Separation of spring viraemia of carp virus from large-volume samples using immunomagnetic beads.

A method for separation of spring viraemia of carp virus (SVCV) from large-volume samples using immunomagnetic beads (IMBs) coated with a polyclonal antibody against SVCV was developed. The optimum amount of IMBs was 2 mg in 100 mL. After IMB treatment, the detection limit of SVCV in reverse transcription quantitative PCR (RT-qPCR) was 103 times the 50% tissue culture infectious dose per mL in 100-mL samples. The concentration of viral RNA extracted from SVCV that had been separated using IMBs was 5.18 × 103-fold higher than that of the unseparated SVCV. When fish samples were tested, the concordance rates of the IMBs/RT-qPCR and RT-qPCR were 100% and 67.5%, respectively.

Development of a Blocking ELISA Kit for Detection of ASFV Antibody Based on a Monoclonal Antibody Against Full-Length p72.

BACKGROUND: African swine fever virus (ASFV) is the etiologic agent of African swine fever (ASF), a disease of highly contagious and significant threat to pork production. At present, the sensitive detection methods are the keys to the disease control. OBJECTIVE: Full-length p72 is produced by a eukaryotic system, and its monoclonal antibody (mAb) 34C10 is subsequently recovered. A blocking ELISA kit for detection of ASFV antibody is developed based on p72 trimers and 34C10. METHODS: Full-length p72 is expressed and is used as an immunogen to prepare a panel of monoclonal antibodies. The mAb 34C10 is verified by immunofluorescence and tested by ELISAs with positive serums. The constant affinity of 34C10 is then confirmed. A blocking ELISA kit is further developed and is compared with two commercial kits. RESULTS: The mAb 34C10 is specifically bound to p72 protein, and it exhibits a blocking effect to positive serum. The immunofluorescence assay experiment shows that 34C10 could bind to p72 expressed by baculoviruses, and the binding affinity of 34C10 is found to be as high as 1.85 × 1011 L/mol. The blocking ELISA kit shows high coincidence with a commercial ELISA kit. The sensitivity between these two kits is 97.6% (95%, CI: 90.65-99.58), and the specificity between them is 100% (95%, CI: 98.34-100). CONCLUSIONS: The blocking ELISA developed in this study may have great potential for diagnosis of ASF. The structure of the antigen p72 is found to be a key factor for the performance of the kit. HIGHLIGHTS: For the first time, the eukaryotic expressed full-length p72 protein is used to recover the monoclonal antibody, and it is coated as antigen during the development of the blocking ELISA kit. This study sheds new light on the development of the blocking ELISA kits, especially for the development of a diagnostic kit for the contagious virus with bio-safety problems.

Identification and Molecular Analysis of Ixodid Ticks (Acari: Ixodidae) Infesting Domestic Animals and Tick-Borne Pathogens at the Tarim Basin of Southern Xinjiang, China

Livestock husbandry is vital to economy of the Tarim Basin, Xinjiang Autonomous Region, China. However, there have been few surveys of the distribution of ixodid ticks (Acari: Ixodidae) and tick-borne pathogens affecting domestic animals at these locations. In this study, 3,916 adult ixodid ticks infesting domestic animals were collected from 23 sampling sites during 2012-2016. Ticks were identified to species based on morphology, and the identification was confirmed based on mitochondrial 16S and 12S rRNA sequences. Ten tick species belonging to 4 genera were identified, including Rhipicephalus turanicus, Hyalomma anatolicum, Rh. bursa, H. asiaticum asiaticum, and Rh. sanguineus. DNA sequences of Rickettsia spp. (spotted fever group) and Anaplasma spp. were detected in these ticks. Phylogenetic analyses revealed possible existence of undescribed Babesia spp. and Borrelia spp. This study illustrates potential threat to domestic animals and humans from tick-borne pathogens.

A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus.

A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 µL of each inner primer, RV-FIP (20 pmol/µL) and RV-BIP (20 pmol/µL), 0.5 µL of each outer primer, RV-F3 (10 pmol/µL) and RV-B3 (10 pmol/µL), 1.25 µL of each loop primer, RV-LF (20 pmol/µL) and RV-LB (20 pmol/µL), 3.5 µL dNTP mix (10 mM each), 8 µL MgSO4 (25 mM), 1 µL of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 µL 10 × supplied buffer, and 1 µL of template DNA in a final volume of 25 µL. The optimum reaction conditions were 63 °C for 60 min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added.

Effective observation on edaravone combined with fibrinolysin for treating acute cerebral infarction / 重庆医学

Objective To observe the clinical effect of edaravone combined with fibrinolysin for treating acute cerebral infarc-tion .Methods 80 cases of acute cerebral infarction were randomly divided into the two groups .The treatment group(40 cases) was treated by edaravone injection 30mg plus normal saline 100 mL by intravenous drip ,twice daily ,fibrinolysin injection 100 U plus 5%glucose injection 250 mL by intravenous drip on 1 d ,subsequently 200U plus 5% glucose 250 mL by intravenous drip from the next day ,for 14 d;the control group(40 cases ) used fibrinolysin alone ,the dose and usage were same to the treatment group .The neuro-logical impairment scale scores(NIHSS) were monitored before and after treatment in the two groups .Results The NIHSS scores and Barthel index on 7 ,14 ,30 ,90 d after treatment in the treatment group were superior to those in the control group (P<0 .05) ,no statistical difference before treatment between the two group existed .Conclusion Edaravone combined with fibrinolysin has better effect for treating acute cerebral infarction .

Analysis of the T lymphocyte receptor beta chain complementarity determining region 3 spectratyping in the peripheral blood and hepatic tissue of patients with chronic hepatitis B / 中华传染病杂志

Objective To analyse the spectral patterns of complementarity determining region 3 (CDR3) length distribution of T lymphocyte receptor beta chain variable (TRBV) gene families in infiltrating T cells of the liver tissues and the peripheral blood samples of patients with chronic hepatitis B (CHB) in order to evaluate the characteristics of T cell clonal expansion. Methods The spectral patterns drift of TRBV gene families (the monoclonal/oligoclonal TCR β T cells) in the peripheral blood and hepatic tissues from 11 cases of CHB patients were analyzed by the real-time fluorescence quantitative reverse transcription polymerase chain reaction (FQ-PCR) with DNA melting curve analysis, and abnormal rates of TRBV gene families were compared between CHB patients and healthy control. The comparison of rates was done by chi square test. Results The gene melting spectral pattern of 26 TRBV families of the 11 CHB patients, no matter in the peripheral blood or hepatic tissue, showed either a single peak or prominent melting peaks, even disappeared for certain TRBV families. The abnormal rate of TRBV gene families in the hepatic tissues was significantly higher than that in the peripheral blood samples (x2 = 23. 246, P＜0. 01). What is more interesting was that some parts of TRBV families were identical in both the peripheral blood and the hepatic tissue in certain patients. TCR BV13.1, TCR BV17 and TCR BV22 fragments were found to be restricted used in both the peripheral blood and hepatic tissue by some CHB patients. Conclusions T cells in the peripheral blood and the hepatic tissues of CHB patients can develop clonal expansion to some extent.Parts of TRBV families are restricted used in the peripheral blood and hepatic tissue in some CHB patients, which offers a foundation for further studying the common specific spectral drift patterns of TRBV CDR3 gene in CHB patients.

[Oligonucleotide microarray for subtyping avian influenza virus].

OBJECTIVE: Avian influenza viruses are important human and animal respiratory pathogens and rapid diagnosis of novel emerging avian influenza viruses is vital for effective global influenza surveillance. We developed an oligonucleotide microarray-based method for subtyping all avian influenza virus (16 HA and 9 NA subtypes). METHODS: In total 25 pairs of primers specific for different subtypes and 1 pair of universal primers were carefully designed based on the genomic sequences of influenza A viruses retrieved from GenBank database. Several multiplex RT-PCR methods were then developed, and the target cDNAs of 25 subtype viruses were amplified by RT-PCR or overlapping PCR for evaluating the microarray. Further 52 oligonucleotide probes specific for all 25 subtype viruses were designed according to published gene sequences of avian influenza viruses in amplified target cDNAs domains, and a microarray for subtyping influenza A virus was developed. Then its specificity and sensitivity were validated by using different subtype strains and 2653 samples from 49 different areas. RESULTS: The results showed that all the subtypes of influenza virus could be identified simultaneously on this microarray with high sensitivity, which could reach to 2.47 pfu/mL virus or 2.5 ng target DNA. Furthermore, there was no cross reaction with other avian respiratory virus. CONCLUSION: An oligonucleotide microarray-based strategy for detection of avian influenza viruses has been developed. Such a diagnostic microarray will be useful in discovering and identifying all subtypes of avian influenza virus.