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Objective: To observe and compare the clinical effects of different electroacupuncture waveforms on primary dysmenorrhea. Methods: This was a prospective, randomized, three-group, parallel-controlled trial. Participants with primary dysmenorrhea were randomly divided into dense-sparse wave, continuous wave, and discontinuous wave groups in a 1:1:1 ratio. Two lateral Ciliao (BL 32) points were used. All three groups started treatment 3–5 days before menstruation, once a day for six sessions per course of treatment, one course of treatment per menstrual cycle, and three menstrual cycles. The primary outcome measure was the proportion with an average visual analog scale (VAS) score reduction of ≥50% from baseline for dysmenorrhea in the third menstrual cycle during treatment. The secondary outcome measures included changes in dysmenorrhea VAS scores, Cox Menstrual Symptom Scale scores and the proportion of patients taking analgesic drugs. Results: The proportion of cases where the average VAS score for dysmenorrhea decreased by ≥50% from baseline in the third menstrual cycle was not statistically significant (P > .05). Precisely 30 min after acupuncture and regarding immediate analgesia on the most severe day of dysmenorrhea, there was a statistically significant difference in the dense-sparse wave group compared with the other two groups during the third menstrual cycle (P

ABSTRACT: Milk coagulation is an important step in the production of fermented dairy products such as yogurt and cheese. Jujube is gaining popularity and acceptance as a food ingredient. In China, jujube yogurt is popular among consumers. However, there is limited information on the effect of jujube on acid- and rennet-induced coagulation properties of milk. The objective of this study was to evaluate the effects of jujube pulp at different concentrations on acid- and rennet-induced coagulation kinetics of milk and the microstructure of acid- and rennet-induced gels. During acid-induced coagulation, with increasing jujube pulp concentration, the initial pH value decreased; however, the final pH value increased. The initial elasticity index (EI) value increased, and the time point at which the mean square displacement curves lost the linear trend advanced. The sample with 10% jujube pulp had the densest structure and highest EI value. During rennet-induced coagulation, with increasing jujube pulp concentration, the production rate and amount of caseinomacropeptide decreased, and the final EI value increased. Protein aggregates in rennet-induced gels became rough, and the sample with 20% jujube pulp had the highest EI value. This study provides a new perspective and understanding of the application of jujube in fermented dairy products.

With the continuous development of technology, traditional marketing methods no longer meet the needs of the main forces of social consumption, and people urgently need more innovative and personalized marketing strategies. E-commerce companies must develop a comprehensive customer-oriented marketing strategy based on big data and multi-channel to achieve their long-term healthy development. This paper first investigated the impact of the digital economy on e-commerce enterprises, focused on the transformation of the digital economy on the marketing model, expounded the development analysis of e-commerce in the digital economy era, and described the development trend of e-commerce marketing in the digital economy era. Then, this paper expounded the current problems faced by e-commerce enterprises, and discussed the lack of integrity, homogeneity, large-scale marketing strategies, and the lack of analysis and application of big data. After that, this paper put forward the marketing strategy of e-commerce enterprises in the digital economy era, and studied it from three aspects, namely, building a reasonable product management structure, marketing strategy based on customized marketing content, and social media marketing strategy based on information sharing. Then this paper proposed to use genetic algorithm to strengthen the marketing strategy of e-commerce enterprises. Finally, based on experiments and surveys, this paper used genetic algorithms to strengthen the construction of e-commerce enterprise marketing strategy in the digital economy, and concluded that the new e-commerce enterprise marketing strategy was 21% more satisfactory than the traditional new e-commerce enterprise marketing strategy. Through comparison, it can see that the integrity of the marketing plan of the new e-commerce enterprise's marketing strategy was 0.33 higher than that of the traditional e-commerce enterprise's marketing strategy, and the integrity of the promotion strategy was 0.34 higher than that of the traditional e-commerce enterprise's marketing strategy. After using the new e-commerce enterprise marketing strategy, the improved management structure was 0.29 higher than that of the traditional monitoring system, and the high quality of products was 0.18 higher than that of the traditional system.

The global e-commerce market is expanding rapidly as the big data era advances and the e-commerce industry thrives. This paper aims to discuss the application of computer security technology in constructing an e-commerce platform business model. The research aims to find effective security technology solutions to strengthen the security of e-commerce platforms, protect user information and rights, and enhance the sustainable development of business models. Big Data-assisted E-Commerce Business Model (BD-ECBM) construction is discussed to overcome the e-commerce platform issues and positively impact marketing strategy decision-makers by raising their level of awareness. The business decision-making process is a series of steps that help businesses overcome obstacles by collecting relevant data, analyzing all relevant alternatives, and settling on a course of action. Big data analytics can improve business management with data fusion technology in many ways. The system's framework of the information service layer, the application-level layer, and the client session layer was developed using the Business model paradigm for accounting for e-past, commerce's capabilities of the platform, and an analysis of supply and demand. It can monitor its rivals in near real-time, adjusting prices, offering more attractive deals, and analyzing negative customer comments to see if it can outperform its rivals. The trial results demonstrate the effectiveness of this method of making marketing decisions and formulating a strategy. Hence, the BD-ECBM technique improves customers' overall satisfaction and experience with the brand while raising the latter's profile.

Abstract Objective To explore the molecular etiology of Waardenburg syndrome type II (WS2) in a family from Yunnan province, China. Methods A total of 406 genes related to hereditary hearing loss were sequenced using next‐generation sequencing. DNA samples were isolated from the peripheral blood DNA of probands. Those pathogenic mutations detected by next‐generation sequencing in probands and their parents were validated by Sanger sequencing. The conservatism of variation sites in genes was also analyzed. The protein expression was detected by flow cytometry. Results A heterozygous mutation c.178delG (p.D60fs*49) in the SOX10 gene was identified in the proband, which is a frameshift mutation and may cause protein loss of function and considered to be a pathogenic mutation. This was determined to be a de novo mutation because her family were demonstrated to be wild‐type and symptom free. SOX10, FGFR3, SOX2, and PAX3 protein levels were reduced as determined by flow cytometry. Conclusion A novel frameshift mutation in SOX10 gene was identified in this study, which may be the cause of WS2 in proband. In addition, FGFR3, SOX2, and PAX3 might also participate in promoting the progression of WS2.

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.

Abstract Background Single-cell RNA sequencing (scRNA-seq) enables the high-throughput profiling of gene expression at the single-cell level. However, overwhelming dropouts within data may obscure meaningful biological signals. Various imputation methods have recently been developed to address this problem. Therefore, it is important to perform a systematic evaluation of different imputation algorithms. Results In this study, we evaluated 11 of the most recent imputation methods on 12 real biological datasets from immunological studies and 4 simulated datasets. The performance of these methods was compared, based on numerical recovery, cell clustering and marker gene analysis. Most of the methods brought some benefits on numerical recovery. To some extent, the performance of imputation methods varied among protocols. In the cell clustering analysis, no method performed consistently well across all datasets. Some methods performed poorly on real datasets but excellent on simulated datasets. Surprisingly and importantly, some methods had a negative effect on cell clustering. In marker gene analysis, some methods identified potentially novel cell subsets. However, not all of the marker genes were successfully imputed in gene expression, suggesting that imputation challenges remain. Conclusions In summary, different imputation methods showed different effects on different datasets, suggesting that imputation may have dataset specificity. Our study reveals the benefits and limitations of various imputation methods and provides a data-driven guidance for scRNA-seq data analysis.

Abstract Hepatoid adenocarcinoma of stomach (HAS) is a special subtype of gastric cancer with poor prognosis. Immunohistochemical analysis could provide important clues for the treatment of HAS. A total of 159 patients were diagnosed as HAS and 139 were enrolled in this study. Statistical differences were determined using relative test methods and survival analyses were performed by the Kaplan–Meier method to find survival differences. All tumors in this study were negative for Epstein–Barr virus‐encoded small RNAs (EBERs) and almost all showed no loss of mismatch repair (MMR) proteins and were positive for alpha fetoprotein (AFP or spalt like transcription factor 4 (SALL4). About half of the tumors had a positive programmed death‐ligand 1 combined positive score (CPS) and 17.3% were positive for human epidermal growth factor receptor 2 (HER2). In addition, there was a relatively high proportion of cmet expression. We also found that HAS patients with recurrent disease treated by emerging therapy had a better survival than those treated with traditional chemotherapy (p = 0.002, median recurrence‐to‐death survival: 23 months versus 6 months); HAS patients who received anti‐HER2 therapy or harbored MMR deficiency had favorable prognosis. Overall, high proportions of MMR protein proficiency, positivity for AFP or SALL4, overexpression of HER2, CPS and cmet, as well as negative EBER findings, are distinctive characteristics of HAS patients. While negative EBER and MMR proficiency indicate molecular features of HAS, positivity for AFP or SALL4 could aid in the diagnosis of HAS. In addition, HAS patients could benefit from anti‐HER2 therapy, immunotherapy, and anti‐angiogenesis therapy.

Our previous studies have shown that the introduction of structurally diverse benzyl side chains at the C5-NH2 position of oseltamivir to occupy 150-cavity contributes to the binding affinity with neuraminidase and anti-influenza activity. To obtain broad-spectrum neuraminidase inhibitors, we designed and synthesised a series of novel oseltamivir derivatives bearing different N-heterocycles substituents that have been proved to induce opening of the 150-loop of group-2 neuraminidases. Among them, compound 6k bearing 4-((r)-2-methylpyrrolidin-1-yl) benzyl group exhibited antiviral activities similar to or weaker than those of oseltamivir carboxylate against H1N1, H3N2, H5N1, H5N6 and H5N1-H274Y mutant neuraminidases. More encouragingly, 6k displayed nearly 3-fold activity enhancement against H3N2 virus over oseltamivir carboxylate and 2-fold activity enhancement over zanamivir. Molecular docking studies provided insights into the explanation of its broad-spectrum potency against wild-type neuraminidases. Overall, as a promising lead compound, 6k deserves further optimisation by fully considering the ligand induced flexibility of the 150-loop.

Abstract Background At present, the hereditary hearing loss homepage, ( https://hereditaryhearingloss.org/ ), includes 258 deafness genes and more than 500 genes that have been reported to cause deafness. With few exceptions, the region-specific distributions are unclear for many of the identified variants and genes. Methods Here, we used a custom capture panel to perform targeted sequencing of 518 genes in a cohort of 879 deaf Chinese probands who lived in Yunnan. Mutation sites of the parents were performed by high-throughput sequencing and validated by Sanger sequencing. Results The ratio of male to female patients was close to 1:1 (441:438) and the age of onset was mainly under six. Most patients (93.5%) were diagnosed with moderate to severe deafness. Four hundred and twenty-eight patients had variants in a deafness gene, with a detection rate of 48.7%. Pathogenic variants were detected in 98 genes and a number of these were recurrent within the cohort. However, many of the variants were rarely observed in the cohort. In accordance with the American College of Medical Genetics and Genomics, pathogenic, likely pathogenic and variants of uncertain significance accounted for 34.3%, 19.3% and 46.4% of all detected variants, respectively. The most common genes included GJB2, SLC26A4, MYO15A, MYO7A, TMC1, CDH23, USH2A and WFS1, which contained variants in more than ten cases. The two genes with the highest mutation frequency were GJB2 and SLC26A4, which accounted for 28.5% (122/428) of positive patients. We showed that more than 60.3% of coding variants were rare and novel. Of the variants that we detected, 80.0% were in coding regions, 17.9% were in introns and 2.1% were copy number variants. Conclusion The common mutation genes and loci detected in this study were different from those detected in other regions or ethnic groups, which suggested that genetic screening or testing programs for deafness should be formulated in accordance with the genetic characteristics of the region.

As an important therapeutic target in breast cancer, HER2 expression assessed by immunohistochemistry plays a critical role in breast cancer treatment. Recent advances in HER2 antibody–drug conjugate therapy have enabled patients with HER2-low expression breast cancer to benefit from the drugs. However, it is not known whether the HER2-low expression in breast cancer FFPE blocks would be lost as storage time increased. In this study, we aimed to assess the loss of HER2 antigenicity in stored FFPE blocks of breast cancer and the rescue effect of modifying the protocol of antigen staining. We selected archived HER2-low breast cancer FFPE blocks with stored time ranging from 1 year to over 15 years and re-detected the expression of HER2. Our study showed that HER2 antigenicity loss increased with storage time and could cause false negativity in HER2-low detection. Moreover, we showed that by either increasing the antigen retrieval time or applying the tyramide signal amplification (TSA) kit, the HER2 signal can be rescued and detected in about half of the cases with HER2-low loss without causing false positivity.

ObjectiveTo screen and analyze the genetic mutations in the PPP1CB gene in a patient with Noonan syndrome with loose anagen hair-2 (NSLH2) in Yunnan Province, China and explore the possible molecular pathogenesis.MethodsAfter obtaining informed consent, we collected the patient's medical history and carried out physical and laboratory examinations for the NSLH2 proband and the family members. Genomic DNA was extracted from the peripheral blood of all individuals. The coding regions including all pathogenic exons, parts of introns, and promoters of genes were sequenced by next-generation sequencing. Pathogenic mutations, which were detected in the probands and their parents, were verified by Sanger sequencing.ResultsThe clinical manifestations of NSLH2 included prominent forehead, yellowish hair, slightly wide eye distance, sparse eyebrows, bilateral auricle deformity, reduced muscle tension, and cardiac and visual abnormalities. The proband carried a c.371A>G mutation in exon 3 of PPP1CB, which is a missense mutation. This was a de novo mutation as the parents of the proband showed no mutation at this site.ConclusionIn this study, we identified a novel mutation of PPP1CB, which enriched the mutation spectrum of the PPP1CB gene and provided a basis for the diagnosis of NSLH2.

Abstract To study the pathogenicity of new duck reovirus (NDRV) to chickens, eighty 3-day-old SPF chickens were equally divided into two groups. The experimental group was inoculated with a NDRV challenge strain of 100 μL (10–5.00 ELD50/0.1 mL) by the subcutaneous (s.c.) route, and the control group was inoculated with 100 μL of sterile phosphate-buffered saline (PBS) by the same route. In the experimental group, chickens exhibited introflexion of claws, performing of splits, stunting syndrome, weight loss and death. Gross lesions such as enlargement and yellowish-white focal necroses were observed in the liver and spleen. Microscopic changes were typical including varying degrees of hepatocyte steatosis and necrosis, splenic lymphocyte necrosis, interstitial pneumonia. Viral loads were detected in lung, liver, heart, spleen, duodenum, burse and kidney. The liver and spleen viral loads remained a much higher level and maintained for a longer time, suggesting that these tissues might be the target organs. In summary, NDRV can cause systemic infections and death in chickens, which indicated that chickens may be infected by NDRV in poultry production.

Stress granules (SGs) are dynamic cytoplasmic protein-RNA structures that form in response to various stress conditions, including viral infection. Porcine epidemic diarrhea virus (PEDV) variant-related diarrhea has caused devastating economic losses to the swine industry worldwide. In this study, we found that the percentage of PEDV-infected cells containing SGs is nearly 20%; meanwhile, PEDV-infected cells were resistant to sodium arsenite (SA)-induced SGs formation, as demonstrated by the recruitment of SGs marker proteins, including G3BP1 and TIA1. Moreover, the formation of SGs induced by SA treatment was suppressed by PEDV papain-like protease confirmed by confocal microscopy. Further study showed that PEDV infection disrupted SGs formation by downregulating G3BP1 expression. Additionally, PEDV replication was significantly enhanced when SGs' assembly was impaired by silencing G3BP1. Taken together, our findings attempt to illuminate the specific interaction mechanism between SGs and PEDV, which will help us to elucidate the pathogenesis of PEDV infection in the near future.

Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1. We discovered an accumulation of ceramides in the AdipoR1–/– retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1–/– mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1-related retinopathy.

To address drug resistance to influenza virus neuraminidase inhibitors (NAIs), a series of novel boron-containing N-substituted oseltamivir derivatives were designed and synthesized to target the 150-cavity of neuraminidase (NA). In NA inhibitory assays, it was found that most of the new compounds exhibited moderate inhibitory potency against the wild-type NAs. Among them, compound 2c bearing 4-(3-boronic acid benzyloxy)benzyl group displayed weaker or slightly improved activities against group-1 NAs (H1N1, H5N1, H5N8 and H5N1-H274Y) compared to that of oseltamivir carboxylate (OSC). Encouragingly, 2c showed 4.6 times greater activity than OSC toward H5N1-H274Y NA. Moreover, 2c exerted equivalent or more potent antiviral activities than OSC against H1N1, H5N1 and H5N8. Additionally, 2c demonstrated low cytotoxicity in vitro and no acute toxicity at the dose of 1000 mg/kg in mice. Molecular docking of 2c was employed to provide a possible explanation for the improved anti-H274Y NA activity, which may be due to the formation of key additional hydrogen bonds with surrounding amino acid residues, such as Arg152, Gln136 and Val149. Taken together, 2c appeared to be a promising lead compound for further optimization.

Recent studies provide compelling evidence to suggest that the tight junction protein claudin 1, aberrantly expressed in several cancer types, plays an important role in cancer progression. Dysregulation of claudin 1 has been shown to induce epithelial mesenchymal transition (EMT). Furthermore, activation of the ERK signaling pathway by protein kinase C (PKC) was shown to be necessary for EMT induction. Whether PKC is involved in regulating breast cancer progression has not been addressed. The PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) was used to investigate the effect of PKC activity on claudin 1 transcription and protein levels, subcellular distribution, and alterations in EMT markers in human breast cancer (HBC) cell lines. As well, tissue microarray analysis (TMA) of a large cohort of invasive HBC biopsies was conducted to investigate correlations between claudin 1 and PKC isomers. TPA upregulated claudin 1 levels in all HBC cell lines analyzed. In particular, a high induction of claudin 1 protein was observed in the MCF7 cell line. TPA treatment also led to an accumulation of claudin 1 in the cytoplasm. Additionally, we demonstrated that the upregulation of claudin 1 was through the ERK signaling pathway. In patient biopsies, we identified a significant positive correlation between claudin 1, PKCα, and PKCε in ER+ tumors. A similar correlation between claudin 1 and PKCε was identified in ER− tumors, and high PKCε was associated with shorter disease-free survival. Collectively, these studies demonstrate that claudin 1 and the ERK signaling pathway are important players in HBC progression.

This paper assesses the explanatory power of the liquidity-risk-based pricing models relative to the Fama–French three-factor model (FF3) and the extensions to the FF3. We find that the liquidity-augmented capital asset pricing model (LCAPM) performs no worse but generally better than other models considered in describing liquidity risk and a variety of anomaly portfolios. Our finding remains intact relative to the troublesome portfolios related to small, value, and aggressive investment.. This study highlights that liquidity risk is not negligible, which is in contrast to some recent findings that the price-impact-based liquidity risk factor contributes little to explain average returns.

We investigate the differential diagnostic value of tremor analysis of EMG on Parkinson’s disease (PD), essential tremor (ET), and enhanced physiological tremor (EPT). Clinical data from 25 patients with PD, 20 patients with ET, and 20 patients with EPT were collected. The tremor frequency and muscle contraction pattern of the resting, posture, and 500 g and 1000 g overload were recorded. The frequency of PD tremor was 4–6 Hz, and the frequency of ET was also in this range; the frequency of EPT is 6–12 hz having some overlap with PD. The muscle contraction patterns of the ET and EPT group were mainly synchronous contraction, and the muscle contraction mode of the PD group was mainly alternating contraction. Having tremor latency from rest to postural position and having changes in tremor amplitude after mental concentration in PD might distinguish ET. Tremor analysis of EMG was able to distinguish PD from ET and EPT by varying the tremor frequency and muscle contraction pattern. It can also differentiate between PD and ET by the latency and concentration effect and ET and EPT by weight load effect.

Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA and TcdB mediated cellular responses are complex, it has been shown that these toxins can alter chemotactic responses of neutrophils and intestinal epithelial cells leading to innate immune responses and tissue damages. The effects of C. difficile toxins on the migration and trafficking of other leukocyte subsets, such as T lymphocytes, are not clear and may have potential implications for adaptive immunity. We investigated here the direct and indirect effects of TcdA and TcdB on the migration of human blood T cells using conventional cell migration assays and microfluidic devices. It has been found that, although both toxins decrease T cell motility, only TcdA but not TcdB decreases T cell chemotaxis. Similar effects are observed in T cell migration toward the TcdA- or TcdB-treated human epithelial cells. Our study demonstrated the primary role of TcdA (compared to TcdB) in altering T cell migration and chemotaxis, suggesting possible implications for C. difficile toxin mediated adaptive immune responses in CDAD.

The duck hepatitis A virus (DHAV), a member of the family Picornaviridae, is the major cause of outbreaks with high mortality rates in young ducklings. It has three distinctive serotypes and among them, serotypes 1 (DHAV-1) and 3 (DHAV-3) were recognized in China. To investigate evolutionary and antigenic properties of the major capsid protein VP1 of these two serotypes, a primary target of neutralizing antibodies, we determined the VP1 coding sequences of 19 DHAV-1 (spanning 2000-2012) and 11 DHAV-3 isolates (spanning 2008-2014) associated with disease outbreaks. By bioinformatics analysis of VP1 sequences of these isolates and other DHAV strains reported previously, we demonstrated that DHAV-1 viruses evolved into two genetic lineages, while DHAV-3 viruses exhibited three distinct lineages. The rate of nucleotide substitution for DHAV-1 VP1 genes was estimated to be 5.57 x 10(-4) per site per year, which was about one-third times slower than that for DHAV-3 VP1 genes. The population dynamics analysis showed an upward trend for infection of DHAV-1 viruses over time with little change observed for DHAV-3 viruses. Antigenic study of representative DHAV-1 and DHAV-3 strains covering all observed major lineages revealed no detectable changes in viral neutralization properties within the serotype, despite the lack of cross-neutralization between serotypes 1 and 3 strains. Structural analysis identified VP1 mutations in DHAV-1 and DHAV-3 viruses that underpin the observed antigenic phenotypes. Results of our experiments described here shall give novel insights into evolution and antigenicity of duck picornaviruses.

Since the first reported cases of ducks infected with a previously unknown flavivirus in eastern China in April 2010, the virus, provisionally designated Duck Tembusu Virus (DTMUV), has spread widely in domestic ducks in China and caused significant economic losses to poultry industry. In this study, we examined in detail structural, antigenic, and evolutionary properties of envelope (E) proteins of six DTMUV isolates spanning 2010-2012, each being isolated from individual farms with different geographical locations where disease outbreaks were documented. Structural analysis showed that E proteins of DTMUV and its closely related flavivirus (Japanese Encephalitis Virus) shared a conserved array of predicted functional domains and motifs. Among the six DTMUV strains, mutations were observed only at thirteen amino acid positions across three separate domains of the E protein. Interestingly, these genetic polymorphisms resulted in no detectable change in viral neutralization properties as demonstrated in a serum neutralization assay. Furthermore, phylogenetic analysis of the nucleotide sequences of the E proteins showed that viruses evolved into two distinct genotypes, termed as DTMUV.I and DTMUV.II, with II emerging as the dominant genotype. New findings described here shall give insights into the antigenicity and evolution of this new pathogen and provide guidance for further functional studies of the E protein for which no effective vaccine has yet been developed.