Search Results (58)

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

Porcine rotavirus (PoRV), a primary etiological agent of viral diarrhea in piglets, frequently co-infects with other enteric pathogens, exacerbating disease severity and causing substantial economic losses. Its genetic recombination capability enables cross-species transmission potential, posing public health risks. Globally, twelve G genotypes and thirteen P genotypes have been identified, with G9, G5, G3, and G4 emerging as predominant circulating strains. The limited cross-protective immunity between genotypes compromises vaccine efficacy, necessitating genotype surveillance to guide vaccine development. While conventional molecular assays demonstrate sensitivity, they lack rapid genotyping capacity and face technical limitations. To address this, we developed a novel diagnostic platform integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR–Cas12a. This system employs universal primers for the simultaneous amplification of G4/G5/G9 genotypes in a single reaction, coupled with sequence-specific CRISPR recognition, achieving genotyping within 50 min at 37 °C with 10⁰ copies/μL sensitivity. Clinical validation showed a high concordance with reverse transcription quantitative polymerase chain reaction (RT-qPCR). This advancement provides an efficient tool for rapid viral genotyping, vaccine compatibility evaluation, and optimized epidemic control strategies. Full article

PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a’s collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10² copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management. Full article

Pigs serve as critical reservoirs and amplifiers for numerous zoonotic viral diseases, presenting substantial public health challenges in India. This study highlights the epidemiology and emerging trends of key zoonotic viruses associated with pigs, emphasizing their role in endemic and emerging disease dynamics. Japanese encephalitis virus (JEV) persists as a major concern, with pigs acting as amplifying host, while hepatitis E virus (HEV) remains a prominent cause of viral hepatitis, transmitted via contaminated water and pork products. Emerging high-fatality viral zoonoses caused by Nipah virus (NiV) and recurrent threats from swine influenza virus (SIV) demonstrate that the zoonotic landscape is evolving. Furthermore, zoonotic viruses like rotavirus, pseudorabies (ADV or SuHV-1), porcine astrovirus (PAstV), and Torque teno sus virus (TTSuV) reflect the expanding diversity of pig-associated pathogens in India. Emerging evidence also implicates viruses such as Chandipura virus (CHPV) in localized outbreaks, indicating broader zoonotic potential. Novel risks such as swine acute diarrhea syndrome coronavirus (SADS-CoV) and SARS-CoV-2 emphasize the role of pigs as potential intermediaries for pandemic-prone viruses. This comprehensive study evaluates the prevalence, outbreak dynamics, and public health implications of zoonotic viral diseases of pigs in India, providing valuable direction for developing effective control measures. Full article

Porcine viral diarrhea has always been one of the main obstacles to the healthy development of the pig industry in China with its variety of pathogens and complexity of co-infections. Analysis of the dominant mixed-infection model is a fundamental step in boosting the prevention and control of porcine diarrhea. In this study, 3256 porcine fecal samples were collected from 17 pig herds in Shanghai, China, from 2015 to 2023 to identify novel pathogenic infection patterns. The results confirmed that porcine astrovirus (PAstV), porcine sapelovirus (PSV), and porcine epidemic diarrhea virus (PEDV) were the top three agents with positive rates of 28.47%, 20.71%, and 20.23%, respectively. Porcine rotavirus (PoRV) and transmissible gastroenteritis virus (TGEV) accounted for only 8.12% and 1.12%, respectively. Importantly, mixed infection rates were high and complicated. The double infection rate was higher than that of a single infection. Next, the mixed-infection model of PEDV and emerging diarrheal pathogens was explored. The predominant dual-infection models were PEDV/PKoV (porcine kobuvirus) (14.18%), PEDV/PAstV (10.02%), and PEDV/PSV (9.29%). The predominant triple infection models were PEDV/PKoV/PAstV (18.93%), PEDV/PSV/PAstV (10.65%), and PEDV/PKoV/PSV (7.10%). The dominant quadruple-infection model was PEDV/PAstV/PSV/PKoV (46.82%). In conclusion, PEDV is mainly mix-infected with PAstV, PSV, and PKoV in clinical settings. Furthermore, multiple-factor logistic regression analysis confirmed that PAstV, PKoV, bovine viral diarrhea virus (BVDV), and PEDV were closely related to porcine diarrhea. PEDV/PKoV, PEDV/porcine sapovirus (PoSaV), PKoV/BVDV, PoSaV/BVDV, and porcine deltacoronavirus (PDCoV)/PoSaV had great co-infection dominance, which will be helpful for porcine co-infection research. Full article

(1) Background: Rotavirus (RV) is the primary pathogen causing diarrhea in infants and young children. The G9-type is an emerging genotype; however, its isolation is challenging, and its immunogenicity to piglets is poorly understood. This study aimed to isolate, characterize, and evaluate the immunogenicity of a PoRVA strain, RHeN2, obtained from the diarrhea samples of piglets from a pig farm in Henan Province, China. (2) Methods: Porcine rotavirus A (PoRVA) was isolated from diarrheal samples of piglets on a farm in Henan Province, China, through sequential passaging on MA-104 cells. Its immunogenicity and cross-neutralization potential were evaluated. Inactivated vaccines were prepared using two agents (formaldehyde and binary ethyleneimine, BEI) and administered intramuscularly to 28-day-old piglets. Dulbecco’s Modified Eagle Medium (DMEM) served as the control. (3) Results: A PoRVA strain, G9P[23] type, was successfully isolated and named RHeN2. Full-genome Sanger sequencing revealed the genetic constellation of this strain as G9-P[23]-I5-R1-C1- M1-A8-N1-T1-E1-H1. In addition, the titers of neutralizing antibodies peaked at 45 days post-vaccination, and the immunogenicity of the BEI-inactivated group of vaccines was better than that of the formaldehyde-inactivated group. The RHeN2 (G9P[23]) strain demonstrated a strong cross-neutralization ability against the same G9P[23] and G4P[23] strains and a stronger cross-neutralizing ability against the P[23] strains than against the P[7] strains. (4) Conclusion: The newly isolated G9P[23] strain demonstrates favorable immunogenicity, holding potential as a G9-type vaccine candidate. Full article

Porcine epidemic diarrhea virus (PEDV) is a highly contagious pathogen responsible for devastating enteric disease and lethal watery diarrhea, leading to significant economic losses in the global swine industry. Understanding the epidemiology and genetic diversity of PEDV over the past decade is crucial for the effective prevention and treatment of porcine epidemic diarrhea. In this study, 1851 fecal samples were collected from pigs exhibiting diarrhea symptoms across 11 cities in Yunnan Province between 2013 and 2022. The prevalence of PEDV, along with other common swine diarrhea viruses, including porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine Sapporo virus (PoSaV), porcine stellate virus (PaStV), and porcine delta coronavirus (PDCoV) was assessed using a polymerase chain reaction (PCR) assay. The results revealed a total detection rate of 52.94% (980/1851) for the six viruses, with PEDV accounting for 25.93% (480/1851) of cases. Further analysis showed that weaned piglets were more susceptible to PEDV than fattening pigs, with the highest prevalence observed in spring (61.52%, 275/447) and the lowest in summer (12.68%, 97/765). Dual infections were also identified, with PEDV + PoSaV being the most common combination (2.81%, 52/1851), followed by PEDV + PoRV, with a detection rate of 1.67% (31/1851). Phylogenetic analysis of the PEDV S genes revealed that the 28 epidemic strains in Yunnan Province shared a nucleotide sequence homology from 91.4% to 98.4% and an amino acid sequence homology ranging from 85.6% to 99.3%. All strains were classified as GII variant strains. This study provides a comprehensive overview of the epidemiology of PEDV and its co-infection patterns with other common diarrhea-causing viruses in the swine herds of Yunnan Province over the past decade. These findings offer valuable insights for the development of effective prevention and control strategies to mitigate the impact of PEDV and other enteroviruses on the swine industry in Yunnan Province. Full article

In 2020, severe diarrhea occurred in four-month-old fattening pigs from nine farms in Shandong Province, China. Fecal samples were collected from diseased pigs and tested by PCR for the presence of mammalian orthoreovirus (MRV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine rotavirus A (PoRVA), transmissible gastroenteritis virus (TGEV), porcine kobuvirus (PKV), and pseudorabies virus (PRV). The viral RNA of MRV and PEDV was detected in the fecal samples. The genome sequences of MRV and PEDV were successfully amplified from the same fecal sample. Genomic and phylogenetic analysis showed that the MRV isolate named MRV2-SD/2020 belongs to serotype 2 MRV (MRV2) and may originate from the reassortment of human and porcine MRVs. Compared with other MRV2 strains, there were four other unique amino acid mutations (L274I, F302L, V347I, and T440M) in the receptor binding region. For the PEDV isolate named PEDV-SD/2020, the nearly complete genome was amplified from the positive fecal samples. Phylogenetic analysis showed that it was classified into the G2a genotype. Compared with CV777 and other PEDV variant strains, its spike (S) protein exhibited two unique mutations (S663T and L966M). This study first reports the co-infection of PEDV and MRV2 in the pigs and provides a new direction for the prevention and control of the diarrhea diseases. Full article

Porcine astrovirus (PoAstV), porcine sapovirus (PoSaV), porcine norovirus (PoNoV), and porcine rotavirus A (PoRVA) are newly discovered important porcine diarrhea viruses with a wide range of hosts and zoonotic potential, and their co-infections are often found in pig herds. In this study, the specific primers and probes were designed targeting the ORF1 gene of PoAstV, PoSaV, and PoNoV, and the VP6 gene of PoRVA. The recombinant standard plasmids were constructed, the reaction conditions (concentration of primers and probes, annealing temperature, and reaction cycle) were optimized, and the specificity, sensitivity, and reproducibility were analyzed to establish a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay for the detection of these four diarrheal viruses. The results demonstrated that the assay effectively tested PoAstV, PoSaV, PoNoV, and PoRVA without cross-reactivity with other swine viruses, and had limits of detection (LODs) of 138.001, 135.167, 140.732, and 132.199 (copies/reaction) for PoAstV, PoSaV, PoNoV, and PoRVA, respectively, exhibiting high specificity and sensitivity. Additionally, it displayed good reproducibility, with coefficients of variation (CVs) of 0.09–1.24% for intra-assay and 0.08–1.03% for inter-assay. The 1578 clinical fecal samples from 14 cities in Guangxi Province, China, were analyzed via the developed assay. The results indicated that the clinical samples from Guangxi Province exhibited the prevalence of PoAstV (35.93%, 567/1578), PoSaV (8.37%, 132/1578), PoNoV (2.98%, 47/1578), and PoRVA (14.32%, 226/1578), and had a notable incidence of mixed infections of 18.31% (289/1578). Simultaneously, the 1578 clinical samples were analyzed with the previously established assays, and the coincidence rates of these two approaches exceeded 99.43%. This study developed an efficient and precise diagnostic method for the detection and differentiation of PoAstV, PoSaV, PoNoV, and PoRVA, enabling the successful diagnosis of these four diseases. Full article

Porcine rotavirus A (RVA) is one of the major etiological agents of diarrhea in piglets and constitutes a significant threat to the swine industry. A molecular epidemiological investigation was conducted on 2422 diarrhea samples from Chinese pig farms to enhance our understanding of the molecular epidemiology and evolutionary diversity of RVA. The findings revealed an average RVA positivity rate of 42% (943/2422), and the study included data from 26 provinces, primarily in the eastern, southern and southwestern regions. Genetic evolutionary analysis revealed that G9 was the predominant genotype among the G-type genotypes, accounting for 25.32% of the total. The VP4 genotypes were P[7] (36.49%) and P[23] (36.49%). The predominant genotypic combinations of RVA were G9P[23] and G9P[7]. Eleven RVA strains were obtained via MA104 cell isolation. A rat model was established to assess the pathogenicity of these strains, with three strains exhibiting high pathogenicity in the model. Specifically, the RVA Porcine CHN HUBEI 2022 (Q-1), RVA Porcine CHN SHANXI 2022 (3.14-E), and RVA Porcine CHN HUBEI 2022 (5.11-U) strains were shown to cause diarrhea in the rats and damage the intestinal villi during the proliferation phase of the infection, leading to characteristic lesions in the small intestine. These data indicate that continuous monitoring of RVA can provide essential data for the prevention and control of this virus. Full article

Piglet diarrhea poses significant economic losses to the pig industry, posing a worldwide challenge that urgently needs to be addressed in pig breeding practices. Porcine rotavirus (PoRV) is an important viral diarrhea pathogen in piglets, with a high incidence rate and a tendency to cause growth retardation. To enhance the sensitivity and specificity of PoRV detection, we sequenced the NSP3 gene of G5 and G9 genotypes of rotavirus A (RVA), enabling simultaneous detection of the two serotypes. Subsequently, we developed a rapid PoRV detection method using a combination of recombinase-aided amplification (RAA) and CRISPR/Cas12a. In this method, Cas12a binds to RAA amplification products, guided by CRISPR-derived RNA (crRNA), which activates its cleavage activity and releases fluorescence by cutting FAM-BHQ-labeled single-stranded DNA (ssDNA). In the optimized reaction system, the recombinant plasmid PoRV can achieve a highly sensitive reaction within 30 min at 37 °C, with a detection limit as low as 2.43 copies/μL, which is ten times higher in sensitivity compared to the qPCR method. Results from specificity testing indicate that no cross-reactivity was observed between the RAA-CRISPR/Cas12a analysis of PoRV and other viral pathogens, including PoRV G3, PoRV G4, porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV). In the clinical sample detection using the RAA-CRISPR/Cas12a method and qPCR, Cohen’s Kappa value reached as high as 0.952. Furthermore, this approach eliminates the need for large-scale instrumentation, offering a visual result under an ultraviolet lamp through fluorescence signal output. Full article

Bacteroides fragilis is a new generation of probiotics, and its probiotic effects on humans and some animals have been verified. However, research on B. fragilis in cattle is still lacking. In this study, 24 stool samples were collected from two large-scale cattle farms in Wuzhong, Ningxia, including 12 diarrheal and 12 normal stools. A non-toxigenic Bacteroides fragilis (NTBF) was isolated and identified by 16S rRNA high-throughput sequencing and named BF-1153; genome composition and genome functional analyses were carried out to reflect the biological characteristics of the BF-1153 strain. A cluster analysis of BF-1153 was performed using Mega X to explore its genetic relationship. In addition, Cell Counting Kit-8 (CCK8) was used to determine the toxic effects of the strain on human ileocecal colorectal adenocarcinoma cell line cells (HCT-8), Madin-Darby bovine kidney cells (MDBK), and intestinal porcine epithelial cells (IPECs). The results showed that BF-1153 conformed to the biological characteristics of B. fragilis. BF-1153 had no toxic effects on HCT-8, MDBK, and IPEC. Animal experiments have shown that BF-1153 has no toxic effects on healthy SPF Kunming mice. Notably, the supernatant of BF-1153 enhanced cell activity and promoted cell growth in all three cell lines. At the same time, a cluster analysis of the isolated strains showed that the BF-1153 strain belonged to the same branch as the B. fragilis strain 23212, and B. fragilis strain 22998. The results of the animal experiments showed that BF-1153 had a certain preventive effect on diarrhea symptoms in SPF Kunming mice caused by a bovine rotavirus (BRV). In summary, the strain BF-1153 isolated in this experiment is NTBF, which has no toxic effect on MDBK, HCT-8, and IPEC, and has obvious cell growth-promoting effects, especially on MDBK. BF-1153 promotes the growth and development of SPF Kunming mice when compared with the control group. At the same time, BF-1153 alleviated the diarrhea symptoms caused by BRV in SPF Kunming mice. Therefore, BF-1153 has the potential to be a probiotic for cattle. Full article

Porcine rotavirus (PoRV) is a significant enteric pathogen causing gastroenteritis in piglets, which causes huge economic loss to the Chinese pig industry. In this study, six porcine rotavirus A strains were isolated from three adjacent sow farms belonging to the same company within one year, which suffered severe diarrhea outbreaks. AHBZ2303 (G11P[7]) and AHBZ2305 (G9P[23]), AHBZ2304 (G9P[23]) and AHBZ2312 (G4P[6]), AHBZ2310 (G9P[23]) and AHBZ2402 (G5P[23]) were isolated from Farm A, B and C, respectively. All six isolates were related to human rotavirus through complete genome analysis, suggesting the potential cross–species infection between humans and pigs. Evolutionary analysis revealed that AHBZ2303 and AHBZ2304 likely emerged simultaneously in Farm A and B, and then AHBZ2304 was introduced to Farm A and C, leading to the emergence of AHBZ2305 and AHBZ2310. Recombination and large variation were identified for AHBZ2312 and AHBZ2402. These findings provided insights into the transmission and evolution of PoRV among farms and underscored the need for enhanced monitoring to mitigate the risk of outbreaks from novel variants. Full article

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 10² copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases. Full article

Rotavirus is a major causative agent of diarrhoea in children, infants, and young animals around the world. The associated zoonotic risk necessitates the serious consideration of the complete genetic information of rotavirus. A segmented genome makes rotavirus prone to rearrangement and the formation of a new viral strain. Monitoring the molecular epidemiology of rotavirus is essential for its prevention and control. The quantitative RT-PCR targeting the NSP5 gene was used to detect rotavirus group A (RVA) in pig faecal samples, and two pairs of universal primers and protocols were used for amplifying the G and P genotype. The genotyping and phylogenetic analysis of 11 genes were performed by RT-PCR and a basic bioinformatics method. A unique G4P[6] rotavirus strain, designated S2CF (RVA/Pig-tc/CHN/S2CF/2023/G4P[6]), was identified in one faecal sample from a piglet with severe diarrhoea in Guangdong, China. Whole genome sequencing and analysis suggested that the 11 segments of the S2CF strain showed a unique Wa-like genotype constellation and a typical porcine RVA genomic configuration of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. Notably, 4 of the 11 gene segments (VP4, VP6, VP2, and NSP5) clustered consistently with human-like RVAs, suggesting independent human-to-porcine interspecies transmission. Moreover, a unique 344-nt duplicated sequence was identified for the first time in the untranslated region of NSP5. This study further reveals the genetic diversity and potential inter-species transmission of porcine rotavirus. Full article