Establishment of a recombined mannose-binding lectin protein-magnetic beads-enriched binding recombinant enzyme-assisted polymerase chain reaction assay for Candida in blood samples / 中华流行病学杂志

Objective: To establish a nested recombinant enzyme-assisted polymerase chain reaction (RAP) technique combined with recombined mannose-binding lectin protein (M1 protein)-magnetic beads enrichment for the detection of Candida albicans (C. albicans) and Candida tropicalis (C. tropicalis) in blood samples for the early diagnosis of candidemia albicans and candidiemia tropicalis. Methods: The primer probes for highly conserved regions of the internal transcribed spacerregions of C. albicans and C. tropicalis were deigned to establish RAP assays for the detections of C. albicans and C. tropicalis; The sensitivity and reproducibility of nucleic acid tests with gradient dilutions of standard strains and specificity of nucleic acid tests with common clinical pathogens causing bloodstream infection were condcuted. M1 protein-magnetic bead enriched plasma C. albicans and C. tropicalis were used for RAP and PCR in with simulated samples and the results were compared. Results: The sensitivity of the established dual RAP assay was 2.4-2.8 copies/reaction, with higher reproducibility and specificity. M1 protein-magnetic bead enrichment of pathogen combined with the dual RAP assay could complete the detections of C. albicans and C. tropicalis in plasma within 4 hours. Fie the pathogen samples at concentration <10 CFU/ml, the number of the samples tested by RAP was higher than that tested by PCR after enrichment. Conclusion: In this study, a dual RAP assay for the detections of C. albicans and C. tropicalis in blood sample was developed, which has the advantages of accuracy, rapidity, and less contaminants and has great potential for rapid detection of Candidemia.

Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection / 生物医学与环境科学（英文）

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (

A Reverse-transcription Recombinase-aided Amplification Assay for the Rapid Detection of the Far-Eastern Subtype of Tick-borne Encephalitis Virus / 生物医学与环境科学（英文）

OBJECTIVE@#Tick-borne encephalitis virus (TBEV) is an emerging pathogen in Europe and North Asia that causes tick-borne encephalitis (TBE). A simple, rapid method for detecting TBEV RNA is needed to control this disease.@*METHODS@#A reverse-transcription recombinase-aided amplification (RT-RAA) assay was developed. This assay can be completed in one closed tube at 39 °C within 30 minutes. The sensitivity and specificity of RT-RAA were validated using non-infectious synthetic RNA representing a fragment of the NS5 region of the wild-type (WT) TBEV genome and the Senzhang strain. Additionally, 10 batches of tick samples were used to evaluate the performance of the RT-RAA assay.@*RESULTS@#The analytical limit of detection of the assay was 20 copies per reaction of the TBEV synthetic transcript and 3 plaque-forming units (pfu) per reaction of TBEV titers. With the specific assay, no signal due to other arboviruses was observed. Of the 10 batches of tick samples obtained from the Changbai Mountains of China, three were TBEV-positive, which was consistent with the results of the quantitative real-time PCR assay.@*CONCLUSION@#A rapid, highly sensitive, specific, and easy-to-use method was developed for the detection of the TBEV Far-Eastern subtype.

Development of an Internally Controlled Reverse Transcription Recombinase-aided Amplification Assay for the Rapid and Visual Detection of West Nile Virus / 生物医学与环境科学（英文）

West Nile virus (WNV) causes West Nile fever and West Nile encephalitis. Because infection by WNV creates serious public health problems, its simple, rapid, and visual detection is very important in clinical practice, especially in resource-limited laboratories. We have developed a rapid, specific, and highly sensitive internally controlled reverse transcription recombinase-aided amplification (RTRAA) assay to detect WNV, using both real-time fluorescence and the lateral flow dipstick (LFD) at 39.0 °C for 30 min. The analytical sensitivity of the RT-RAA assay was 10 plasmid copies and 1.6 pfu per reaction with real-time fluorescence, and 1,000 plasmid copies per reaction with the LFD. No crossreaction with other control viruses was observed. Compared with the RT-qPCR assay, the RT-RAA assay demonstrated 100% sensitivity and 100% specificity for WNV.

VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences.</p><p><b>METHODS</b>A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis.</p><p><b>RESULTS</b>A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin.</p><p><b>CONCLUSION</b>The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.</p>

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.</p><p><b>METHODS</b>In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.</p><p><b>RESULTS</b>Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.</p><p><b>CONCLUSION</b>MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.</p>

Study on the B cell linear epitopes of rabies virus CVS-11 nucleoprotein / 病毒学报

To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes.

Factors influencing the number of rabies cases in children in China / 生物医学与环境科学（英文）

To understand the epidemic situation and factors influencing rabies cases in children in China, we obtained an overview of the current epidemic based on individual data of rabies cases in children and a descriptive analysis was carried on the prevalence and related factors. The results showed that the rabies cases in children accounted for 21.3% of the total number of rabies cases in China, 97.0% of these cases occurred in rural areas, they were mainly caused by dogs (81.5%), and were primarily level III exposure (47.7%). More than half of the cases were not treated with wound care, vaccination rate was extremely low (15.7%), and only 5.9% of cases were injected with antibodies. Furthermore, 25.4% of cases adopted incorrect treatments such as extruding bleed and wound closure, cases vaccinated with 5 injections accounted for only 22.5%. In conclusion, the prevalence of rabies cases in children in China remains a serious concern, the number and immune status of dogs in rural areas, and knowledge of rabies by risk populations should be considered in future rabies prevention and control programs.

Comparative analysis of the pathogenic mechanisms of street rabies virus strains with different virulence levels / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To characterize two strains of street rabies virus (RABV) isolated from the brain tissue of cattle from Inner Mongolia. Differences in the histopathological and ultrastructural changes in the brain tissue of infected mice were determined to reveal variation in the pathogenesis of infection between street rabies virus strains.</p><p><b>METHODS</b>Ten-day-old mice were intracranially inoculated with one of three virus strains and brain tissue harvested when the mice were moribund. Various histopathological and ultrastructural markers of disease were then compared between the groups.</p><p><b>RESULTS</b>Infection with the street virus strain CNM1101C resulted in severe neuronal dendrites damage, but only mild cell apoptosis, T lymphocyte infiltration and microglial activation. Infection with the other street virus strain, CNM1103C, was characterized by cell apoptosis, T lymphocyte infiltration and microglial activation as well as dendrites damage. However, in comparison, infection with the attenuated virus strain CTN caused severe T lymphocyte infiltration, microglial activation and cell apoptosis, but left the neuronal dendrites intact.</p><p><b>CONCLUSION</b>The two street rabies virus strains isolated from cattle from Inner Mongolia had different levels of virulence and caused distinct pathological changes in infected mice. Therefore, we concluded that different pathogenic mechanisms exist between different RABV strains.</p>

Identification of animal rabies in Inner Mongolia and analysis of the etiologic characteristics / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To perform pathological observation and etiological identification of specimens collected from dairy cows, beef cattle and dogs which were suspected of rabies in Inner Mongolia in 2011, and analyze their etiological characteristics.</p><p><b>METHODS</b>Pathological observation was conducted on the brain specimens of three infected animals with Hematoxylin-Eosin staining, followed by confirmation using immunofluorescence and nested RT-PCR methods. Finally, phylogenetic analysis was conducted using the virus N gene sequence amplified from three specimens.</p><p><b>RESULTS</b>Eosinophilic and cytoplasmic inclusion bodies were seen in neuronal cells of the CNS; and rabies non-characteristic histopathological changes were also detected in the CNS. The three brain specimens were detected positive. N gene nucleotide sequence of these three isolates showed distinct sequence identity, therefore they fell into different groups in the phylogenetic analysis. N gene in the cow and dog had higher homology with that in Hebei isolate, but that in the beef cattle had higher homology with that in Mongolian lupine isolate and Russian red fox isolate.</p><p><b>CONCLUSION</b>Rabies were observed in the dairy cow, beef cattle and canine in the farm in Inner Mongolia, in 2011, which led to a different etiologic characteristics of the epidemic situation.</p>

Follicular fluid levels of prostaglandin E2 and the effect of prostaglandin E2 on steroidogenesis in granulosa-lutein cells in women with moderate and severe endometriosis undergoing in vitro fertilization and embryo transfer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The mechanisms of endometriosis with infertility have not been fully studied. The present study aimed to assess the follicular fluid (FF) levels of prostaglandin E2 (PGE2), which plays a critical role within the ovary, and to investigate the effect of PGE2 on steroidogenesis in granulosa-lutein cells (GLCs) from women with and without endometriosis.</p><p><b>METHODS</b>Thirty-three women with laparoscopically documented endometriosis and 40 controls undergoing in vitro fertilization (IVF) were studied. We assayed the concentrations of PGE2 in FF, the production of E2 and progesterone in FF and in culture medium, and the expression of steroidogenic acute regulatory protein (StAR) and CYP19A1 in GLCs with the intervention of PGE2.</p><p><b>RESULTS</b>PGE2 and progesterone concentrations were increased and displayed positive correlation in endometriotic FF. PGE2 induced the expression of StAR and the production of progesterone in GLCs from women with endometriosis, and the expression of StAR and the production of progesterone were increased in GLCs from women with endometriosis. However, there were no significant effects of PGE2 on promoting the production of E2 or the expression of CYP19A1 in GLCs. Moreover, the production of E2 and the expression of CYP19A1 in GLCs from women with endometriosis were significantly decreased compared to the controls.</p><p><b>CONCLUSIONS</b>PGE2 concentrations are increased in endometriotic FF, along with concomitant increases in progesterone and StAR. In contrast, the E2 and CYP19A1 are decreased in GLCs, which may delay the development of the follicles and cause an imbalance in the follicular steroid hormone levels. These changes may have close relationship with endometriosis-associated infertility.</p>

Preparation and initial application of a monoclonal antibody specific for a newly discovered conserved linear epitope of rabies virus nucleoprotein / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test.</p><p><b>METHODS</b>Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT).</p><p><b>RESULTS</b>Two positive hybridoma cell lines, RVNP-mAb1-CL and RVNP-mAb2-CL, were obtained. RVNP- mAb1-CL produced a higher concentration of monoclonal antibody RVNP-mAb1 in Balb/c ascites. FITC-labeled RVNP-mAb1 showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR).</p><p><b>CONCLUSION</b>FITC-labeled RVNP-mAb1 has potential application for laboratory diagnosis of rabies.</p>

Epidemiological analysis of rabies in 2010, China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the related factors of rabies epidemic and provide the basic data for rabies control and prevention in China by statistic and retrospective analysis of rabies surveillance data in 2010.</p><p><b>METHODS</b>We used descriptive epidemiology method and statistic analysis to analyze the epidemiological characteristics of rabies in 2010 of China.</p><p><b>RESULTS</b>2048 rabies cases were rabies cases were reported in 817 counties (districts) in 2010, which dropped 7.46% compares to 2009. The incidences in children and elder people were high; farmers are main occupation of the cases, the male to female ratio of the cases was 2.44:1. Children and older people are higher acquired rabies than other age population. 640 cases reported through national rabies sentinel surveillance system, 87.50% cases were caused by exposed to dogs, bite was the main exposure reason. The situation of deposing wounds was poor, and the use of vaccine was still low in individual cases, but in the rabies clinic cases under surveillance, the vaccine usage can reach 98%, the usage of immunoglobulin (RIG) or anti-serum for category III exposure in either group cases was not high.</p><p><b>CONCLUSION</b>The epidemic of the rabies in 2010 was eased, Out-patient post-exposure prophylaxis was in good station, but there are still lots of problem existed: post-exposure prophylaxis of individual case was not desirable yet.</p>

The establishment of a rapid fluorescent focus inhibition test for testing rabies virus neutralizing antibody / 中华流行病学杂志

Objective To establish a rapid fluorescent inhibition test (RFFIT) for testing rabies virus neutralizing antibody and the titer of rabies virus neutralizing antibody.CVS-11 was used as the standard challenge virus,and three generations prepared for the establishment of the virus library.Methods International standard for rabies immunoglobulin was used as the reference serum.RFFIT test was established under consulting the protocol of Institute of Pasteur,and its specificity,stability and reproducibility were validated.Results We established the RFFIT which showed both good specificity ( 100% ) and reproducibility (P＞0.5).Conclusion The establishment of RFFIT test perfected the rabies laboratory techniques and would enhance the overall ability in detecting rabies in China.

Analysis of human rabies high-occurrence factors in Guangxi from 2004 to 2008 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Analysis the epidemiological characteristics on rabies cases occurred in Guangxi from 2004 to 2008 and summarize the result of healthy-dog infection rabies virus investigation from 2006 to 2008. Exploring the high-occurrence and correlated factors on rabies in Guangxi.</p><p><b>METHODS</b>Data collected from the National Disease Surveillance System and the National Active Surveillance System for Rabies from 2004 to 2008 and Data of healthy-dog infection rabies virus investigation from 2006 to 2008 were analyzed.</p><p><b>RESULTS</b>The total rabies cases were 2463 in Guangxi from 2004-2008 and average incidence rate was 0.98 per 100 thousand per year. There were 95 counties had rabies case reported, anyway more than 10 cases occurred county number was declined while less than 5 cases rose year by year. The rabies case incidence area was expanded and the cases in middle and west area of Guangxi rose significantly. Rabies cases were reported whole year and no seasonal peak. Human rabies cases mainly were farmers, students and children. Yanger than 20 years old and elder 40 years old were the highest age groups in the population of the investigation, 83.79% cases were attacked by dogs. 78.5% cases classification category III. 83.17% cases had exposed on the upper and lower limbs, 10.56% exposed to the head, face or neck. But 67.88% cases did not receive any PEP and only 18.31% cases vaccinated and 3.63% category III exposure cases combined administration of RIG. The incubation median was 60 days. The rabies virus infection rate among randomly collection healthy-dog brain samples from 2006 to 2008 was 1.92%, 0.93% and 0.89%.</p><p><b>CONCLUSION</b>Unsuccessful and inadequate PEP of patients were the main factors leading to the high-occurrence of human rabies in Guangxi. And there are a lot of infection rabies virus healthy-dogs alive in Guangxi also as a high-occurrence factor.</p>

The comparison of the WHO standard rabies immunoglobulin and the national standard human rabies immunoglobulin used in the rapid fluorescent focus inhibition test (RFFIT) / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Compare the difference of the results referred to the WHO standard rabies immunoglobulin and the national standard human rabies immunoglobulin used in the rapid fluorescent focus inhibition test (RFFIT).</p><p><b>METHODS</b>Setting the WHO standard immunoglobulin and the national standard immunoglobulin in the same system and testing 12 human serum at the same time. Compare the fluorescence percentage of the two different standard immunoglobulin; compare the 12 serum results calculated from the two different standard immunoglobulin used the calculation formula of neutralization antibody titer.</p><p><b>RESULTS</b>The Results display that the 50% percent of the two standard immunoglobulin are all between the fifth and the sixth well, but the percentage of the national standard immunoglobulin is lower than the WHO one. The same testserum result calculated from the WHO standard immunoglobulin is little higher than the national one.</p><p><b>CONCLUSION</b>There is difference in the WHO standard immunoglobulin and the national one, but there is no influence in the results.</p>

Study on the status of infection and distribution of rabies virus in China / 中华流行病学杂志

Objective To investigate the status of infection and distribution of rabies virus (RV) in different epidemic areas in China. Methods Brain specimens from animals and suspected patients were collected at the districts of high-, medium- and low incidence rates of human rabies and detected by both direct Immunofluorescence assay (DFA) and RT-PCR. Results 254 of 3007 specimens of dog brains showed RV positive by DFA (positive rate of 8.4% ). Among these 254 samples, 78 showed positive (positive rate of 30.7% ) by RT-PCR. 93 specimens from dogs and cats that had attacked human beings, 63 of them showed positive by DFA (positive rate of 67.7%) and all of them were also positive by RT-PCR. In addition, RV could also be detected in Apodemus agrarius,ferret badger, and suspected patients specimens from the districts under survey. There was no statistical difference between the infection rates of RV in different provinces and regions with different incidence of rabies. Conclusion There might be a relatively high infection rate of RV among the domestic dogs/cats in the endemic areas in China. Wild animals might have been infected with RV in the districts under survey.