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. ​.

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.

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.

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.

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.

Rapid and sensitive lateral flow biosensor for the detection of GII human norovirus based on immunofluorescent nanomagnetic microspheres.

Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point-of-care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino-functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti-HuNoV monoclonal antibody was then conjugated with QDs-MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL-1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross-reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM-LFIC and RT-qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point-of-care testing in the future.

[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.

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.

Safety, Immunogenicity, and Mechanism of a Rotavirus mRNA-LNP Vaccine in Mice.

Rotaviruses (RVs) are a major cause of diarrhea in young children worldwide. The currently available and licensed vaccines contain live attenuated RVs. Optimization of live attenuated RV vaccines or developing non-replicating RV (e.g., mRNA) vaccines is crucial for reducing the morbidity and mortality from RV infections. Herein, a nucleoside-modified mRNA vaccine encapsulated in lipid nanoparticles (LNP) and encoding the VP7 protein from the G1 type of RV was developed. The 5' untranslated region of an isolated human RV was utilized for the mRNA vaccine. After undergoing quality inspection, the VP7-mRNA vaccine was injected by subcutaneous or intramuscular routes into mice. Mice received three injections in 21 d intervals. IgG antibodies, neutralizing antibodies, cellular immunity, and gene expression from peripheral blood mononuclear cells were evaluated. Significant differences in levels of IgG antibodies were not observed in groups with adjuvant but were observed in groups without adjuvant. The vaccine without adjuvant induced the highest antibody titers after intramuscular injection. The vaccine elicited a potent antiviral immune response characterized by antiviral clusters of differentiation CD8+ T cells. VP7-mRNA induced interferon-γ secretion to mediate cellular immune responses. Chemokine-mediated signaling pathways and immune response were activated by VP7-mRNA vaccine injection. The mRNA LNP vaccine will require testing for protective efficacy, and it is an option for preventing rotavirus infection.

Genetic Profile of Rotavirus Type A in Children under 5 Years Old in Africa: A Systematic Review of Prevalence.

Human type A rotavirus (RV-A) is world-recognized as the major pathogen causing viral gastroenteritis in children under 5 years of age. The literature indicates a substantial increase in the diversity of rotavirus strains across continents, especially in Africa, which can pose significant challenges including an increase of disease burden and a reduction of vaccines' effectiveness. However, few studies have mapped the variety of circulating virus strains in different regions, which may hamper decisions on epidemiological surveillance and preventive public health measures. Thus, our aim was to compile the most updated available evidence on the genetic profile of RV-A among children in Africa and determine the prevalence of different genotypes according to the geographical regions by means of a broad systematic review. Systematic searches were performed in PubMed, Scopus, Web of Science, and Scielo without language, time limits, or geographical restrictions within the African continent. We selected full-text peer-reviewed articles assessing the genetic profile (i.e., genotyping) of RV-A in children up to 5 years old in Africa. Overall, 682 records were retrieved, resulting in 75 studies included for evidence synthesis. These studies were published between 1999 and 2022, were conducted in 28 countries from the five African regions, and 48% of the studies were carried out for 24 months or more. Most studies (n = 55; 73.3%) evaluated RV-A cases before the introduction of the vaccines, while around 20% of studies (n = 13) presented data after the vaccine approval in each country. Only seven (9.3%) studies compared evidence from both periods (pre- and post-vaccine introduction). Genotyping methods to assess RV-A varied between RT-PCR, nested or multiplex RT-PCR, testing only the most common P and G-types. We observed G1 and P[8] to be the most prevalent strains in Africa, with values around 31% and 43%, respectively. Yet if all the genotypes with the following highest prevalence were added ((G1 + G2, G3, G9) and (P[8] + P[6], P[4])), these figures would represent 80% and 99% of the total prevalence. The combination G1P[8] was the most reported in the studies (around 22%). This review study demonstrated an increased strain diversity in the past two decades, which could represent a challenge to the efficacy of the current vaccine.

Enhancing enteric pathogen detection: implementation and impact of multiplex PCR for improved diagnosis and surveillance.

BACKGROUND: Syndromic surveillance of acute gastroenteritis plays a significant role in the diagnosis and management of gastrointestinal infections that are responsible for a substantial number of deaths globally, especially in developing countries. In Lebanon, there is a lack of national surveillance for acute gastroenteritis, and limited data exists regarding the prevalence of pathogens causing diarrhea. The one-year study aims to investigate the epidemiology of common gastrointestinal pathogens and compare our findings with causative agents of diarrhea reported by our study collaborative centers. METHODS: A multicenter, cross-sectional study was conducted over a one-year period. A total of 271 samples were obtained from outpatients and inpatients presenting with symptoms of acute gastroenteritis at various healthcare facilities. The samples were then analyzed using Allplex gastrointestinal assay that identifies a panel of enteric pathogens. RESULTS: Overall, enteropathogens were detected in 71% of the enrolled cases, 46% of those were identified in patients as single and 54% as mixed infections. Bacteria were observed in 48%, parasites in 12% and viruses in 11%. Bacterial infections were the most prevalent in all age groups. Enteroaggregative E. coli (26.5%), Enterotoxigenic E. coli (23.2%) and Enteropathogenic E. coli (20.3%) were the most frequently identified followed by Blastocystis hominis (15.5%) and Rotavirus (7.7%). Highest hospitalization rate occurred with rotavirus (63%), Enterotoxigenic E. coli (50%), Blastocystis hominis (45%) and Enteropathogenic E. coli (43%). Enteric pathogens were prevalent during summer, fall and winter seasons. CONCLUSIONS: The adoption of multiplex real-time PCR assays in the diagnosis of gastrointestinal infections has identified gaps and improved the rates of detection for multiple pathogens. Our findings highlight the importance of conducting comprehensive surveillance to monitor enteric infections. The implementation of a syndromic testing panel can therefore provide healthcare professionals with timely and accurate information for more effective treatment and public health interventions.

Identification of a novel intra-genotype reassortant G1P[8] rotavirus in Italy, 2021.

OBJECTIVES: Rotaviruses G1P[8] are epidemiologically relevant and are targeted by vaccines. The introduction of vaccines has altered rotavirus epidemiology. Hospital-based surveillance conducted in Sicily, Italy, showed a progressive decline in rotavirus prevalence since 2014, along with an increasing vaccine coverage (63.8% in 2020), and a marked decrease in circulation of G1P[8] strains. Surprisingly in 2021, G1P[8] viruses accounted for 90.5% (19/21) of rotavirus infections. This study aimed to understand if the increased activity of G1P[8]'s was related to virus-related peculiarities. DESIGN: In 2021, 266 patients <15 years of age were hospitalized with acute gastroenteritis (AGE) and included in rotavirus surveillance. Viral proteins (VP7 and VP4) genotyping and sequence data were generated from all rotavirus-positive samples. The genetic makeup of G1P[8] rotaviruses was investigated by full-genome sequencing. RESULTS: Peculiar G1P[8] rotaviruses, with VP7 and VP4 belonging to novel sub-lineages, circulated in 2021, accounting for 76.2% (16/21) of all rotavirus infections. On full-genome analysis, the novel G1P[8] variant displayed an intra-genotype (Wa-like) reassortant constellation, involving G12 and G1 strains, into a unique arrangement never observed before. The novel G1P[8] variant showed peculiar amino acid substitutions in 8-1 and 8-3 epitopes of the VP4 with respect to the Rotarix strain. CONCLUSIONS: Prompt identification of virus variants circulating in the human population is pivotal to understanding epidemiological trends and assessing vaccine efficacy.

Genetic diversity of G9, G3, G8 and G1 rotavirus group A strains circulating among children with acute gastroenteritis in Vietnam from 2016 to 2021.

Rotavirus group A (RVA) is the most common cause of severe childhood diarrhea worldwide. The introduction of rotavirus vaccination programs has contributed to a reduction in hospitalizations and mortality caused by RVA. From 2016 to 2021, we conducted surveillance to monitor RVA prevalence and genotype distribution in Nam Dinh and Thua Thien Hue (TT Hue) provinces where a pilot Rotavin-M1 vaccine (Vietnam) implementation took place from 2017 to 2020. Out of 6626 stool samples, RVA was detected in 2164 (32.6%) by ELISA. RT-PCR using type-specific primers were used to determine the G and P genotypes of RVA-positive specimens. Whole genome sequences of a subset of 52 specimens randomly selected from 2016 to 2021 were mapped using next-generation sequencing. From 2016 to 2021, the G9, G3 and G8 strains dominated, with detected frequencies of 39%, 23%, and 19%, respectively; of which, the most common genotypes identified were G9P[8], G3P[8] and G8P[8]. G1 strains re-emerged in Nam Dinh and TT Hue (29.5% and 11.9%, respectively) from 2020 to 2021. G3 prevalence decreased from 74% to 20% in TT Hue and from 21% to 13% in Nam Dinh province between 2017 and 2021. The G3 strains consisted of 52% human typical G3 (hG3) and 47% equine-like G3 (eG3). Full genome analysis showed substantial diversity among the circulating G3 strains with different backgrounds relating to equine and feline viruses. G9 prevalence decreased sharply from 2016 to 2021 in both provinces. G8 strains peaked during 2019-2020 in Nam Dinh and TT Hue provinces (68% and 46%, respectively). Most G8 and G9 strains had no genetic differences over the surveillance period with very high nucleotide similarities of 99.2-99.9% and 99.1-99.7%, respectively. The G1 strains were not derived from the RVA vaccine. Changes in the genotype distribution and substantial diversity among circulating strains were detected throughout the surveillance period and differed between the two provinces. Determining vaccine effectiveness against circulating strains over time will be important to ensure that observed changes are due to natural secular variation and not from vaccine pressure.

Multicenter Study of Rotavirus Infection, Diversity of Circulating Genotypes and Clinical Outcomes in Children ≤5 Years Old in Iran.

BACKGROUND: To determine the epidemiology of rotavirus group A (RVA) infection in symptomatic children, and analyze genotype diversity in association with clinical characteristics, geographical and seasonal changes. METHODS: The stool samples of symptomatic children 5≥ years old were collected from 5 different hospitals during December 2020 and March 2022. Rotavirus stool antigen test was done and G and P genotypes of the positive samples were determined. Associations of the infection and genotype diversity with demographical and clinical data were assessed by statistical methods. RESULTS: RVA infection was detected in 32.1% (300/934) of the patients (Ranges between 28.4% and 47.4%). An inverse association with age was detected, where the highest frequency was measured in children ≤12 months of age (175/482, 36.3%). The infection was more frequent during winter (124/284, 43.7%) and spring (64/187, 34.2%). Children who were exclusively fed with breast milk showed a lower rate of infection (72/251, 28.6%). Among the 46 characterized genotypes (17 single- and 29 mixed-genotype infections), G1P[8] and G9P[4] were more frequently detected in children <36 (67/234, 28.63%) and 36-60 (7/24, 29.16%) months of age children, respectively. A seasonal diversity in the circulating genotypes was detected in different cities. Children with G1P[8], G1P[6], and mixed-genotype infection experienced a shorter duration of hospitalization, and a higher frequency of nausea and severe diarrhea, respectively. CONCLUSIONS: In this study high frequency of RVA infection was detected in symptomatic children in Iran. Moreover, genotype diversity according to geographic area, seasons, age groups, and clinical features of disease was detected.