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.

Fecal leukocyte frequency in children with acute viral gastroenteritis: a single-center experience.

OBJECTIVE: Our objective was to determine the frequency of rotavirus, adenovirus, and rota-adenovirus co-infections and investigate the fecal leukocyte rate associated with these infections in patients with gastroenteritis. METHODS: This is a retrospective study. We identified patients who were admitted to the pediatric emergency department with acute gastroenteritis and had their stool samples tested for rotavirus and/or adenovirus antigens. Among them, we determined the individuals who underwent stool microscopy tests on the same day and recorded their results. RESULTS: A total of 1,577 patients who underwent testing for rotavirus and/or adenovirus antigens in their stool samples were identified. Among these patients, 583 individuals had concurrent fecal microscopy results. The prevalence of solely rotavirus antigen positivity was 16.4%, solely adenovirus antigen positivity was 2.9%, and rota-adenovirus co-infections were detected in 1.8% of the children. The fecal leukocyte rates in children infected with rotavirus, adenovirus, and rota-adenovirus co-infections were 4.8, 13.3, and 88.9%, respectively. CONCLUSION: The presence of fecal leukocytes was detected at a high rate in cases of viral gastroenteritis, especially in rota-adenovirus co-infections. Therefore, clinicians should not consider only bacterial pathogens in the presence of fecal leukocytes.

Rotavirus trends and distribution of genotypes before and during COVID-19 pandemic era: Bangladesh, 2017-2021.

Rotavirus gastroenteritis is accountable for an estimated 128 500 deaths among children younger than 5 years worldwide, and the majority occur in low-income countries. Although the clinical trials of rotavirus vaccines in Bangladesh revealed a significant reduction of severe rotavirus disease by around 50%, the vaccines are not yet included in the routine immunization program. The present study was designed to provide data on rotavirus diarrhea with clinical profiles and genotypes before (2017-2019) and during the COVID-19 pandemic period (2020-2021). Fecal samples were collected from 2% of the diarrheal patients at icddr,b Dhaka hospital of all ages between January 2017 and December 2021 and were tested for VP6 rotavirus antigen using ELISA. The clinical manifestations such as fever, duration of diarrhea and hospitalization, number of stools, and dehydration and so on were collected from the surveillance database (n = 3127). Of the positive samples, 10% were randomly selected for genotyping using Sanger sequencing method. A total of 12 705 fecal samples were screened for rotavirus A antigen by enzyme immunoassay. Overall, 3369 (27%) were rotavirus antigen-positive, of whom children <2 years had the highest prevalence (88.6%). The risk of rotavirus A infection was 4.2 times higher in winter than in summer. Overall, G3P[8] was the most prominent genotype (45.3%), followed by G1P[8] (32.1%), G9P[8] (6.8%), and G2P[4] (6.1%). The other unusual combinations, such as G1P[4], G1P[6], G2P[6], G3P[4], G3P[6], and G9P[6], were also present. Genetic analysis on Bangladeshi strains revealed that the selection pressure (dN/dS) was estimated as <1. The number of hospital visits showed a 37% drop during the COVID-19 pandemic relative to the years before the pandemic. Conversely, there was a notable increase in the rate of rotavirus positivity during the pandemic (34%, p < 0.00) compared to the period before COVID-19 (23%). Among the various clinical symptoms, only the occurrence of watery stool significantly increased during the pandemic. The G2P[4] strain showed a sudden rise (19%) in 2020, which then declined in 2021. In the same year, G1P[8] was more prevalent than G3P[8] (40% vs. 38%, respectively). The remaining genotypes were negligible and did not exhibit much fluctuation. This study reveals that the rotavirus burden remained high during the COVID-19 prepandemic and pandemic in Bangladesh. Considering the lack of antigenic variations between the circulating and vaccine-targeted strains, integrating the vaccine into the national immunization program could reduce the prevalence of the disease, the number of hospitalizations, and the severity of cases.

Ten different viral agents infecting and co-infecting children with acute gastroenteritis in Southern Italy: Role of known pathogens and emerging viruses during and after COVID-19 pandemic.

Acute gastroenteritis (AGE) represents a world public health relevant problem especially in children. Enteric viruses are the pathogens mainly involved in the episodes of AGE, causing about 70.00% of the cases. Apart from well-known rotavirus (RVA), adenovirus (AdV) and norovirus (NoV), there are various emerging viral pathogens potentially associated with AGE episodes. In this study, the presence of ten different enteric viruses was investigated in 152 fecal samples collected from children hospitalized for gastroenteritis. Real time PCR results showed that 49.3% of them were positive for viral detection with the following prevalence: norovirus GII 19.7%, AdV 15.8%, RVA 10.5%, human parechovirus (HPeV) 5.3%, enterovirus (EV) 3.3%, sapovirus (SaV) 2.6%. Salivirus (SalV), norovirus GI and astrovirus (AstV) 1.3% each, aichivirus (AiV) found in only one patient. In 38.2% of feces only one virus was detected, while co-infections were identified in 11.8% of the cases. Among young patients, 105 were ≤5 years old and 56.0% tested positive for viral detection, while 47 were >5 years old with 40.0% of them infected. Results obtained confirm a complex plethora of viruses potentially implicated in gastroenteritis in children, with some of them previously known for other etiologies but detectable in fecal samples. Subsequent studies should investigate the role of these viruses in causing gastroenteritis and explore the possibility that other symptoms may be ascribed to multiple infections.

Enteropathogenic viruses associated with acute gastroenteritis among African children under 5 years of age: A systematic review and meta-analysis.

Gastroenteritis viruses are the leading etiologic agents of diarrhea in children worldwide. We present data from thirty-three (33) eligible studies published between 2003 and 2023 from African countries bearing the brunt of the virus-associated diarrheal mortality. Random effects meta-analysis with proportion, subgroups, and meta-regression analyses were employed. Overall, rotavirus with estimated pooled prevalence of 31.0 % (95 % CI 24.0-39.0) predominated in all primary care visits and hospitalizations, followed by norovirus, adenovirus, sapovirus, astrovirus, and aichivirus with pooled prevalence estimated at 15.0 % (95 % CI 12.0-20.0), 10 % (95 % CI 6-15), 4.0 % (95 % CI 2.0-6.0), 4 % (95 % CI 3-6), and 2.3 % (95 % CI 1-3), respectively. Predominant rotavirus genotype was G1P[8] (39 %), followed by G3P[8] (11.7 %), G9P[8] (8.7 %), and G2P[4] (7.1 %); although, unusual genotypes were also observed, including G3P[6] (2.7 %), G8P[6] (1.7 %), G1P[6] (1.5 %), G10P[8] (0.9 %), G8P[4] (0.5 %), and G4P[8] (0.4 %). The genogroup II norovirus predominated over the genogroup I-associated infections (84.6 %, 613/725 vs 14.9 %, 108/725), with the GII.4 (79.3 %) being the most prevalent circulating genotype. In conclusion, this review showed that rotavirus remains the leading driver of viral diarrhea requiring health care visits and hospitalization among under-five years children in Africa. Thus, improved rotavirus vaccination in the region and surveillance to determine the residual burden of rotavirus and the evolving trend of other enteric viruses are needed for effective control and management of cases.

Pepper mild mottle virus intended for use as a process indicator for drinking water treatment: Present forms and quantitative relations to norovirus and rotavirus in surface water.

Pepper mild mottle virus (PMMoV) has been proposed as a potential indicator of human enteric viruses in environmental water and for viral removal during drinking water treatment. To investigate the occurrence and present forms of PMMoV and quantitative relations to norovirus GII and rotavirus A (RVA) in surface waters, 147 source water samples were collected from 21 drinking water treatment plants (DWTPs) in Japan between January 2018 and January 2021, and the concentrations of viruses in suspended and dissolved fractions were measured using real-time RT-PCR. PMMoV was detected in 81-100 % of samples in each sample month and observed concentrations ranged from 3.0 to 7.0 log10 copies/L. The concentrations of PMMoV were higher in dissolved fraction compared to suspended fractions, while different partitioning was observed for NoV GII depending on seasons. The concentrations of PMMoV were basically higher than those of norovirus GII (1.9-5.3 log10 copies/L) and RVA (1.9-6.6 log10 copies/L), while in 18 samples, RVA presented higher concentrations than PMMoV. Partial regions of VP7, VP4, and VP6 of the RVA in the 18 samples were amplified using nested PCR, and the genotypes were determined using an amplicon-based next-generation sequencing approach. We found that these source water samples included not only human RVA but also various animal RVA and high genetic diversity due to the existence of animal RVA was associated with a higher RVA concentration than PMMoV. Our findings suggest that PMMoV can be used as an indicator of norovirus GII and human RVA in drinking water sources and that the indicator performance should be evaluated by comparing to zoonotic viruses as well as human viruses.

Trends in the detection of viruses causing gastroenteritis over a 10-year period and impact of nonpharmaceutical interventions.

BACKGROUND: Viral gastroenteritis continues to be a leading cause of death in low-income countries. The impact of nonpharmaceutical interventions (NPIs) on the transmission of gastroenteritis-causing viruses during the COVID-19 pandemic is understudied. OBJECTIVES: To investigate the 10-year trends of enteric viruses and estimate the impact of implementing and mitigating NPIs. STUDY DESIGN: Data regarding norovirus, rotavirus, adenovirus, astrovirus, and sapovirus detection were collected from five Korean hospitals between January 2013 and April 2023. We compared positivity between the pre-pandemic, pandemic, and post-pandemic periods. The causal effects of implementing and mitigating NPIs were quantified using the Bayesian Structural Time Series (BSTS) model. RESULTS: Norovirus was most frequently detected (9.9 %), followed by rotavirus (6.7 %), adenovirus (3.3 %), astrovirus (1.4 %), and sapovirus (0.6 %). During the pandemic, the positivity of all five viruses decreased, ranging from -1.0 % to -8.1 %, with rotavirus showing the greatest decrease. In the post-pandemic period, positivity rebounded for all viruses except for rotavirus. The BSTS model revealed that NPI implementation negatively affected the detection of all five viruses, resulting in reductions ranging from -73.0 % to -91.0 % compared to the prediction, with rotavirus being the least affected. Conversely, NPI mitigation positively affected the detection of all viruses, ranging from 79.0 % to 200.0 %, except for rotavirus. CONCLUSIONS: Trends observed over 10 years show that NPIs have had a major impact on changes in enteric virus detection. The effect of vaccines, in addition to NPIs, on rotavirus detection requires further investigation. Our findings emphasize the importance of NPIs in infection control and prevention.

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.

Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.

PreK-12 school and citywide wastewater monitoring of the enteric viruses astrovirus, rotavirus, and sapovirus.

Wastewater monitoring is an efficient and effective way to surveil for various pathogens in communities. This is especially beneficial in areas of high transmission, such as preK-12 schools, where infections may otherwise go unreported. In this work, we apply wastewater disease surveillance using school and community wastewater from across Houston, Texas to monitor three major enteric viruses: astrovirus, sapovirus genogroup GI, and group A rotavirus. We present the results of a 10-week study that included the analysis of 164 wastewater samples for astrovirus, rotavirus, and sapovirus in 10 preK-12 schools, 6 wastewater treatment plants, and 2 lift stations using newly designed RT-ddPCR assays. We show that the RT-ddPCR assays were able to detect astrovirus, rotavirus, and sapovirus in school, lift station, and wastewater treatment plant (WWTP) wastewater, and that a positive detection of a virus in a school sample was paired with a positive detection of the same virus at a downstream lift station or wastewater treatment plant over 97 % of the time. Additionally, we show how wastewater detections of rotavirus in schools and WWTPs were significantly associated with citywide viral intestinal infections. School wastewater can play a role in the monitoring of enteric viruses and in the detection of outbreaks, potentially allowing public health officials to quickly implement mitigation strategies to prevent viral spread into surrounding communities.

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.

Quadruplex Real-Time TaqMan® RT-qPCR Assay for Differentiation of Equine Group A and B Rotaviruses and Identification of Group A G3 and G14 Genotypes.

Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan® RT-qPCR assay for differentiation of ERVA and ERVB and simultaneous G-typing of ERVA strains, evaluated its analytical and clinical performance, and compared it to (1) a previously established ERVA triplex RT-qPCR assay and (2) standard RT-PCR assay and Sanger sequencing of PCR products. This quadruplex RT-qPCR assay demonstrated high sensitivity (>90%)/specificity (100%) for every target and high overall agreement (>96%). Comparison between the triplex and quadruplex assays revealed only a slightly higher sensitivity for the ERVA NSP3 target using the triplex format (p-value 0.008) while no significant differences were detected for other targets. This quadruplex RT-qPCR assay will significantly enhance rapid surveillance of both ERVA and ERVB circulating and emerging strains with potential for interspecies transmission.

Molecular epidemiology of rotavirus among children in Western Canada: Dynamic changes in genotype prevalence in four consecutive seasons.

Rotavirus molecular surveillance remains important in the postvaccine era to monitor the changes in transmission patterns, identify vaccine-induced antigenic changes and discover potentially pathogenic vaccine-related strains. The Canadian province of Alberta introduced rotavirus vaccination into its provincial vaccination schedule in June 2015. To evaluate the impact of this program on stool rotavirus positivity rate, strain diversity, and seasonal trends, we analyzed a prospective cohort of children with acute gastroenteritis recruited between December 2014 and August 2018. We identified dynamic changes in rotavirus positivity and genotype trends during pre- and post-rotavirus vaccine introduction periods. Genotypes G9P[8], G1P[8], G2P[4], and G12P[8] predominated consecutively each season with overall lower rotavirus incidence rates in 2016 and 2017. The demographic and clinical features of rotavirus gastroenteritis were comparable among wild-type rotaviruses; however, children with G12P[8] infections were older (p < 0.001). Continued efforts to monitor changes in the molecular epidemiology of rotavirus using whole genome sequence characterization are needed to further understand the impact of the selection pressure of vaccination on rotavirus evolution.

Isolation and Characterization of Distinct Rotavirus A in Bat and Rodent Hosts.

Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.

Isolation and Pathogenicity Analysis of a G5P[23] Porcine Rotavirus Strain.

(1) Background: Group A rotaviruses (RVAs) are the primary cause of severe intestinal diseases in piglets. Porcine rotaviruses (PoRVs) are widely prevalent in Chinese farms, resulting in significant economic losses to the livestock industry. However, isolation of PoRVs is challenging, and their pathogenicity in piglets is not well understood. (2) Methods: We conducted clinical testing on a farm in Jiangsu Province, China, and isolated PoRV by continuously passaging on MA104 cells. Subsequently, the pathogenicity of the isolated strain in piglets was investigated. The piglets of the PoRV-infection group were orally inoculated with 1 mL of 1.0 × 106 TCID50 PoRV, whereas those of the mock-infection group were fed with an equivalent amount of DMEM. (3) Results: A G5P[23] genotype PoRV strain was successfully isolated from one of the positive samples and named RVA/Pig/China/JS/2023/G5P[23](JS). The genomic constellation of this strain was G5-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1. Sequence analysis revealed that the genes VP3, VP7, NSP2, and NSP4 of the JS strain were closely related to human RVAs, whereas the remaining gene segments were closely related to porcine RVAs, indicating a reassortment between porcine and human strains. Furthermore, infection of 15-day-old piglets with the JS strain resulted in a diarrheal rate of 100% (8 of 8) and a mortality rate of 37.5% (3 of 8). (4) Conclusions: The isolated G5P[23] genotype rotavirus strain, which exhibited strong pathogenicity in piglets, may have resulted from recombination between porcine and human strains. It may serve as a potential candidate strain for developing vaccines, and its immunogenicity can be tested in future studies.

A TaqMan Probe-Based Multiplex Real-Time PCR for Simultaneous Detection of Porcine Epidemic Diarrhea Virus Subtypes G1 and G2, and Porcine Rotavirus Groups A and C.

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/µL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/µL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.

Risk factors for bovine rotavirus infection and genotyping of bovine rotavirus in diarrheic calves in Bangladesh.

Bovine rotavirus (BRV) is considered the leading cause of calf diarrhea worldwide, including Bangladesh. In this study we aimed to identify risk factors for BRV infection and determine the G and P genotypes of BRV strains in diarrheic calves. Fecal samples were collected from 200 diarrheic calves in three districts between January 2014 and October 2015. These samples were screened to detect the presence of BRV using rapid test-strips BIO K 152 (RTSBK). The RTSBK positive samples were further tested by polyacrylamide gel electrophoresis and the silver staining technique to detect rotavirus dsRNA. Risk factors were identified by multivariable logistic regression analysis. The G and P genotypes of BRV were determined by RT-PCR and sequencing. A phylogenetic tree was constructed based on the neighbor-joining method using CLC sequence viewer 8.0. About 23% of the diarrheic calves were BRV positive. The odds of BRV infection were 3.8- (95% confidence interval [95% CI]: 1.0-14.7) and 3.9-times (95% CI:1.1-14.2) higher in Barisal and Madaripur districts, respectively, than Sirrajganj. The risk of BRV infection was 3.1-times (95% CI: 1.5-6.5) higher in calves aged ≤ 5 weeks than those aged >5 weeks. Moreover, the risk of BRV infection was 2.6-times (95% CI:1.1-5.8) higher in crossbred (Holstein Friesian, Shahiwal) than indigenous calves. G6P[11] was the predominant genotype (94.4%), followed by G10P[11] (5.6%). The BRV G6 strains were found to be closest (98.9-99.9%) to Indian strains, and BRV G10 strains showed 99.9% identities with Indian strain. The VP4 gene of all P[11] strains showed >90% identities to each other and also with Indian strains. The most frequently identified BRV genotype was G6P[11]. About 23% of calf diarrhea cases were associated with BRV. To control disease, high-risk areas and younger crossbred calves should be targeted for surveillance and management. The predominant genotype could be utilized as the future vaccine candidate or vaccines with the dominant genotype should be used to control BRV diarrhea in Bangladesh.

A shift in circulating rotaviral genotypes among hospitalized neonates.

In neonates, rotavirus (RV) infection is generally nosocomial. The control of rotaviral infection within hospital settings is challenging due to prolonged shedding of the virus and contamination of the surrounding environment. There are few studies that have reported asymptomatic infection within neonates. In this study, neonates were screened for RV infection and possible clinical manifestations that may play a role in RV acquisition were analysed. Stool samples were collected from 523 hospitalized neonates admitted for > 48 h in a low-cost and higher-cost tertiary centre. RV antigen was screened using ELISA and the samples which tested positive were confirmed by semi-nested RT-PCR. RV was detected in 34% of participants and genotypes identified included G12P[11] (44.4%), G10 P[11] (42.6%), G10G12P[11] (10.1%) and G3P[8] (2.9%). ICU admissions were associated with higher viral shedding (p < 0.05). Hospitalization in the low-cost facility ICU was associated with higher RV acquisition risk (p < 0.05). RV was detected in higher rates (36.9%) among neonates with gastrointestinal manifestations. G10P[11] was the predominant genotype for several years (1988-2016) among neonates within India. The preponderance of an emerging G12P[11] genotype and heterotypic distribution was documented. RV surveillance is important to identify emerging strains and establish the road ahead in managing RV infection.

Detection of a novel species A, DS-1-like, G4P[6] rotavirus strain from a Brazilian child with gastroenteritis.

Rotaviruses belonging to species A (RVA) remain among the most common causes of severe gastroenteritis in children aged <5 years, leading to substantial morbidity and mortality worldwide. Genome reassortment events between two human strains or human and animal strains represent one of the mechanisms which appear to generate the broad genetic variability of circulating. According to a nucleotide, sequence-based classification system, RVA strains are currently classified into three genotype constellations including Wa-like (genogroup I), DS-1-like (genogroup II), and AU-like (genogroup III). The present study reports the detection of an unusual RVA G4P[6] strain (coded as strain HSE005), which might have originated from a natural reassortment event between human and animal RVA strains. Molecular characterization of this isolate showed that it belonged to genogroup II, genotype G4P[6]. In addition, two genes (VP3 and NSP4) of this strain denoted evidence of reassortment events involving strains of distinct zoonotic evolutionary origins. Therefore, we propose that a new G4P[6] strain was identified, highlighting a possible first zoonotic transmission including a reassortment event that involved the VP3 gene.