Varied immune responses of HBV-specific B cells in patients undergoing pegylated interferon-alpha treatment for chronic hepatitis B.

BACKGROUND & AIMS: The changes of HBV-specific B-cells in chronic hepatitis B (CHB) patients underwent pegylated interferon-alfa (PEG-IFNα) treatment and achieved functional cure remain unclear. We aimed to evaluate the alterations in HBV-specific B-cells during treatment and therefore explored the mechanism of functional recovery of HBsAg-specific B-cells. METHODS: We included 39 nucleos(t)ide analogues-treated CHB patients who received sequential combination therapy with PEG-IFNα and 8 treatment-naive CHB patients. HBV-specific B-cells were characterized ex vivo using fluorescent labeled HBsAg and HBcAg. The frequency, phenotype, and subsets of HBV-specific B-cells and follicular helper T cells (Tfh-cells) were detected using flow cytometry. The functionality of HBV-specific B-cells was quantified through ELISpot assays. RESULTS: During treatment, the fraction of activated memory B-cells (MBCs) among HBsAg-specific B-cells and the expression of IgG, CXCR3, and CD38 increased. Antibody-secretion capacity of HBsAg-specific B-cell was restored after treatment only in patients with a functional cure and it showed a positive correlation with serum hepatitis B surface antibody levels. The phenotype and function of HBsAg-specific B-cells differed between patients with and without functional cure. Patients with functional cure exhibited IgG+ classical MBCs and plasmablasts in HBsAg-specific B-cells. HBcAg-specific B-cells displayed both attenuated antibody secretion with reduced IgG expression and an IgM+ atypical type of MBCs after treatment, irrespective of with and without functional cure. The number of CD40L+ Tfh-cells increased after PEG-IFNα treatment and positively correlated with HBsAg-specific B-cell activation. CONCLUSIONS: After PEG-IFNα treatment, HBsAg- and HBcAg-specific B-cells exhibit various changes in antibody secretion. Their functional differences are reflected in the alterations in phenotypes and subtypes. The presence of CD40L+ Tfh-cells is associated with the active recovery of HBsAg-specific B-cells. IMPACT AND IMPLICATIONS: HBV-related complications and hepatocellular carcinoma remain the leading causes of mortality from chronic liver disease worldwide, and a cure is rarely achieved with antiviral therapies. Elucidating the immunological mechanisms underlying the functional cure of CHB patients offers a promising therapeutic strategy for viral clearance, such as therapeutic vaccine. We analyzed the alterations in HBV-specific B-cells in patients treated with PEG-IFNα and identified novel pathways for immunotherapeutic boosting of B cell immunity.

Exosomes derived from cardiac fibroblasts with angiotensin II stimulation provoke hypertrophy and autophagy inhibition in cardiomyocytes.

Although accumulating evidence has revealed that autophagy inhibition contributes to the development of pathological cardiac hypertrophy, the mechanisms leading to declined autophagy activity in the hypertrophic heart remain to be elucidated. Exosomes are known to be important mediators of intercellular communication, and the involvement of exosomes in cardiovascular abnormities has attracted increasing attentions. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. Here, we investigated the potential role of CFs-derived exosomes in regulating cardiomyocyte hypertrophy and autophagy. Exosomes from rat CFs treated with angiotensin II (Ang II-CFs-exosomes) were collected and characterized. Our experiments showed that these exosomes could induce hypertrophic responses and impair autophagy activity in primary neonatal rat cardiomyocytes (NRCMs). Ang II-CFs-exosomes blocked the autophagic flux of NRCMs via inhibiting the formation of autolysosomes. Moreover, the pro-hypertrophic effects and autophagy inhibition induced by Ang II-CFs-exosomes was validated in mice receiving injection of the exosomes. These findings highlight a novel role of Ang II-CFs-exosomes in suppressing cardiomyocyte autophagy, which may help to better understand the pathogenesis of cardiac hypertrophy.

Inhibition of miR-214-3p attenuates ferroptosis in myocardial infarction via regulating ME2.

Myocardial infarction (MI) contributes to an increased risk of incident heart failure and sudden death, but there is still a lack of effective treatment in clinic. Recently, growing evidence has indicated that abnormal expression of microRNAs (miRNAs) plays a crucial role in cardiovascular diseases. In this research, the involvement of miRNA-214-3p in MI was explored. A mouse model of MI was established by ligation of the left anterior descending coronary artery, and primary cultures of neonatal rat cardiomyocytes (NRCMs) were submitted to hypoxic treatment to stimulate cellular injury in vitro. Our results showed that miR-214-3p level was significantly upregulated in the infarcted region of mouse hearts and in NRCMs exposed to hypoxia, accompanying with an obvious elevation of ferroptosis. Inhibition of miR-214-3p by antagomir injection improved cardiac function, decreased infarct size, and attenuated iron accumulation and oxidant stress in myocardial tissues. MiR-214-3p could also promote ferroptosis and cellular impairments in NRCMs, while miR-214-3p inhibitor effectively protected cells from hypoxia. Furthermore, dual luciferase reporter gene assay revealed that malic enzyme 2 (ME2) is a direct target of miR-214-3p. In cardiomyocytes, overexpression of ME2 ameliorated the detrimental effects and excessive ferroptosis induced by miR-214-3p mimic, whereas ME2 depletion compromised the protective role of miR-214-3p inhibitor against hypoxic injury and ferroptosis. These findings suggest that miR-214-3p contributes to enhanced ferroptosis during MI at least partially via suppressing ME2. Inhibition of miR-214-3p may be a new approach for tackling MI.

Study of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in different organs of Penaeus vannamei.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured shrimp. It is generally believed that the target organs of IHHNV in shrimp include tissues of ectodermal and mesodermal origin, but do not normally include organ systems of endodermal origin, such as hepatopancreas. In this study, the feeding challenge of IHHNV in different organs (pleopods, muscles, gills, and hepatopancreas) of Penaeus vannamei was studied. The PCR results showed that hepatopancreas of P. vannamei had the strongest IHHNV positivity (100% positive, 19.4 copies/mg) in the feeding challenge experiment. Gills and pleopods had similar infectivity to IHHNV (86.7% positive, 10.6 and 10.5 copies/mg). Among the four organs tested in this study, the IHHNV positivity of muscles was the weakest (33.3% positive, 4.7 copies/mg). The IHHNV infection to hepatopancreas of P. vannamei was also histological confirmed. Our current data indicated that the shrimp tissues derived from the endoderm such as hepatopancreas could also be infected by IHHNV.

Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Factors affecting periodontal healing of the adjacent second molar after lower third molar surgery: a systematic review and meta-analysis.

OBJECTIVES: This systematic review and meta-analysis aim to evaluate the effect modifiers which may influence the periodontal healing of the adjacent second molar after lower third molar surgery. MATERIALS AND METHODS: Prospective studies including patients with third molars requiring removal, with at least 6 months follow-up were considered. Outcomes were periodontal probing depth (PPD) reduction, final PPD, clinical attachment level (CAL) change, final CAL, alveolar bone defect (ABD) reduction, and final ABD depth. Meta-regression was performed to identify factors. RESULTS: Fourteen studies were included in the quantitative synthesis. At 6 months, the PPD reduction was 1.06 mm (95% CI, 0.72 to 1.39 mm) and the remaining PPD was 3.81 mm (95% CI, 3.00 to 4.62 mm). Baseline PPD was strongly correlated with the remaining PPD at 6 months (p < 0.001, adjusted R2 = 70.05%). CONCLUSIONS: Lower third molar surgery results in a modest reduction in PPD, CAL, and ABD; however, periodontal defects still remain at 6 months and beyond. Baseline PPD is strongly correlated with final PPD. While age is not shown to have effect on most of the periodontal parameters, a larger baseline PPD is more likely to manifest with increasing age and third molar surgery should be done before severe periodontitis occurs to avoid persistent periodontal defects. CLINICAL RELEVANCE: This study shows that lower third molar surgery results in modest improvement in periodontal parameters on adjacent second molars and identified factors such as baseline PPD which may affect the healing, which may guide the pre-, peri-, and post-operative management of lower third molar surgery.

Pre-Epiglottic Baton Plate in the Management of Upper Airway Obstruction in an Infant with Femoral Facial Syndrome: A Case Report.

Femoral facial syndrome (FFS) is a rare condition which may present with hypoplasia or aplasia of the femora and unusual facies characterized by long philtrum, thin upper lip and micrognathia. We present the case of a ten-month old infant with FFS who had retroglossal obstruction and who was treated with a pre-epiglottic baton plate. The pre-epiglottic baton plate can be a simple, non-invasive and effective tool for the clinical management of syndromic patients with mild-to-moderate upper airway obstruction due to micrognathia.

Factors related to microimplant-assisted rapid palatal expansion in teenagers and young adults: A cone-beam computed tomography study.

INTRODUCTION: For patients with maxillary transverse deficiency, selecting an appropriate therapeutic method is important for the treatment effect and prognosis. Our study aimed to explore factors related to microimplant-assisted rapid palatal expansion (MARPE) in teenagers and young adults using cone-beam computed tomography. METHODS: Twenty-five patients who underwent MARPE were included in this retrospective study from February 2014 to June 2019. Midpalatal suture density (MPSD) ratio, midpalatal suture maturation (MPSM), bone effect, dentoalveolar effect, and dental effect in maxillary first molar were evaluated using cone-beam computed tomography. Spearman correlation analysis was used to analyze the correlation between the MPSD ratio, MPSM, age, and the expansion amount generated by MARPE. RESULTS: Twenty-five patients (mean age, 19.84 ± 3.96 years; range, 15-29 years) with maxillary transverse deficiency were analyzed. Age was negatively correlated with bone expansion, alveolar expansion, and alveolar change (all P <0.05). There was a negative correlation between MPSM and nasal cavity variation, bone expansion, and alveolar change (all P <0.05). The bone expansion was negatively correlated with MPSD ratio 3 (r = -0.417; P <0.05) and MPSD ratio 4 (all P <0.05). CONCLUSIONS: Age, MPSM, and MPSD ratio were significantly related to the MARPE effect. Age, MPSM, and MPSD ratio should be considered when choosing MARPE.

Airborne Avian Influenza Virus in Ambient Air in the Winter Habitats of Migratory Birds.

Outbreaks of avian influenza virus (AIV) have raised public concerns recently. Airborne AIV has been evaluated in live poultry markets and case farms; however, no study has discussed airborne AIV in ambient air in the winter habitats of migratory birds. Therefore, this study aimed to evaluate airborne AIV, specifically H5, H7, and H9, in a critical winter habitat of migratory birds and assess the factors influencing airborne AIV transmission in ambient air to provide novel insights into the epidemiology of avian influenza. A total of 357 ambient air samples were collected in the Aogu Wetland, Taiwan, Republic of China, between October 2017 and December 2019 and analyzed using quantitative real-time polymerase chain reaction. The effects of environmental factors including air pollutants, meteorological factors, and the species of the observed migratory birds on the concentration of airborne AIV were also analyzed. To our knowledge, this is the first study to investigate the relationship between airborne AIV in ambient air and the influence factors in the winter habitats of migratory birds, demonstrating the benefits of environmental sampling for infectious disease epidemiology. The positive rate of airborne H7 (12%) was higher than that of H5 (8%) and H9 (10%). The daily mean temperature and daily maximum temperature had a significant negative correlation with influenza A, H7, and H9. Cold air masses and bird migration were significantly associated with airborne H9 and H7, respectively. In addition, we observed a significant correlation between AIV and the number of pintails, common teals, Indian spot-billed ducks, northern shovelers, Eurasian wigeons, tufted ducks, pied avocets, black-faced spoonbills, and great cormorants. In conclusion, we demonstrated the potential for alternative surveillance approaches (monitoring bird species) as an indicator for influenza-related risks and identified cold air masses and the presence of specific bird species as potential drivers of the presence and/or the airborne concentration of AIV.

Pediatric Rheumatic Heart Disease in a Middle-Income Country: A Population-Based Study.

INTRODUCTION: Rheumatic heart disease (RHD), a debilitating complication from rheumatic fever, remains a problem in low- and middle-income countries. This study describes the incidence, prevalence, modifiable risk factors for severe RHD and immediate outcome of pediatric RHD. METHODS: This population-based and observational cohort study reviewed pediatric RHD patients (0-18 years) from the Sabah Pediatric Rheumatic Heart Registry from 2015 till 2018. RESULTS: A total of 188 RHD were reviewed with 120 new cases. The incidence of RHD is 2.19 [95% confidence interval (CI): 1.83-2.61] per 100 000 population, with a rising trend over time. Meanwhile, the prevalence of RHD was 13.78 (95% CI: 11.92-15.86) per 100 000 pediatric population. The majority of patients were from indigenous groups (59.0%), male (56.4%) with a mean age of 14.3 (3.31) years. About 77.9% had the lowest household income, and a significant proportion lived in overcrowded conditions. At diagnosis, 59% were diagnosed with severe RHD. There is heightened risk but no statistical significance between modifiable factors (low weight and height percentile at diagnosis, lowest income group, renting a house, overcrowding and healthcare access of more than 5 km) with severe RHD. Severe RHD is significantly associated with risk for intervention (p = 0.016). Sixteen (13.8%) patients required surgical intervention. About 97.6% of patients were on intramuscular benzathine penicillin G with 84.5% compliance. CONCLUSION: The rising prevalence and incidence of pediatric RHD in Sabah, with the most being severe RHD at diagnosis, necessitates the development of an echocardiographic screening and a comprehensive national disease program.

Life cycle assessment of paper mill wastewater: a case study in Viet Nam.

Although in a critical position in the economy, the paper industry releases a lot of wastewater that requires adequate treatment for sustainable development. This study presents an application of Life cycle assessment (LCA) with the ReCiPe tool on the wastewater treatment plant (WWTP) of a paper factory in Vietnam to evaluate the environmental effect of the individual techniques in WWTP, especially the internal circulation (IC) reactor, a pioneer and practical anaerobic technology. Both Midpoint and Endpoint categories results demonstrated that chemical use and electricity consumption mainly contributed to the environmental impact in the WWTP. The Dissolved air flotation (DAF) and Moving bed biofilm reactor (MBBR) are classified as effective techniques to reduce the impacts on the environment. Moreover, the comparison of LCA between IC and up-flow anaerobic sludge bed (UASB) shows that IC is the better practically green technique for the environment.

Cell-based high-throughput screening of polysaccharide biosynthesis hosts.

Valuable polysaccharides are usually produced using wild-type or metabolically-engineered host microbial strains through fermentation. These hosts act as cell factories that convert carbohydrates, such as monosaccharides or starch, into bioactive polysaccharides. It is desirable to develop effective in vivo high-throughput approaches to screen cells that display high-level synthesis of the desired polysaccharides. Uses of single or dual fluorophore labeling, fluorescence quenching, or biosensors are effective strategies for cell sorting of a library that can be applied during the domestication of industrial engineered strains and metabolic pathway optimization of polysaccharide synthesis in engineered cells. Meanwhile, high-throughput screening strategies using each individual whole cell as a sorting section are playing growing roles in the discovery and directed evolution of enzymes involved in polysaccharide biosynthesis, such as glycosyltransferases. These enzymes and their mutants are in high demand as tool catalysts for synthesis of saccharides in vitro and in vivo. This review provides an introduction to the methodologies of using cell-based high-throughput screening for desired polysaccharide-biosynthesizing cells, followed by a brief discussion of potential applications of these approaches in glycoengineering.

Colloidal-sized zirconium porphyrin metal-organic frameworks with improved peroxidase-mimicking catalytic activity, stability and dispersity.

Metal-organic frameworks (MOFs) have attracted great attention as enzyme mimic materials in colorimetric hydrogen peroxide (H2O2) detection. At present, it is highly desirable but remains challenging to prepare MOFs with high stability and dispersity to further improve their peroxidase-mimicking catalytic activity. In this work, we developed a new and facile method for the synthesis of a sub-100 nm peroxidase-mimicking zirconium porphyrin metal-organic framework (Zr-PorMOF) via a solvothermal method. The experimental results indicated that compared with the micron-sized crystals obtained using a classical synthesis method, the catalytic activity, stability and dispersity in water of the colloidal Zr-PorMOF were obviously enhanced. The as-synthesized colloidal Zr-PorMOF was further successfully applied in colorimetric H2O2 detection, and satisfactory detection performance was obtained. Furthermore, the colloidal Zr-PorMOF was also successfully employed in the construction of a peroxidase-based tandem catalysis system. Taking glucose oxidase as an example, this system was successfully applied for glucose sensing in real human serum samples, which proved its practical feasibility in diabetes diagnosis and indicates its high potential feasibility in peroxidase-related applications in complex biomatrix.

Application of Hepatitis E Virus-Related Markers on Samples from a Developing Country.

BACKGROUND: Nepal is an endemic area for hepatitis E virus (HEV) epidemics. The research on viral hepatitis in Nepal is limited. METHODS: Serum samples from 170 patients presenting with symptoms of hepatitis were collected from April to May 2014 in Biratnagar, Nepal, and then transported to Xiamen, China, for further evaluation. All samples were tested for HEV RNA, HEV antigen, anti-HEV IgM, anti-HEV IgG and anti-HBc IgM, anti-HCV IgG, and anti-HAV IgM. RESULTS: Sixteen patients were identified as acute hepatitis E with the presence of ≥ 2 HEV acute phase markers (antigen, RNA, and anti-HEV IgM). HEV infection was the major cause of potential active viral hepatitis (59.2%, 16 of 27), followed by HBV (25.9%, 7 of 27, anti-HBc IgM positive), HAV (18.5%, 5 of 27, anti-HAV IgM positive), and HCV (3.7%, 1 of 27, anti-HCV antibodies). All 16 confirmed HE cases were positive for HEV antigen, while 5 cases were HEV RNA positive, as well as 15 anti-HEV IgM positive. The low positive rate of RNA might be related to the collection and/or the transportation of these samples. CONCLUSIONS: This study showed that HEV is a major cause of acute hepatitis in developing countries and regions. Application of immunoassay diagnostic kits, especially the HEV antigen tests, showed great potential for HE detection in these countries and regions.

User acceptance of an app-based adherence intervention: Perspectives from patients taking oral anticancer medications.

BACKGROUND: Widespread adoption by patients is imperative for the success of app-based interventions for enhancing adherence to oral anticancer medications. Patients' attitudes and beliefs should be evaluated to understand determinants of their acceptance and adoption of such interventions. OBJECTIVE: To identify factors that influence cancer patients' intention to adopt an app-based system for enhancing oral anticancer medication adherence. METHODS: This study was conducted as part of the usability evaluation of an app-based system for enhancing adherence. We followed the grounded theory approach employing audio-recorded face-to-face interviews for data collection from patients taking oral anticancer medications (n = 15) and caregivers of such patients (n = 3). Data analysis involved verbatim transcription of all interviews, coding of the transcripts and field notes, detailed memo writing, and constant comparative evaluation of emergent categories. RESULTS: A conceptual framework of facilitating and hindering factors for users' adoption intention for an oral anticancer medication adherence app was developed. Findings suggest that facing difficulties in maintaining adherence and patients' perceived superiority of the app over their current methods facilitate adoption intention. In contrast, having to pay, lack of language options and users' perception of low competence in using an app were the hindrance factors. CONCLUSION: This study showed that adoption of adherence apps could be explained by technology acceptance constructs, such as performance expectancy. Adoption intention was also facilitated by patients perceived vulnerabilities in maintaining adherence to their medications, which was a health behaviour construct. Implementation of app-based programs should address patients' perceived vulnerabilities and relative advantage of the app over their current methods. Clinicians and app developers should also consider the financial, technological and language barriers for end users.

Design Considerations in the Development of App-Based Oral Anticancer Medication Management Systems: a Qualitative Evaluation of Pharmacists' and Patients' Perspectives.

Smartphone apps can potentially help in enhancing oral anticancer medication (OAM) adherence. Patient adoption and efficacy of such apps depends on inclusion of user-centred and evidence-based features. The objective of this study was to identify important design considerations from the perspectives of patients taking OAMs, caregivers and oncology pharmacists. The study employed a qualitative study design. Data were collected using in-depth interviews with patients (n = 15), caregivers (n = 3) and pharmacists (n = 16). Interviews were audio-recorded, transcribed verbatim and inductive thematic analysis approach was used in data analysis. Monitoring medication-related problems, medication information, replacement of or integration with current systems and accessibility of app content on devices other than smartphones were the key themes identified in the analysis. Flexible input methods for monitored data, glanceability of monitored reports/information, near real-time adherence enhancing and symptom management interventions and customisable reminder options were design considerations identified under the monitoring medication-related problems theme. Participants suggested the provision of focused and easily understandable medication information with a potential for personalisation. Integration of app-based adherence systems with patients' electronic medical records with added mechanisms for alerts in the dispensing system was also suggested as a key design requirement to improve quality of patient care and facilitate adoption by clinicians. Finally, smartphones were the most favoured platform with optional accessibility of app content on other devices. In conclusion, important design considerations were identified through a user-centred design approach. The findings will help developers and clinicians in the design of new app-based systems and evaluation of existing ones.

A novel electrochemiluminescence biosensor based on S-doped yttrium oxide ultrathin nanosheets for the detection of anti-Dig antibodies.

New S-doped yttrium oxide ultrathin nanosheets (NSs) were synthesized which had good electrochemiluminescence (ECL) properties. Through combining these NSs with small molecule linked DNA as the substrate, a novel ECL biosensor was constructed for the detection of protein biomarkers with an acceptable performance.

Fluorescence Regulation of Poly(thymine)-Templated Copper Nanoparticles via an Enzyme-Triggered Reaction toward Sensitive and Selective Detection of Alkaline Phosphatase.

The activity of alkaline phosphatase (ALP) is a crucial index of blood routine examinations, since the concentration of ALP is highly associated with various human diseases. To address the demands of clinical tests, efforts should be made to develop more approaches that can sense ALP in real samples. Recently, we find that fluorescence of poly(30T)-templated copper nanoparticles (CuNPs) can be directly and effectively quenched by pyrophosphate ion (PPi), providing new perspective in designing sensitive biosensors based on DNA-templated CuNPs. In addition, it has been confirmed that phosphate ion (Pi), product of PPi hydrolysis, does not affect the intense fluorescence of CuNPs. Since ALP can specifically hydrolyze PPi into Pi, fluorescence of CuNPs is thus regulated by an ALP-triggered reaction, and a novel ALP biosensor is successfully developed. As a result, ALP is sensitively and selectively quantified with a wide linear range of 6.0 × 10-2 U/L to 6.0 × 102 U/L and a low detection limit of 3.5 × 10-2 U/L. Besides, two typical inhibitors of ALP are evaluated by this analytical method, and different inhibitory effects are indicated. More importantly, by challenging this biosensor with real human serums, the obtained results get a fine match with the data from clinical tests, and the serum sample from a patient with liver disease is clearly distinguished, suggesting promising applications of this biosensor in clinical diagnosis.

Classification of human and zoonotic group hepatitis E virus (HEV) using antigen detection.

Hepatitis E virus (HEV) is one of the major pathogens that cause acute viral hepatitis. The human (genotypes 1 and 2) and zoonotic (genotypes 3 and 4) groups of HEV present different epidemiology and clinical features. In this study, we developed a classification method for rapidly classifying HEV into human or zoonotic groups that combines a general antigen test with a zoonotic group-specific antigen test. Evaluation of serial samples from HEV-infected rhesus monkeys indicated that HEV antigen-positive samples can be classified using the antigen-based classification method. The antigen-based classification method was evaluated further on 55 genotyped samples from acute hepatitis E patients, including 9 human and 46 zoonotic groups. The novel method was completely consistent with the sequencing results: 9/9 for the human groups (100%, 95% confidence interval [CI] 66.4-100%) and 46/46 for the zoonotic groups (100%, 95% CI 92.3-100%). This method was also successfully used for the clustering of some samples that could not be clustered by sequencing. Compared with the sequencing-based method, this method is less time-consuming, less expensive, and less technically complex and is therefore ideal for large numbers of samples. In conclusion, this study provides a convenient and sensitive method for classifying different groups of HEV, and it has potentially important public health applications, especially in underdeveloped areas that cannot afford the high cost of nucleic acid testing.