Genetic diversity and evolution of G12P[6] DS-1-like and G12P[9] AU-1-like Rotavirus strains in Brazil.

In the early 2000s, the global emergence of rotavirus (RVA) G12P[8] genotype was noted, while G12P[6] and G12P[9] combinations remained rare in humans. This study aimed to characterize and phylogenetically analyze three Brazilian G12P[9] and four G12P[6] RVA strains from 2011 to 2020, through RT-PCR and sequencing, in order to enhance our understanding of the genetic relationship between human and animal-origin RVA strains. G12P[6] strains displayed a DS-1-like backbone, showing a distinct genetic clustering. G12P[6] IAL-R52/2020, IAL-R95/2020 and IAL-R465/2019 strains clustered with 2019 Northeastern G12P[6] Brazilian strains and a 2018 Benin strain, whereas IAL-R86/2011 strain grouped with 2010 Northern G12P[6] Brazilian strains and G2P[4] strains from the United States and Belgium. These findings suggest an African genetic ancestry and reassortments with co-circulating American strains sharing the same DS-1-like constellation. No recent zoonotic reassortment was observed, and the DS-1-like constellation detected in Brazilian G12P[6] strains does not seem to be genetically linked to globally reported intergenogroup G1/G3/G9/G8P[8] DS-1-like human strains. G12P[9] strains exhibited an AU-1-like backbone with two different genotype-lineage constellations: IAL-R566/2011 and IAL-R1151/2012 belonged to a VP3/M3.V Lineage, and IAL-R870/2013 to a VP3/M3.II Lineage, suggesting two co-circulating strains in Brazil. This genetic diversity is not observed elsewhere, and the VP3/M3.II Lineage in G12P[9] strains seems to be exclusive to Brazil, indicating its evolution within the country. All three G12P[9] AU-1-like strains were closely relate to G12P[9] strains from Paraguay (2006-2007) and Brazil (2010). Phylogenetic analysis also highlighted that all South American G12P[9] AU-1-like strains had a common origin and supports the hypothesis of their importation from Asia, with no recent introduction from globally circulating G12P[9] strains or reassortments with local G12 strains P[8] or P[6]. Notably, certain genes in the Brazilian G12P[9] AU-1-like strains share ancestry with feline/canine RVAs (VP3/M3.II, NSP4/E3.IV and NSP2/N3.II), whereas NSP1/A3.VI likely originated from artiodactyls, suggesting a history of zoonotic transmission with human strains. This genomic data adds understanding to the molecular epidemiology of G12P[6] and G12P[9] RVA strains in Brazil, offering insights into their genetic diversity and evolution.

Comprehensive genetic analysis of the first near-complete genome of bovine coronavirus and partial genome of bovine rotavirus in Türkiye through metagenomics.

Obtaining the complete or near-complete genome sequence of pathogens is becoming increasingly crucial for epidemiology, virology, clinical science and practice. This study aimed to detect viruses and conduct genetic characterization of genomes using metagenomics in order to identify the viral agents responsible for a calf's diarrhoea. The findings showed that bovine coronavirus (BCoV) and bovine rotavirus (BRV) are the primary viral agents responsible for the calf's diarrhoea. The current study successfully obtained the first-ever near-complete genome sequence of a bovine coronavirus (BCoV) from Türkiye. The G+C content was 36.31% and the genetic analysis revealed that the Turkish BCoV strain is closely related to respiratory BCoV strains from France and Ireland, with high nucleotide sequence and amino acid identity and similarity. In the present study, analysis of the S protein of the Turkish BCoV strain revealed the presence of 13 amino acid insertions, one of which was found to be shared with the French respiratory BCoV. The study also identified a BRV strain through metagenomic analysis and detected multiple mutations within the structural and non-structural proteins of the BRV strain, suggesting that the BRV Kirikkale strain may serve as an ancestor for reassortants with interspecies transmission, especially involving rotaviruses that infect rabbits and giraffes.

Major changes in prevalence and genotypes of rotavirus diarrhea in Beijing, China after RV5 rotavirus vaccine introduction.

To analyze the epidemiological characteristics of group A rotavirus (RVA) diarrhea in Beijing between 2019 and 2022 and evaluate the effectiveness of the RV5 vaccine. Stool specimens were collected from patients with acute diarrhea, and RVA was detected and genotyped. The whole genome of RVA was sequenced by fragment amplification and Sanger sequencing. Phylogenetic trees were constructed using Bayesian and maximum likelihood methods. Descriptive epidemiological methods were used to analyze the characteristics of RVA diarrhea. Test-negative design was used to evaluate the vaccine effectiveness (VE) of the RV5. Compared with 2011-2018, RVA-positive rates in patients with acute diarrhea under 5 years of age and adults decreased significantly between 2019 and 2022, to 9.45% (249/634) and 3.66% (220/6016), respectively. The predominant genotype of RVA had changed from G9-VIP[8]-III between 2019 and 2021 to G8-VP[8]-III in 2022, and P[8] sequences from G8-VP[8]-III strains formed a new branch called P[8]-IIIb. The complete genotype of G8-VP[8]-III was G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. The VE of 3 doses of RV5 was 90.4% (95% CI: 28.8%-98.7%) against RVA diarrhea. The prevalence of RVA decreased in Beijing between 2019 and 2022, and the predominant genotype changed to G8P[8], which may be related to RV5 vaccination. Continuous surveillance is necessary to evaluate vaccine effectiveness and improve vaccine design.

Prevalence and genetic diversity of rotavirus among children under 5 years of age in China: a meta-analysis.

Background: This meta-analysis was performed to assess the prevalence and circulating strains of rotavirus (RV) among Chinese children under 5 years of age after the implantation of the RV vaccine. Material and methods: Studies published between 2019 and 2023, focused on RV-based diarrhea among children less than 5 years were systematically reviewed using PubMed, Embase, Web of Science, CNKI, Wanfang and SinoMed Data. We synthesized their findings to examine prevalence and genetic diversity of RV after the RV vaccine implementation using a fixed-effects or random-effects model. Results: Seventeen studies met the inclusion criteria for this meta-analysis. The overall prevalence of RV was found to be 19.00%. The highest infection rate was noted in children aged 12-23months (25.79%), followed by those aged 24-35 months (23.91%), and 6-11 months (22.08%). The serotype G9 emerged as the most predominant RV genotype, accounting for 85.48% of infections, followed by G2 (7.70%), G8 (5.74%), G1 (4.86%), and G3 (3.21%). The most common P type was P[8], representing 64.02% of RV cases. Among G-P combinations, G9P[8] was the most frequent, responsible for 78.46% of RV infections, succeeded by G8P[8] (31.22%) and G3P[8] (8.11%). Conclusion: Despite the variation of serotypes observed in China, the G1, G2, G3, G8 and G9 serotypes accounted for most RV strains. The genetic diversity analysis highlights the dynamic nature of RV genotypes, necessitating ongoing surveillance to monitor changes in strain distribution and inform future vaccine strategies.

VP4 Mutation Boosts Replication of Recombinant Human/Simian Rotavirus in Cell Culture.

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains.

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 characteristics and genotype trends of rotavirus diarrhea in China from 2009 to 2020].

Objective: To investigate the epidemiological characteristics and genotype trends of rotavirus infection among the population with diarrhea in China, from 2009 to 2020 and provide evidence for strategic surveillance and prevention. Methods: Surveillance data on diarrhea syndrome from 252 sentinel hospitals across 28 provinces (municipalities, autonomous regions) were obtained from the information management system of the Infectious Disease Surveillance Technology Platform of the National Science and Technology Major Project. Descriptive epidemiological methods were employed to analyze the distribution of rotavirus diarrhea cases in different climatic zones, populations, and times from 2009 to 2020, as well as the genotyping characteristics and changing trends of group A rotavirus diarrhea cases. Results: From 2009 to 2020, a total of 114 606 diarrhea cases were tested for rotavirus, and the positive rate was 19.1% (21 872/114 606); group A rotavirus was dominant (98.2%, 21 471/21 872). The positive rate of rotavirus was the highest in 2009 (36.9%, 2 436/6 604) and 2010 (30.6%, 5 130/16 790), fluctuated between 14.0% to 18.0% from 2011 to 2017, raised slightly in 2018 (20.3%, 2 211/10 900), and declined continuously in the following two years (15.5%, 2 262/14 611 and 9.5%, 470/4 963). The positive rate of males (20.2%, 13 660/67 471) was significantly higher than that of females (17.4%, 8 212/47 135). Children under five had the highest positive rate (28.4%, 18 261/64 300), more than four times that of adults. The positive rate peaked from December to February in the mediate temperate zone, warm temperate zone, and subtropical zone, while there were two peaks from November to January and May to June in the frigid zone of the plateau. The dominant genotype of group A rotavirus gradually changed from G3P[8] and G1P[8] to G9P[8] during 2009-2020. Conclusions: The overall rotavirus infection rate in China was on a downward trend. Meanwhile, significant variations of positive rates were observed in seasonal epidemics and different age groups from 2009 to 2020. Rotavirus diarrhea in children was still a prominent concern. Vaccination of rotavirus vaccine should be promoted, and the epidemiological characteristics and genotypes of rotavirus diarrhea should be continuously monitored.

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.

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.

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.

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.

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.

[Pathogenic characteristics of viral diarrhea in children under five years of age in sentinel surveillance in Lulong County of Hebei Province, 2010-2020].

Objective: To analyze pathogenic characteristics of viral diarrhea in children aged <5 years in Hebei Province and provide reference for the prevention and control of viral diarrhea in children. Methods: Stool samples were collected from in-patients with diarrhea under five years old from sentinel hospitals in Lulong County of Hebei between 2010 and 2020. ELISA detected rotavirus antigen, and then positive samples were genotyped by semi nested reverse transcription PCR of two rounds. Calicivirus, genotyping astrovirus, and adenovirus were detected by real-time fluorescence quantification PCR. The data were analyzed by using software SPSS 20.0. Results: In 2 925 detected stool samples, 1 919 (65.61%) were positive. The positive rates of rotavirus, calicivirus, adenovirus, and astrovirus were 42.80% (1 252/2 925), 22.12% (647/2 925), 6.19% (181/2 925), 3.56% (104/2 925). Viral diarrhea was mainly caused by rotavirus infection, accounting for 59.30% (1 017/1 715) between 2010 and 2017, and by calicivirus infection accounting for 53.43% (109/204) between 2018 and 2020. The peak positive rate of rotavirus occurred in winter, with the highest rate in infants aged 12 to 17 months (52.96%,483/912). In the rotavirus positive samples, G9P[8] was mainly detected strains (58.31%,730/1 252), followed by G3P[8] (8.15%,102/1 252). The calicivirus-positive samples were mainly infected with norovirus GⅡ. Sequence analysis indicated that the main type was GⅡ.4 [P31] between 2011 and 2016 and GⅡ.3 [P12] in 2018. Conclusions: Rotavirus and calicivirus were the main pathogens causing infant diarrhea in children under five years old in Hebei from 2010 to 2020. Winter was the main epidemic season.

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.

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.

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.

Genomic diversity of group A rotaviruses from wild boars and domestic pigs in Japan: wide prevalence of NSP5 carrying the H2 genotype.

Group A rotavirus (RVA) sequences were detected in 10.8% (23/212) and 20.7% (87/421) of fecal samples collected in 2017-2022 from wild boars and domestic pigs, using next-generation sequencing. Complete genome sequence analysis of one wild boar and 13 domestic pig RVAs revealed that six of them carried the rare H2 NSP5 genotype. Out of the 39 samples for which the NSP5 genotype could be determined, 23 (59.0%) were of genotype H2. H2 porcine RVAs consist exclusively of Japanese porcine RVAs and exhibit sequence diversity in each segment, suggesting that H2 porcine RVAs may have evolved through reassortment within the Japanese pig population.