in Brazilian territory, stool samples collected from 11 Brazilian states (n = 919) were genotyped by RT-qPCR and proceeded to sequence the VP7 gene (n = 102, 79 being newly generated) of the G3P[8] samples with pronounced viral loads. Our phylogenetic genotyping showed that G3P[8] became the dominant strain in Brazil between 2017 and 2020, with equine-like variants representing 75%-100% of VP7 samples in this period. A Bayesian discrete phylogeographic analysis strongly suggests that the equine-like G3P[8] strain originated in Asia during the early 2010s and subsequently spread to Europe, the Caribbean, and South America. Multiple introductions were detected in Brazil between 2014 and 2017, resulting in five national clusters. The reconstruction of the effective population size of the largest Brazilian cluster showed an expansion until 2017, followed by a plateau phase until 2019 and subsequent contraction. Our study also supports that most mutations fixed during equine-like G3P[8] evolution were synonymous, suggesting that adaptive evolution was not an important driving force during viral dissemination in humans, potentially increasing its susceptibility to acquired immunity. This research emphasizes the need for comprehensive rotavirus genomic surveillance that allows close monitoring of its ever-shifting composition and informs more effective public health policies.IMPORTANCEOur original article demonstrated the origin and spread in a short time of equine-like G3P[8] in Brazil and the world. Due to its segmented genome, it allows numerous mechanisms including genetic drift and reassortment contribute substantially to the genetic diversity of rotavirus. Although the effectiveness and increasing implementation of vaccination have not been questioned, a matter of concern is its impact on the emergence of escape mutants or even the spread of unusual strains of zoonotic transmission that could drive epidemic patterns worldwide. This research emphasizes the need for comprehensive rotavirus genomic surveillance, which could facilitate the formulation of public policies aimed at preventing and mitigating its transmission.

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.

Novel Universal Recombinant Rotavirus A Vaccine Candidate: Evaluation of Immunological Properties.

Rotavirus infection is a leading cause of severe dehydrating gastroenteritis in children under 5 years of age. Although rotavirus-associated mortality has decreased considerably because of the introduction of the worldwide rotavirus vaccination, the global burden of rotavirus-associated gastroenteritis remains high. Current vaccines have a number of disadvantages; therefore, there is a need for innovative approaches in rotavirus vaccine development. In the current study, a universal recombinant rotavirus antigen (URRA) for a novel recombinant vaccine candidate against rotavirus A was obtained and characterised. This antigen included sequences of the VP8* subunit of rotavirus spike protein VP4. For the URRA, for the first time, two approaches were implemented simultaneously-the application of a highly conserved neutralising epitope and the use of the consensus of the extended protein's fragment. The recognition of URRA by antisera to patient-derived field rotavirus isolates was proven. Plant virus-based spherical particles (SPs), a novel, effective and safe adjuvant, considerably enhanced the immunogenicity of the URRA in a mouse model. Given these facts, a URRA + SPs vaccine candidate is regarded as a prospective basis for a universal vaccine against rotavirus.

Recent Molecular Characterization of Porcine Rotaviruses Detected in China and Their Phylogenetic Relationships with Human Rotaviruses.

Porcine rotavirus A (PoRVA) is an enteric pathogen capable of causing severe diarrhea in suckling piglets. Investigating the prevalence and molecular characteristics of PoRVA in the world, including China, is of significance for disease prevention. In 2022, a total of 25,768 samples were collected from 230 farms across China, undergoing porcine RVA positivity testing. The results showed that 86.52% of the pig farms tested positive for porcine RVA, with an overall positive rate of 51.15%. Through the genetic evolution analysis of VP7, VP4 and VP6 genes, it was revealed that G9 is the predominant genotype within the VP7 segment, constituting 56.55%. VP4 genotypes were identified as P[13] (42.22%), P[23] (25.56%) and P[7] (22.22%). VP6 exhibited only two genotypes, namely I5 (88.81%) and I1 (11.19%). The prevailing genotype combination for RVA was determined as G9P[23]I5. Additionally, some RVA strains demonstrated significant homology between VP7, VP4 and VP6 genes and human RV strains, indicating the potential for human RV infection in pigs. Based on complete genome sequencing analysis, a special PoRVA strain, CHN/SD/LYXH2/2022/G4P[6]I1, had high homology with human RV strains, revealing genetic reassortment between human and porcine RV strains in vivo. Our data indicate the high prevalence, major genotypes, and cross-species transmission of porcine RVA in China. Therefore, the continuous monitoring of porcine RVA prevalence is essential, providing valuable insights for virus prevention and control, and supporting the development of candidate vaccines against porcine RVA.

Bacterial-derived sialidases inhibit porcine rotavirus OSU replication by interfering with the early steps of infection.

Rotavirus infections in suckling and weaning piglets cause severe dehydration and death, resulting in significant economic losses in the pig breeding industry. With the continuous emergence of porcine rotavirus (PoRV) variants and poor vaccine cross-protection among various genotypes, there is an urgent need to develop alternative strategies such as seeking effective antiviral products from nature, microbial metabolites and virus-host protein interaction. Sialidases play a crucial role in various physiopathological processes and offer a promising target for developing antivirus drugs. However, the effect of bacterial-derived sialidases on the infection of PoRVs remains largely unknown. Herein, we investigated the impact of bacterial-derived sialidases (sialidase Cp and Vc) on PoRV strain OSU(Group A) infection, using differentiated epithelial monkey kidney cells (MA104) as a model. Our results indicated that the pretreatment of MA104 with exogenous sialidases effectively suppressed PoRV OSU in a concentration-dependent manner. Notably, even at a concentration of 0.01 µU/mL, sialidases significantly inhibited the virus (MOI = 0.01). Meanwhile, we found that sialidase Vc pretreatment sharply reduced the binding rate of PoRV OSU. Last, we demonstrated that PoRV OSU might recognize α-2,3-linked sialic acid as the primary attachment factor in MA104. Our findings provide new insights into the underlying mechanism of PoRV OSU infections, shedding lights on the development of alternative antivirus approaches based on bacteria-virus interaction.

Isolation and characterization of a G9P[23] porcine rotavirus strain AHFY2022 in China.

Rotavirus group A (RVA) is a main pathogen causing diarrheal diseases in humans and animals. Various genotypes are prevalent in the Chinese pig herd. The genetic diversity of RVA lead to distinctly characteristics. In the present study, a porcine RVA strain, named AHFY2022, was successfully isolated from the small intestine tissue of piglets with severe diarrhea. The AHFY2022 strain was identified by cytopathic effects (CPE) observation, indirect immunofluorescence assay (IFA), electron microscopy (EM), high-throughput sequencing, and pathogenesis to piglets. The genomic investigation using NGS data revealed that AHFY2022 exhibited the genotypes G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1, using the online platform the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/). Moreover, experimental inoculation in 5-day-old and 27-day-old piglets demonstrated that AHFY2022 caused severe diarrhea, fecal shedding, small intestinal villi damage, and colonization in all challenged piglets. Taken together, our results detailed the virological features of the porcine rotavirus G9P[23] from China, including the whole-genome sequences, genotypes, growth kinetics in MA104 cells and the pathogenicity in suckling piglets.

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

Rotavirus (RV) replication takes place in the viroplasms, cytosolic inclusions that allow the synthesis of virus genome segments and their encapsidation in the core shell, followed by the addition of the second layer of the virion. The viroplasms are composed of several viral proteins, including NSP5, which serves as the main building block. Microtubules, lipid droplets, and miRNA-7 are among the host components recruited in viroplasms. We investigated the interaction between RV proteins and host components of the viroplasms by performing a pull-down assay of lysates from RV-infected cells expressing NSP5-BiolD2. Subsequent tandem mass spectrometry identified all eight subunits of the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for folding at least 10% of the cytosolic proteins. Our confirmed findings reveal that TRiC is brought into viroplasms and wraps around newly formed double-layered particles. Chemical inhibition of TRiC and silencing of its subunits drastically reduced virus progeny production. Through direct RNA sequencing, we show that TRiC is critical for RV replication by controlling dsRNA genome segment synthesis, particularly negative-sense single-stranded RNA. Importantly, cryo-electron microscopy analysis shows that TRiC inhibition results in defective virus particles lacking genome segments and polymerase complex (VP1/VP3). Moreover, TRiC associates with VP2 and NSP5 but not with VP1. Also, VP2 is shown to be essential for recruiting TRiC in viroplasms and preserving their globular morphology. This study highlights the essential role of TRiC in viroplasm formation and in facilitating virion assembly during the RV life cycle. IMPORTANCE: The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.