Soft Electrodes for Electrochemical and Electrophysiological Monitoring of Beating Cardiomyocytes.

Many cells in vivo have their inherent motions, which involve numerous biochemical and biophysical signals synergistically regulating cell behavior and function. However, existing methods offer little information about the concurrently chemical and physical responses of dynamically pulsing cells. Here, we report a soft electrode with an electrospun poly(3,4-ethylenedioxythiophene) (PEDOT)-based nanomesh to fully comply with spontaneous motions of cells. Moreover, this electrode demonstrated excellent electrical conductivity, electrochemical performance and cellular biocompatibility. Cardiomyocytes cultured thereon exhibited autonomous and rhythmic contractility, and synchronously induced mechanical deformation of the underlying electrode, which allowed real-time monitoring of nitric oxide release and electrophysiological activity of cardiomyocytes. This work provides a promising way toward recording chemical and electrical signals of biological systems with their natural motions.

Association between ADAM33 polymorphisms and asthma risk: a systematic review and meta-analysis.

BACKGROUND: Asthma is a common complex chronic, inflammatory polygenic disease with heterogeneous manifestations, affecting individuals of all age groups and posing an immense burden on healthcare resources. A number of studies have identified the association between a disintegrin and metalloprotease 33 (ADAM33) polymorphisms and asthma risk, however, the results still remain inconclusive. The objective of the present study was to identify the effect of ADAM33 variants in asthma susceptibility. METHODS: Eligible case-control studies published between January 2000 and June 2018 was searched and retrieved from online electronic databases. The odds ratio (OR) with its 95% confidence interval (CI) was employed to calculate the effect. RESULTS: A total of 63 case-control studies were finally screened out, including 13,280 asthma patients and 13,340 controls. Eleven SNPs of ADAM33 gene were identified. Our results detected a significant association between ADAM33 T2, Q1, F + 1 and AA genotype of T + 1 polymorphisms and asthma risk in total population. Subgroup analysis by ethnicities showed that the alleles and genotypes of T2, Q1 and F + 1 polymorphisms were associated with asthma susceptibility among Asian populations, while V4 polymorphism was associated with asthma among Caucasian populations. Subgroup analysis by ages showed that T2, F + 1 and ST + 4 polymorphisms were associated with childhood asthma, while Q1 and V4 polymorphisms were associated with asthma risk in adults. Subgroup analysis by asthma severity showed that only the G allele of ADAM33 T1 polymorphism was associated with the severity of asthma when compared with the controls. In addition, T2, Q1 and F + 1 polymorphisms of ADAM33 were significantly associated with increased the asthma risk in Chinese asthma patients. CONCLUSIONS: Our results found that T2, Q1 and F + 1 polymorphisms of ADAM33 gene might contribute to asthma risk. Future well-designed case-control studies with large population and more ethnicities are still needed to estimate the association.

Genetic characterization of H9N2 avian influenza virus in plateau pikas in the Qinghai Lake region of China.

Qinghai Lake is a major migratory-bird breeding site that has experienced several highly pathogenic avian influenza virus (AIV) epizootics. Plateau pikas (Ochotona curzoniae) have previously been implicated in the ecology of avian influenza virus in this region. We first isolated an H9N2 AIV (A/Pika/Menyuan/01/2008) from plateau pikas between November 2008 and October 2009. Sequence analysis showed that the A/Pika/Menyuan/01/2008 AIV was closely related to the H9N2 AIV strain (A/Turkey/Wisconsin/ 1/1966). Our findings suggested that plateau pikas may contribute to AIV epidemiology in the Qinghai Lake region.

Subclinical hypothyroidism is associated with metabolic syndrome and clomiphene citrate resistance in women with polycystic ovary syndrome.

BACKGROUND: To evaluate the possible relationship between subclinical hypothyroidism (SCH) and metabolic syndrome (MS) and the response to clomiphene citrate (CC) stimulation in women with polycystic ovary syndrome (PCOS). METHODS: One hundred and ninety-six women with PCOS were divided into two groups: (1) the SCH group with 92 patients; (2) the euthyroid (EU) group with 104 patients. Physical characteristics and metabolic parameters as well as the reaction to CC stimulating test were compared between these two groups. RESULTS: (1) In the SCH group, significantly higher body mass index, Ferriman-Gallwey score, serum triglyceride, insulin and glucose of oral glucose tolerance test, homeostatic model assessment-insulin resistance (HOMA-IR) and significantly lower serum high-density lipoprotein cholesterol was observed in comparison with those in the EU group (p < 0.05). (2) The prevalence of CC resistance (30.4%), IR (43.5%) and MS (34.8%) in the SCH group was significantly higher than that in the EU group (p < 0.05). CONCLUSIONS: SCH was found associated with IR, MS and CC resistance in women with PCOS. PCOS patients with SCH may have a poorer treatment response to ovulation induction with CC.

Transformation of marginal zone lymphoma into high-grade B-cell lymphoma expressing terminal deoxynucleotidyl transferase: A case report.

BACKGROUND: High-grade B-cell lymphoma (HGBL) is an unusual malignancy that includes myelocytomatosis viral oncogene (MYC), B-cell lymphoma-2 (BCL-2), and/or BCL-6 rearrangements, termed double-hit or triple-hit lymphomas, and HGBL-not otherwise specific (HGBL-NOS), which are morphologically characteristic of HGBL but lack MYC, BCL-2, or BCL-6 rearrangements. HGBL is partially transformed by follicular lymphoma and other indolent lymphoma, with few cases of marginal zone lymphoma (MZL) transformation. HGBL often has a poor prognosis and intensive therapy is currently mainly advocated, but there is no good treatment for these patients who cannot tolerate chemotherapy. CASE SUMMARY: We reported a case of MZL transformed into HGBL-NOS with TP53 mutation and terminal deoxynucleotidyl transferase expression. Gene analysis revealed the gene expression profile was identical in the pre- and post-transformed tissues, suggesting that the two diseases are homologous, not secondary tumors. The chemotherapy was ineffective and the side effect was severe, so we tried combination therapy including venetoclax and obinutuzumab. The patient tolerated treatment well, and reached partial response. The patient had recurrence of hepatocellular carcinoma and died of multifunctional organ failure. He survived for 12 months after diagnosis. CONCLUSION: Venetoclax combined with obinutuzumab might improve the survival in some HGBL patients, who are unsuitable for chemotherapy.

[Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis complex].

OBJECTIVE: To identify and evaluate a new nucleic acid amplification (NAA) test target for specific detection of Mycobacterium tuberculosis (MTB) complex (MTC). METHODS: MTC-specific fragment was obtained by ISSR genotyping technology. Primer pairs were designed based on the sequences of MTC-specific fragment and tested in 211 mycobacterial strains including 107 MTC strains and 104 nontuberculous mycobacteria (NTM) strains. IS6110 element (specific identification of MTC strains) and 16s rRNA gene (specific identification of Mycobacterium) amplification were used as a control to evaluate the efficacy of the NAA test target in the detection of MTC strains. RESULTS: One MTC-specific fragment with the length of 588 bp, located in 315947 - 316534 of the genome from MTB reference strain H(37) Rv, were obtained, cloned and sequenced. MTC-specific primer pairs MTCF/R were designed based on these sequences. All 211 mycobacterial strains accurately produced the genus-specific 16s rRNA amplicon. All MTC strains were positive in the MTCF/R PCR amplification while 99% MTC strains (106/107) were positive in the amplification of IS6110 sequences. All NTM strains were negative in both IS6110 and MTCF/R PCR amplification. CONCLUSIONS: The MTC-specific fragment developed in this study can be used as a new NAA test target to correctly distinguish MTC from NTM.

Mitogenomes provide new insights of evolutionary history of Boreheptagyiini and Diamesini (Diptera: Chironomidae: Diamesinae).

Mitogenomes have been widely used for phylogenetic reconstruction of various Dipteran groups, but specifically for chironomid, they have not been carried out to resolve the relationships. Diamesinae (Diptera: Chironomidae) are important bioindicators for freshwater ecosystem monitoring, but its evolutionary history remains uncertain for lack of information. Here, coupled with one previously published and 30 new mitogenomes of Diamesinae, we carried out comparative mitogenomic analysis and phylogenetic analysis. Mitogenomes of Diamesinae were conserved in structure, and all genes arranged in the same order as the ancestral insect mitogenome. All protein-coding genes in Diamesinae were under stronger purifying selection than those of other nonbiting midge species, which may exhibit signs of adaptation to life at cold living conditions. Phylogenetic analyses strongly supported the monophyly of Diamesinae, with Boreheptagyiini deeply nested within Diamesini. In addition, phylogenetic relationship of selected six genera was resolved, except Sympotthastia remained unstable. Our study revealed that the mitogenomes of Diamesinae are highly conserved, and they are practically useful for phylogenetic inference.

First complete mitogenomes of Diamesinae, Orthocladiinae, Prodiamesinae, Tanypodinae (Diptera: Chironomidae) and their implication in phylogenetics.

BACKGROUND: The mitochondrial genome (mitogenome) has been extensively used for phylogenetic and evolutionary analysis in Diptera, but the study of mitogenome is still scarce in the family Chironomidae. METHODS: Here, the first complete mitochondrial genomes of four Chironomid species representing Diamesinae, Orthocladiinae, Prodiamesinae and Tanypodinae are presented. Coupled with published mitogenomes of two, a comparative mitochondrial genomic analysis between six subfamilies of Chironomidae was carried out. RESULTS: Mitogenomes of Chironomidae are conserved in structure, each contains 37 typical genes and a control region, and all genes arrange the same gene order as the ancestral insect mitogenome. Nucleotide composition is highly biased, the control region displayed the highest A + T content. All protein coding genes are under purifying selection, and the ATP8 evolves at the fastest rate. In addition, the phylogenetic analysis covering six subfamilies within Chironomidae was conducted. The monophyly of Chironomidae is strongly supported. However, the topology of six subfamilies based on mitogenomes in this study is inconsistent with previous morphological and molecular studies. This may be due to the high mutation rate of the mitochondrial genetic markers within Chironomidae. Our results indicate that mitogenomes showed poor signals in phylogenetic reconstructions at the subfamily level of Chironomidae.

Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells.

Recently, stretchable electrochemical sensors have stood out as a powerful tool for the detection of soft cells and tissues, since they could perfectly comply with the deformation of living organisms and synchronously monitor mechanically evoked biomolecule release. However, existing strategies for the fabrication of stretchable electrochemical sensors still face with huge challenges due to scarce electrode materials, demanding processing techniques and great complexity in further functionalization. Herein, we report a novel and facile strategy for one-step preparation of stretchable electrochemical biosensors by doping ionic liquid and catalyst into a conductive polymer (poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate), PEDOT:PSS). Bis(trifluoromethane) sulfonimide lithium salt as a small-molecule plasticizer can significantly improve the stretchability and conductivity of the PEDOT:PSS film, and cobalt phthalocyanine as an electrocatalyst endows the film with excellent electrochemical sensing performance. Moreover, the functionalized PEDOT:PSS retained good cell biocompatibility with two extra dopants. These satisfactory properties allowed the real-time monitoring of stretch-induced transient hydrogen peroxide release from cells. This work presents a versatile strategy to fabricate conductive polymer-based stretchable electrodes with easy processing and excellent performance, which benefits the in-depth exploration of sophisticated life activities by electrochemical sensing.

Correction: Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells.

[This corrects the article DOI: 10.1039/D1SC04138J.].

Characterization of the biochemical properties and identification of amino acids forming the catalytic center of 3C-like proteinase of porcine reproductive and respiratory syndrome virus.

PURPOSE OF WORK: The non-structural protein 4 (Nsp4) of porcine reproductive and respiratory syndrome virus (PRRSV) functions as a 3C-like proteinase (3CLpro) and plays a pivotal role in gene expression and replication. We have examined the biochemical properties of PRRSV 3CLpro and identified those amino acid residues involved in its catalytic activity as a prelude to developing anti-PRRSV strategies. The 3C-like proteinase (3CLpro) of porcine reproductive and respiratory syndrome virus (PRRSV) was expressed in Escherichia coli and characterized. The optimal temperature and pH for its proteolytic activity were 8°C and 7.5, respectively. Na(+) (1000 mM) and K(+) (500 mM) were not inhibitory to its activity but Cu(2+), Zn(2+), PMSF and EDTA were significantly inhibitory. His(39), Asp(64) and Ser(118) residues were identified to form the catalytic triad of PRRSV 3CLpro by a series of site-directed mutagenesis analysis.

Evolutionary insights into bot flies (Insecta: Diptera: Oestridae) from comparative analysis of the mitochondrial genomes.

Bot flies (Oestridae) are obligate endoparasites of mammals, and their extraordinary diversification is of great importance in understanding the evolution of parasitism. However, evolutionary analysis of Oestridae has long been impeded by lack of information. Here, the first three mitochondrial genomes of nasal bot flies (Cephalopina titillator, Cephenemyia trompe and Rhinoestrus usbekistanicus) and a comparative mitochondrial genomic analysis between subfamilies of Oestridae are presented. Contrasting to many other parasites, mitochondrial genomes of oestrids are conserved in structure, and genes have retained the same order and direction as the ancestral insect mitochondrial genome. Nucleotide composition is highly heterogenous, with Gasterophilinae possessing highest GC content and smallest genomic size. Mitochondrial evolutionary rates vary considerably, with Hypodermatinae and Oestrinae exhibiting a faster average rate than Cuterebrinae and Gasterophilinae. In addition, the first phylogenomic analysis covering all four bot fly subfamilies was conducted, supporting monophyly of Oestridae and a sister-group relationship of Hypodermatinae and Oestrinae. The only topological ambiguity is Cuterebrinae being a sister-group of either (Hypodermatinae + Oestridae) or Gasterophilinae. Thus, we suggest that mitochondrial genomes carry a great potential for phylogenetic analysis of Oestridae, and more information of Cuterebrinae is needed to illuminate the early evolutionary radiation and parasite-host coevolution of bot flies.

[Overwintering distribution and its environmental determinants of small yellow croaker based on ensemble habitat suitability modeling].

Small yellow croaker is a trans-boundary fish resource shared by China and South Korea. Information on the distribution and preferred habitats of overwintering populations is lacking, parti-cularly in southern waters of Yellow Sea where the species is regulated together by China and South Korea. We simulated the geographic distribution under current condition with eight species distribution models (SDM) based on the presence-absence data and five environmental variables. The performance of model's prediction was evaluated using the area under the receiver operating characteris-tic curve (AUC) based on 5-fold cross-validation. Ensemble SDMs were constructed using a weighted average of eight habitat suitability model types to identify core areas with high probability of small yellow croaker occurrence. The results suggested that predictions based on presence-absence data generally perform better than those based on presence-only data and classical regression models under-performed compared to machine learning approaches. Among all the approaches that supported presence-absence data, support vector machine was the best performing technique and GLM was the worst. The ensemble model outperformed individual SDM models, demonstrating higher effectiveness of ensemble modelling approaches than individual models in reducing the predictive uncertainty. Salinity and temperature were important factors in predicting the overwintering distribution of small yellow croaker. The core areas with high probability of occurrence were concentrated in three areas, the open waters of southern Yellow Sea, the open waters of northern East China Sea, and the coastal sea of Zhejiang Province. Coastal waters in southern Yellow Sea and open waters in central and southern East China Sea were not suitable for overwintering of small yellow croaker. Our results provided a basis for predicting the potential overwintering distribution to guide spatial planning in support of sustainable utilization of small yellow croaker.

Diuretic Effect and Metabolomics Analysis of Crude and Salt-Processed Plantaginis Semen.

Plantaginis Semen (PS) is well recognized in traditional Chinese medicine (TCM) and health products. Crude PS (CPS) and salt-processed CPS (SPS) are the two most commonly used decoction pieces of PS, and are included in the 2020 edition of Chinese Pharmacopoeia. Although they all have multiple effects, the mechanisms for treating diseases are different and remain unclear, the processing mechanism of SPS is also indeterminate, which hinders their clinical application to a certain extent. In order to solve these problems and further develop PS in the clinical application. Here, we used saline-loaded model rats for experiments, and utilized an integrated approach consisting of pharmacological methods and metabolomics, which could assess the diuretic impact of CPS and SPS ethanol extracts on saline-loaded rats and elucidate the underlying mechanism. The results showed that CPS and SPS both produced increased urine volume excretion and urine electrolyte excretion, but the levels of aldosterone (ALD) and aquaporin 2 (AQP2) were decreased. And 30 differential metabolites such as linoleic acid, lysoPC(O-18:0), sphingosine-1-phosphate, lysoPC(18:0) were found, mainly involving three metabolic pathways. In conclusion, CPS and SPS both have a diuretic effect, and that of SPS is better. This work investigated the possible diuretic mechanisms of CPS and SPS which may also be the mechanism of PS for anti-hypertension. In addition, a holistic approach provided novel and helpful insights into the underlying processing mechanisms of TCM.

Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China.

As pigs are susceptible to both human and avian influenza viruses, they have been proposed to be intermediate hosts or mixing vessels for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we reported avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China. Homology and phylogenetic analyses showed that the H1N1 virus (A/swine/Zhejiang/1/07) was closely to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses, which was for the first time reported in China; and the two H1N2 viruses (A/swine/Shanghai/1/07 and A/swine/Guangxi/13/06) were novel ressortant H1N2 influenza viruses containing genes from the classical swine (HA, NP, M and NS), human (NA and PB1) and avian (PB2 and PA) lineages, which indicted that the reassortment among human, avian, and swine influenza viruses had taken place in pigs in China and resulted in the generation of new viruses. The isolation of avian-like H1N1 influenza virus originated from the European swine H1N1 viruses, especially the emergence of two novel ressortant H1N2 influenza viruses provides further evidence that pigs serve as intermediate hosts or "mixing vessels", and swine influenza virus surveillance in China should be given a high priority.

Further evidence for infection of pigs with human-like H1N1 influenza viruses in China.

Classical swine and avian-like H1N1 influenza viruses were reported widely in swine population worldwide, but human-like H1N1 swine viruses were reported occasionally. In 2006, a human-like H1N1 swine virus (A/swine/Guangdong/96/06) was isolated from pigs in Guangdong province, which was reported in China for the first time. To get further evidence for infection of pigs with human-like H1N1 influenza viruses, we analyzed eight gene segments of three human-like swine H1N1 viruses (A/swine/Guangdong/96/06, A/swine/Tianjin/01/04 and A/swine/Henan/01/06) isolated in China. All the eight genes of the three viruses are highly homologous to recent (about 2000) and early (1980s) human H1N1 influenza viruses, respectively. Phylogenetic analyses revealed that A/Swine/Guangdong/96/06 was directly derived from about 2000 human H1N1 influenza viruses, while A/swine/Tianjin/01/04 and A/swine/Henan/01/06 seemed to be descendants of human H1N1 viruses circulating in 1980s. Seroprevalence of our isolate (A/swine/Guangdong/96/06) confirmed the presence of human-like H1N1 virus in pigs in China. Existence of these influenza viruses, especially older viruses (A/swine/Tianjin/01/04 and A/swine/Henan/01/06), indicates that human-like H1N1 influenza viruses may remain invariant for long periods in pigs and provides the evidence that pigs serve as reservoirs of older influenza viruses for human pandemics.

[Expression of connective tissue growth factor after trabeculectomy in rabbits].

OBJECTIVE: To investigate the role of connective tissue growth factor (CTGF) after trabeculectomy associated with wound healing and to identify the role of CTGF in this process. METHODS: It was a experimental study. Forty-nine rabbits were used and divided into 5 groups: normal eyes without trabeculectomy group (group A), ocular hypertension (OHT) model without trabeculectomy group (group B), OHT model with trabeculectomy group (group C), normal eyes with trabeculectomy group (group D) and normal eyes with sham operation group (group E). Group A and B were as control. CTGF mRNA was detected by RT-PCR using blebs and tissues harvested at day 2, 5, 7, and 14. Three replicates of three blebs per time point in the right eyes were collected. The expression of CTGF protein was detected by immunohistochemistry and inflammatory histopathology was inspected by HE staining using the whole eyeball harvested in the left eyes. RESULTS: Compared to group A and B, the expression of CTGF was significantly increased at day 5 after surgery (F = 19.54, P < 0.05) in group C, D, and E. The expression of CTGF mRNA in group C is significantly higher than that in group D at day 2 and 5 (t = 2.300, 5.140, P < 0.05), while group D is significantly higher than that in group E at day 2, 5, 7, and 14 (t = -2.927, -6.424, -4.176, -4.997, P < 0.05). The expression of CTGF protein in group C is significantly higher than that in group D (t = -7.147, -10.955, -9.900, -6.385, P < 0.05), and group D is higher than that in group E (F = 68.33, P < 0.05) at day 2, 5, 7, and 14, respectively. Inflammatory reaction reached peak at day 5 after surgery in group C, D, and E showing an infiltration of neutrophil, monocytes, macrophages, and the proliferation of fibroblast. CONCLUSIONS: Overexpression of CTGF in the blebs after trabeculectomy demonstrates that CTGF may play an important role in the process of wound healing. Furthermore, ocular hypertension may be involved in the upregulation of CTGF expression.

A new species of Fannia (Diptera, Fanniidae) from Yunnan, China.

A new species of the genus Fannia (Diptera, Fanniidae) is described from Yunnan, China, namely Fanniabaihualingensis sp. nov. The male habitus as well as terminalia are documented with focus-stacked photographs. A detailed comparison of new species with related species is provided.

[Electroacupuncture relieved neuropathic pain by suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord in rats with spared nerve injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on changes of expression of L-Arg transporter 2 (CAT-2) mRNA and nitric oxide synthase (iNOS) mRNA and protein and contents of NO and cGMP of L4-L6 segments of spinal cord in rats with spared nerve injury (SNI), so as to reveal its mechanism underlying reducing neuropathic pain. METHODS: A total of 120 male SD rats were randomly divided into sham operation, model, EA and NOS inhibitor (N omega-Nitro-L-arginine methyl ester hydrochloride, L-NAME) groups, with 30 rats in each group. The neuropathic pain model was established by ligating and cutting the tibial nerve and the common peroneal nerve. EA (2 Hz, 1-3 mA) was applied to "Weizhong" (BL40) and "Huantiao" (GB30)on the damaged hindlimb for 30 min, once daily from day 11 to 17 after SNI. Rats of the L-NAME group received i.p. of L-NAME (60 mg·kg-1·d-1) for 7 consecutive days. The mechanical pain threshold (PT) was determined before and 10 and 16 d after SNI, respectively. The expression le-vels of CAT-2 mRNA and iNOS mRNA, and iNOS protein in the L4-L6 segments of the spinal cord were detected by using reverse transcription - polymerase chain reaction (RT-PCR) and Western blot, respectively, and the contents of NO and cGMP of L4-L6 assayed using nitrate/nitrite reductase method and radioimmunoassay, respectively. RESULTS: After modeling, the PT was significantly decreased on day 10 and 16 after SNI in comparison with the sham operation group and their own baseline data of pre-operation in each group (P<0.01), and remarkably increased in the EA and L-NAME groups relevant to the model group on day 16 (P<0.01, P<0.05). Compared with the sham operation group, the expression levels of CAT-2 mRNA and iNOS mRNA and protein, as well as the contents of NO2－/NO3－and cGMP were signi-ficantly up-regulated in the model group (P<0.05, P<0.01). Following EA intervention, the levels of CAT-2 mRNA and iNOS mRNA and iNOS protein, and NO2－/NO3－and cGMP contents were all reversed in both EA and L-NAME groups (P<0.05, P<0.01). The effect of EA was significantly superior to that of L-NAME in raising the PT on day 16 after SNI (P<0.05), but obviously inferior to that of L-NAME in down-regulating the expression of CAT-2 mRNA and iNOS mRNA and protein (P<0.05). No significant differences were found between the EA and L-NAME groups in down-regulating NO2－/NO3－ andcGMP contents (P>0.05). CONCLUSION: EA intervention can effectively relieve neuropathic pain in SNI rats, which may be closely related to its function in suppressing L-Arg/NO/cGMP pathway in the lumbar spinal cord.

Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.