Shared phylogeographic patterns and environmental responses of co-distributed soybean pests: Insights from comparative phylogeographic studies of Riptortus pedestris and Riptortus linearis in the subtropics of East Asia.

Comparative phylogeographic studies of closely related species sharing co-distribution areas can elucidate the role of shared historical factors and environmental changes in shaping their phylogeographic pattern. The bean bugs, Riptortus pedestris and Riptortus linearis, which both inhabit subtropical regions in East Asia, are recognized as highly destructive soybean pests. Many previous studies have investigated the biological characteristics, pheromones, chemicals and control mechanisms of these two pests, but few studies have explored their phylogeographic patterns and underlying factors. In this study, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) dataset to investigate phylogeographic patterns and construct ecological niche models (ENM) for both Riptortus species. Our findings revealed similar niche occupancies and population genetic structures between the two species, with each comprising two phylogeographic lineages (i.e., the mainland China and the Indochina Peninsula clades) that diverged approximately 0.1 and 0.3 million years ago, respectively. This divergence likely resulted from the combined effects of temperatures variation and geographical barriers in the mountainous regions of Southwest China. Further demographic history and ENM analyses suggested that both pests underwent rapid expansion prior to the Last Glacial Maximum (LGM). Furthermore, ENM predicts a northward shift of both pests into new soybean-producing regions due to global warming. Our study indicated that co-distribution soybean pests with overlapping ecological niches and similar life histories in subtropical regions of East Asia exhibit congruent phylogeographic and demographic patterns in response to shared historical biogeographic drivers.

Identifying a potentially invasive population in the native range of a species: The enlightenment from the phylogeography of the yellow spotted stink bug, Erthesina fullo (Hemiptera: Pentatomidae).

The yellow spotted stink bug (YSSB), Erthesina fullo (Thunberg, 1783) is an important Asian pest that has recently successfully invaded Europe and an excellent material for research on the initial stage of biological invasion. Here, we reported the native evolutionary history, recent invasion history, and potential invasion threats of YSSB for the first time based on population genetic methods [using double digest restriction-site associated DNA (ddRAD) data and mitochondrial COI and CYTB] and ecological niche modelling. The results showed that four lineages (east, west, southwest, and Hainan Island) were established in the native range with a strong east-west differentiation phylogeographical structure, and the violent climate fluctuation might cause population divergence during the Middle and Upper Pleistocene. In addition, land bridges and monsoon promote dispersal and directional genetic exchanging between island populations and neighboring continental populations. The east lineage (EA) was identified as the source of invasion in Albania. EA had the widest geographical distribution among all other lineages, with a star-like haplotype network with the main haplotype as the core. It also had a rapid population expansion history, indicating that the source lineage might have stronger diffusion ability and adaptability. Our findings provided a significant biological basis for fine tracking of invasive source at the lineage or population level and promote early invasion warning of potential invasive species on a much subtler lineage level.

Limits of mitochondrial genes in delimiting species within a Carbula species complex (Hemiptera: Pentatomidae).

Molecular data has become a powerful tool for species delimitation, particularly among those that present limited morphological differences; while the mitochondrial genome, with its moderate length, low cost of sequencing and fast lineage sorting, has emerged as a practical data set. Due to the limited morphological differences among the closely related species of Carbula Stål 1865, the species boundaries between Carbula abbreviata (Motschulsky, 1866), Carbula humerigera (Uhler, 1860), and Carbula putoni (Jakovlev, 1876) have remained particularly unclear. In this study, we applied two phylogenetic reconstruction methods to two data sets (mitogenome and COI) to assess the phylogeny of Carbula distributed in Asia, and five species delimitation methods to determine the boundaries between East Asian Carbula species. Our phylogenetic analyses showed Carbula to be paraphyletic; the seven known species distributed within East Asia to form a single monophyletic group, and within this, C. abbreviata, C. humerigera, C. putoni and middle-type to comprise a C. humerigera species complex. Our results show that mitogenome data alone, while effective in the differentiation of more distantly related Carbula species, is not sufficient to accurately delimit the species within this newly described complex.

Out of East Asia: Early Warning of the Possible Invasion of the Important Bean Pest Stalk-Eyed Seed Bug Chauliops fallax (Heteroptera: Malcidae: Chauliopinae).

The short stay at the beginning of the invasion process is a critical time for invasive species identification and preventing invasive species from developing a wider distribution and significant economic impact. The stalk-eyed seed bug Chauliops fallax is an important agricultural pest of soybean and was first reported to occur outside East Asia. Here, we reported the native evolutionary history, recent invasion history, and potential invasion threats of C. fallax for the first time based on population genetic methods and ecological niche modelling. The results showed that four native East Asian genetic groups (EA, WE, TL, and XZ) were well supported, showing an east-west differentiation pattern consistent with the geographical characteristics of three-step landforms in China. Two main haplotypes existed: Hap1 might have experienced a rapid northwards expansion process after the LGM period, and Hap5 reflected local adaptation to the environment in southeastern China. The Kashmir sample was found to come from the recent invasion of populations in the coastal areas of southern China. Ecological niche modelling results suggested that North America has a high risk of invasion, which might pose a serious threat to local soybean production. In addition, with future global warming, the suitable habitat in Asia will move towards the higher latitude region and gradually deviate from the soybean planting area, which indicates the threat of C. fallax to soybean production in Asia will decrease in the future. The results could provide new insights into the monitoring and management of this agricultural pest in the early invasion stage.

Targeted dexamethasone nano-prodrug for corneal neovascularization management.

BACKGROUND: To overcome the drawbacks of traditional therapy for corneal neovascularization (CNV), we evaluated the efficacy of polyethylene glycol (PEG)-conjugated Ala-Pro-Arg-Pro-Gly (APRPG) peptide modified dexamethasone (Dex), a novel nano-prodrug (Dex-PEG-APRPG, DPA). METHODS: Characterization of DPA nano-prodrug were measured with transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses. Cytotoxicity and effects on cell migration and tube formation of DPA were evaluated in vitro. A murine CNV model was established by cornea alkali burn. The injured corneas were given eye drops of DPA (0.2 mM), Dex solution (0.2 mM), Dexp (2 mM), or normal saline three times a day. After two weeks, eyes were obtained for the analysis of histopathology, immunostaining, and mRNA expression. RESULTS: DPA with an average diameter of 30 nm, presented little cytotoxicity and had good ocular biocompatibility. More importantly, DPA showed specific targeting to vascular endothelial cells with efficient inhibition on cell migration and tube formation. In a mouse CNV model, clinical, histological, and immunohistochemical examination results revealed DPA had a much stronger angiogenesis suppression than Dex, resembling a clinical drug with an order of magnitude higher concentration. This was ascribed to the significant downregulations in the expression of pro-angiogenic and pro-inflammatory factors in the corneas. In vivo imaging results also demonstrated that APRPG could prolong ocular retention time. CONCLUSIONS: This study suggests that DPA nano-prodrug occupies advantages of specific targeting ability and improved bioavailability over conventional therapy, and holds great potential for safe and efficient CNV therapy.

The anthropogenic effect of land use on population genetics of Malcus inconspicuus.

Since the beginning of the Holocene era, human activities have seriously impacted animal habitats and vegetative environments. Species that are dependent on natural habitats or with narrow niches might be more severely affected by habitat changes. Malcus inconspicuus is distributed in subtropical China and highly dependent on the mountain environment. Our study investigated the role of the mountainous landscape in the historical evolution of M. inconspicuus and the impact of Holocene human activities on it. A phylogeographical approach was implemented with integrative datasets including double-digest restriction site-associated DNA (ddRAD), mitochondrial data, and distribution data. Three obvious clades and an east-west phylogeographical pattern were found in subtropical China. Mountainous landscape has "multifaceted" effects on the evolutionary history of M. inconspicuus, it has contributed to population differentiation, provided glacial refuges, and provided population expansion corridors during the postglacial period. The effective population size (Ne) of M. inconspicuus showed a sharp decline during the Holocene era, which revealed a significantly negative correlation with the development of cropland in a hilly area at the same time and space. It supported that the species which are highly dependent on natural habitats might undergo greater impact when the habitat was damaged by agricultural activities and we should pay more attention to them, especially in the land development of their distribution areas.

Working Mechanisms and Experimental Research of Piezoelectric Pump with a Cardiac Valve-like Structure.

In this study, based on the working principle of the cardiac valve structure that prevents blood from flowing back, a piezoelectric pump with a cardiac valve-like structure (PPCVLS) is designed. The operating principles of cardiac-valve-like structures (CVLSs) are introduced. Furthermore, the closure conditions of the CVLSs on both sides of the flow channel are explored. The principle behind the working-state conversion between "valve-based" and "valve-less" of PPCVLS is also analyzed. A high-speed dynamic microscopic image-analysis system was utilized to observe and verify the working-state conversion between "valve-based" and "valve-less" PPCVLSs. The resonant frequency of the piezoelectric pump was measured by Doppler laser vibrometer, and the optimal working frequency of the piezoelectric vibrator was determined as 22.35 Hz. The prototype piezoelectric pump was fabricated by the 3D printing technique, and the output performance of the piezoelectric pump was also evaluated. The experimental results show that the piezoelectric pump is valve-based when the driving voltage is greater than 140V, and the piezoelectric pump is valve-less when the driving voltage is less than 140 V. Furthermore, the maximum output pressure of the piezoelectric pump was 199 mm H2O when driven by the applied voltage of 220 V at 7 Hz, while the maximum flow rate of the piezoelectric pump was 44.5 mL/min when driven by the applied voltage of 220 V at 11 Hz.

Two new stick insect species of Sosibia Stål (Phasmatodea: Lonchodidae: Necrosciinae) from China and the first report on mitochondrial genomes of this genus.

We describe and illustrate two new species of Sosibia from China: Sosibia gibba sp. nov. and Sosibia ovata sp. nov. This report includes a key to Sosibia species from China and a description of the distribution area in China. The two mitochondrial genomes of these new Sosibia species were sequenced and annotated for the first time. The compositional biases, codon usage, nucleotide composition, and construct tRNA secondary structures of the two mitogenomes were analyzed. The phylogenetic relationships based on the mitogenomes using Bayesian inference and maximum likelihood methods supported the monophyly of Necrosciinae and divided it into two distinct clades: A: (Sipyloidea + [Sosibia + Calvisia]); and B: (Neohirasea + Micadina).

The phylogenic position of aschiphasmatidae in euphasmatodea based on mitochondrial genomic evidence.

The mitochondrial genome (mitogenome) has been regarded as significant source of data to better understand the phylogenetic relationships within the Euphasmatodea, but no mitogenome in Aschiphasmatoidea has been sequenced to date. In this study, two mitogenomes of Orthomeria smaragdinum and Nanhuaphasma hamicercum of Aschiphasmatidae were sequenced and annotated for the first time. The same mitochondrial gene rearrangement structure was present in the two mitogenomes sequenced, showing as the translocation of tRNA-Arg and tRNA-Asn, which conformed to the tandem duplication-random loss and could be used as a possible synapomorphy for Aschiphasmatidae. The phylogenetic results based on the maximum likelihood (ML) and bayesian inference (BI) methods both showed that Aschiphasmatidae and Neophasmatodea in Euphasmatodea are sister taxa. Although the monophyly of Oriophasmata, Occidophasmata, Diapheromeridae, Phasmatidae, Lonchodidae and Bacilloidea has not been solved, the monophyly of Neophasmatodea and Phyllioidea was well supported.

Integrated Transcriptomic and Proteomic Analysis Reveals Up-Regulation of Apoptosis and Small Heat Shock Proteins in Lens of Rats Under Low Temperature.

Cold cataract is the reversible opacification of the lens when the temperature decreases. However, we observed that when temperature of the rats' lens was maintained at a lower temperature for a prolonged time, the opacification of lens was only partly reversible. To review the potential molecular mechanism of the irreversible part of opacification under cold stimulation, we applied comparative transcriptomic and proteomic analysis to systematically investigate the molecular changes that occurred in the lens capsules of rats under low temperature treatments. The RNA sequencing based transcriptomic analysis showed a significant up-regulation of genes related to the lens structure and development in the Hypothermia Group. Hub genes were small heat shock proteins (sHSPs). Besides the same findings as the transcriptomic results, the liquid chromatography-tandem mass spectrometry based proteomic analysis also revealed the up-regulation of the apoptotic process. To further analyze the regulatory mechanism in this process, we subsequently performed integrated analysis and identified the down-regulation of Notch3/Hes1 and PI3K/Akt/Xiap signaling axis. Our research revealed the activation of the apoptotic process in rats' lens under cold stimulation, and the sHSP related heat shock response as a potential protective factor through our transcriptomic and proteomic data.

Effects of Concentration and Spin Speed on the Optical and Electrical Properties of Silver Nanowire Transparent Electrodes.

In this paper, silver nanowires (AgNWs) with a diameter of 40 nm and a length of 45 µm were dispersed into an ethanol solution to prepare AgNW solutions with concentrations of 1, 2, and 3 mg/mL, respectively. The AgNW solutions were then deposited on a glass substrate using spin-coating at 1000, 2000, and 3000 rpm for 45 s, respectively, to prepare transparent electrodes. The results showed that the distribution of AgNWs on the substrate increased in density with the increase in the AgNW solution concentration and the decrease in spin speed. The effect of concentration on the distribution of AgNWs was greater than that of the spin speed. The transmittance of each electrode was between 84.19% and 88.12% at 550 nm, the average sheet resistance was between 20.09 and 358.11 Ω/sq, the highest figure of merit (FoM) was 104.42, and the lowest haze value was 1.48%. The electrode prepared at 1000 rpm with a concentration of 2 mg/mL and that prepared at 3000 rpm with a concentration of 3 mg/mL were very similar in terms of the average sheet resistance, transmittance at 550 nm, FoM, and haze value; thus, these two electrodes could be considered equivalent. The haze value of the electrode was positively correlated with the spin speed at low concentration, but that relationship became inverse as the concentration rose. For the AgNWs used in this experiment with an aspect ratio of 1125, the concentration of the AgNW solution should reach at least 2 mg/mL to ensure that the FoM of the electrode is greater than 35.

[Design and Implementation of User-oriented Auxiliary Treatment Instrument for Meibomian Gland Dysfunction].

Dry eye is a common ophthalmic disease caused by eye maladjustment due to meibomian gland dysfunction (MGD), which is often accompanied by symptoms such as increased tear film osmotic pressure and ocular surface inflammation. In the treatment of dry eye patients, dredging gland obstruction caused by meibomian gland secretion is an effective treatment method. Based on electrothermal effect and hyperelasticity of the silicone, an auxiliary treatment instrument for MGD is designed, which can improve the blood circulation of the glands through heat compress and massage to achieve the purpose of dredging the meibomian glands. The therapy device can display the temperature and pressure during the treatment in real time, so that the surgeon can grasp the progress of the treatment in real time. The therapy device constructs a user-oriented interactive interface based on parametric modeling method, which can be customized by 3D printing according to the user's eyeball geometric parameters. The designed therapeutic device was finally tested on New Zealand white rabbits. The experimental results show that the therapeutic device has significant effectiveness and safety, as well as clinical application prospects.

Ultrastructural and immunofluorescence analysis of anterior lens capsules in autosomal recessive Alport syndrome.

Background: To first report and study the ultrastructural and immunofluorescence abnormalities of the lens anterior capsules in a patient with autosomal recessive Alport syndrome.Methods: Two anterior lens capsules were collected in femtosecond laser-assisted cataract surgeries from a 29-year-old male patient with bilateral lenticonus caused by autosomal recessive Alport syndrome. The left capsule was examined by transmission electron microscopy and the right capsule was serial sectioned and stained with antibodies against the α2, α3, and α4 chains of type Ⅳ collagen. Anterior lens capsules of another two uncomplicated age-related cataract patients were collected and treated in the same way as the control.Results: The novel findings are that the mitochondria in lens epithelial cells in autosomal recessive Alport syndrome patients increased, twisted, and exhibited high electron density. Characteristic ultrastructure changes of capsule thinning, vertical dehiscence, and irregular-shaped lens epithelial cells were also observed in the left anterior lens capsule. Normal reactivity against the α2 chain and decreased reactivity against the α3 and α4 chains were observed in the right anterior lens capsule.Conclusions: The homozygous c.4599 T > G mutation of COL4A4 not only affects the formation of type Ⅳ collagen networks in the extracellular matrix, but also affects the morphology and survival of the lens epithelial cells in the patient with autosomal recessive Alport syndrome. This study is the first report of the ultrastructural and immunofluorescence changes of anterior lens capsules in autosomal recessive Alport syndrome.

Cytoprotective role of humanin in lens epithelial cell oxidative stress­induced injury.

Oxidative stress-induced injury and apoptosis of human lens epithelial cells (HLECs) are early events in the development of age­related cataracts (ARCs). Humanin (HN) is a mitochondrial­related peptide that serves a cytoprotective role in various cell types and animal models. Following HN knockdown or overexpression, the level of reactive oxygen species (ROS), mitochondrial membrane potential and mitochondrial DNA copy number, cell viability, LDH activity and apoptosis of HLECs under oxidative stress were detected, and apoptosis and autophagy were detected via transmission electron microscopy. The results suggested that HN may be involved in the response of HLECs to oxidative stress, and that HN expression was significantly upregulated under oxidative stress conditions. Furthermore, exogenous HN reduced intracellular ROS content and mitochondrial damage, and enhanced mitochondrial biosynthesis; however, this protection was lost in an endogenous HN knockdown cell model. In addition, to the best of our knowledge, the present study was the first to identify that HN increased mitochondrial autophagy, which was involved in reducing ROS production under oxidative stress. The present study indicated a potential mechanism underlying the anti­oxidative damage and apoptotic effects of HN under oxidative stress. In conclusion, HN may be a potential therapeutic target for ARCs as it has a significant cellular protective effect on HLECs under oxidative stress; therefore, further study is required to investigate its role in the occurrence and development of ARCs.

[Three-dimensional Reconstruction of Retinal Vessels Based on Binocular Vision].

In robot-assisted eye surgery, such as retinal vascular bypass surgery, precise positioning of operating points is required. In this study, a binocular vision-based 3D reconstruction method is proposed to locate the incision points on retinal vessels. Vessels in the image were extracted by CLAHE algorithm to remove the influence of background, then stereo matching was performed. Finally, the retinal vessel image was reconstructed by using the principle of parallax in binocular vision. Experimental results show that this method can accurately locate the incision points on retinal vessels and meet the requirements of ophthalmic surgery.

Association of IGF1R polymorphisms (rs1546713) with susceptibility to age-related cataract in a Han Chinese population.

AIM: To explore the susceptible association between the insulin-like growth factor-1 receptor (IGF1R) single nucleotide polymorphism (SNP) and age-related cataract (ARC), and investigate the underlying mechanisms in human lens epithelium (HLE) cells. METHODS: Totally 1190 unrelated participants, comprising 690 ARC patients and 500 healthy individuals in Han Chinese population were recruited and genotyped for target SNP. The χ 2-test was used to detect genotypic distribution between the patient and control groups and the logistic regression was performed to adjust the age and gender. Meanwhile, different biological experimental methods, such as cell counting kit 8 (CCK-8) assay, flow cytometry, quantitative real time polymerase chain reaction (Q-PCR) and Western blot, were used to detect cell viability, cell cycle progression and apoptosis in HLE cells or IGF1R knockdown HLE cells. RESULTS: The rs1546713 in IGF1R gene was identified (P=0.046, OR: 1.606, 95%CI: 1.245-2.071), which shown a significant relevance with ARC risk under the dominant model. The results demonstrated that IGF1R knockdown inhibited cell proliferation by inducing cell cycle arrested at S phase and promoting apoptosis. Mechanistically, the cell cycle blocked at S phase was linked with the alterations of cyclin A, cyclin B, cyclin E and P21. The pro-apoptosis function of IGF1R may related with stimulating the activation of Caspase-3 and altering the expression levels of apoptotic proteins, including Bcl-2, Bax and Caspase-3. CONCLUSION: This study first report that IGF1R polymorphisms may affect susceptibility to ARCs in Han Chinese population and provide new clues to understand the pathogenic mechanism of ARCs. Notably, IGF1R is likely a potential target for ARC prevention and treatment.

Scanning and transmission electron microscopy study of anterior lens epithelium in presenile cataract.

PURPOSE: To study the structure of lens epithelial cells (LECs) in the anterior lens epithelium of presenile cataract and to further explore the possible reasons for presenile cataract development. METHODS: The anterior lens capsules (aLCs) of patients with presenile cataracts and patients with ordinary age-related cataracts were obtained from routine cataract surgery, and the 5-5.5 mm circles of the central aLC were cut in half and prepared for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). RESULTS: The most obvious structural changes in the LECs observed in both cataract groups by TEM were uneven thickness of the anterior lens epithelium, vacuolated cytoplasm and elongated nuclei. SEM showed abnormal structural changes in the LECs, with swollen cells and spheres on the anterior lens epithelium observed in both groups and holes formed by the LECs stretching observed only in the presenile cataract patients. The degeneration of the anterior lens epithelium and the structural changes in the LECs were observed more prominently in presenile cataract patients. CONCLUSIONS: Abnormal and prominently affected structural features of LECs were observed in the presenile compared to age-related cataract patients by TEM and SEM. We suppose that ultrastructural pathological changes in the anterior lens epithelial cells are one of the important reasons for the development of presenile and age-related cataract.

Socioeconomic disparities in the global burden of glaucoma: an analysis of trends from 1990 to 2016.

PURPOSE: To assess the global burden of glaucoma by year, age, sex, regions, socioeconomic development, and mean years of schooling (MYS) by using disability-adjusted life year (DALY), then to explore the health inequality with socioeconomic status in glaucoma. METHODS: Global, national, and regional DALY data of glaucoma by year, age, and sex were extracted from the Global Health Data Exchange. Human development index (HDI) and national MYS in 2015 were obtained from the Human Development Report (HDR) 2016. Mann-Whitney U test was performed to explore the sexual difference in global DALYs. Kruskal-Wallis tests were performed to explore the difference of age-standardized DALY rates across WHO regions and HDI-related country groups. Linear regression analyses were performed to explore the association between age-standardized DALY rates with HDI and MYS. Health-related Gini coefficients and concentration indexes were calculated to evaluate the trends in health inequality of glaucoma since 1990. RESULTS: DALY numbers, crude DALY rates, and age-standardized DALY rates increased by 118.0%, 55.22%, and 12.12%, respectively, since 1990. Global DALY numbers and crude DALY rates increased with age, and Mann-Whitney U test revealed no significant sex difference in global DALY numbers (P = 0.807) and global crude DALYs rates (P = 0.976) for each age group in 2016. Africa and Eastern Mediterranean had higher age-standardized DALY rates than the global one in 2016. Kruskal-Wallis test indicated significant difference in age-standardized DALY rates across WHO regions (χ2 = 94.227, P < 0.001). Linear regression analysis indicated that HDI (adjusted R2 = 0.079; F = 16.722, P < 0.001) and MYS (adjusted R2 = 0.108; F = 23.048, P < 0.001) had a significant effect on age-standardized DALY rates. Gini coefficients rose from 0.290 in 1990 to 0.292 in 2015 with a peak value 0.299 in 2005, concentration index declined from 1990 (- 0.099) to 2000 (- 0.077) with reaching a low peak value, then rapidly increased to - 0.097 in 2015. CONCLUSIONS: With population growth and aging, global burden of glaucoma is increasing and older age, lower socioeconomic status, and lower MYS are associated with higher glaucoma burden. Our results help to gain a better understanding of glaucoma and guide future health policies tailored for public.

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang, an Eastern Province of China.

BACKGROUND: Sporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 570 serum specimens were sampled from community population aged from 0 to 92 years old in Xianju county of Zhejiang Province in 2013-2014. Microseroneutralization test results were analyzed to estimate the population immunity and to observe antibody dynamics in vaccinated children. E genes of 28 JEV strains isolated in Zhejiang Province were sequenced for phylogenetic tree construction and molecular characteristics analysis with other selected strains. Positive JE neutralizing antibody rates were higher in residents ≥35 years old (81%~98%) and lower in residents <35 years old (0~57%). 7 or 8 years after the 2nd live attenuated vaccine dose, the antibodies against for 4 different strains with microseroneutralization test were decreased by 55%~73% on seropositive rates and by 25%~38% on GMTs respectively. JEV strains isolated in recent years were all grouped into GI, while those isolated in the 1980s belonged to GIII. On important amino acid sites related to antigenicity, there was no divergence between the Zhejiang JE virus strains and the vaccine strain (SA14-14-2). CONCLUSION/SIGNIFICANCES: JE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.

[Etiology and molecular-epidemiological characteristics of viral meningoencephalitis of Zhejiang province in 2013].

OBJECTIVE: To investigate pathogens and molecular-epidemiology characteristics of viral meningoencephalitis in the monitoring sites of Zhejiang province, 2013. METHODS: Cerebrospinal fluid and/or stool specimens were collected from suspected patients admitted to the monitoring hospitals in southern and northern Zhejiang province. Such specimen were subject to real-time qPCR for the detection of Human enterovirus (HEV), Japanese encephalitis virus (JEV), Mumps virus (MuV), Herpes simplex virus (HSV) and Cytomegalovirus (CMV). HEVs were isolated using the RD and Hep-2 cell lines, while VP1 genes from all HEV-positive isolates or RNA-positive specimen were amplified, sequenced, for homology and evolution analysis. RESULTS: 92 (38.5%) of the 239 samples collected from 229 patients were detected as virus nucleic acid positive, including 87 HEV positive samples, 1 MuV positive, 2 HSV positive, and 2 CMV positive; of the 87 HEV positive samples, 38 were further determined to be Coxsackievirus (CV) and 49 as Echovirus (E). 56 HEV strains were isolated from 239 (23.4%) samples. From the 31 cerebral fluid specimen of nucleic acid positive yet virus isolation negative, the most specimen were identified with E9 (9 specimen), followed by CVA9 (8 specimen); the viral serotype of Zhejiang province HEV were CVA9, CVB4, CVB5, E6, E7, E9, E11, E14, E16, E25 and E30, respectively. Predominant epidemic strains identified at southern and northern Zhejiang province were CVB5 and E6 respectively. The phylogenetic analysis of VP1 gene showed that all the HEV isolates in Zhejiang province were HEV-B. CONCLUSION: The HEV-B was the main pathogen for viral meningoencephalitis in Zhejiang province in 2013, including 11 serotypes, while E7 was the first time to be isolated in Zhejiang province. The predominant isolates were CVB5 and E6 in southern and northern Zhejiang province respectively. The positive rate of viral nucleic acid detection was significantly higher than that of viral isolation. Regular EV isolation method was exposed to the risk of missing-detection of E9 and CVA9.