In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.
Animals ; Chlorocebus aethiops ; Clone Cells ; DNA, Complementary ; Distemper Virus, Canine/genetics* ; Plasmids/genetics* ; Vero Cells

Objective To detect the changes of heart volume during concurrent chemoradiotherapy for esophageal cancer based on repeated enhanced 4DCT.Methods Patients with squamous cell esophageal cancer underwent repeated enhanced 4DCT and 3DCT scans before and after 10,20 and 30 fractions of radiotherapy,respectively.The heart was contoured on 3DCT,end expiratory (EE) and maximum intensity projection (MIP) of 4DCTimages.The changes in theheart volume,blood pressure.and heart rate were statistically compared at different time points.Results A total of forty-six patients completed 4 fractions of 3DCT and enhanced 4DCT scans.Compared with the initial values,the heart volume was significantly decreased by 3.27％,4.45％ and 4.52％ after 10 fractions of radiotherapy,and reduced by 6.05％,5.64％ and 4.51％ following 20 fractions of radiotherapy on 3DCT,EE and MIP,respectively (P=0.000-0.027).The heart volume after 30 fractions of radiotherapy did not significantly differ from the initial volume (P＞ 0.05).After radiotherapy,there was a significant decrease inboth systolic and diastolic blood pressure (P =0.000 and P =0.009) and a significant ‖ increase in the heart rate (P=0.0 0 1) compared with those measured before radiotherapy.Conclusions Enhanced 4DCT scan can clearly reflect the changes of heart volume throughout concurrent chemoradiotherapy.The heart volume starts to shrink during the early stage of radiotherapy and continue to decrease until the middleand late-stage,whereas it restores to the initial volume after radiotherapy.Simultaneously,blood pressure declines and heart rate is accelerated during radiotherapy.

Objective To investigate the click and tone burst evoked auditory brainstem responses (ABR)in normal Wistar rat,and to establish the standards of ABR testing method,and to provide a reference for studies rat audition.Methods Fifteen male Wistar rats(30 ears)were used in this sutdy.The latency and amplitude of ABR e-voked by click and TB at 80,50 and 20 dB SPL were measured.Results The occurrence rate of wave Ⅱand Ⅳat low levels(20 dB SPL)was nearly the same according to the amplitude.The cABR (dB peSPL)threshold was 21.83± 4.45 and tbABR (dB SPL)thresholds were 2.02±0.09,2.88±0.16,3.77±0.25,4.69±0.29,and 5.78±0.41, respectively.80 dB stimulus evoked cABR (peSPL)wave I,I b,II,III,IV and V latency (ms)were 1.76±0.12, 2.13±0.11,2.67±0.16,3.49±0.28,4.39±0.29,and 5.45±0.41,respectively.tbABR (SPL)of wave I,Ib, II,III,IV and V latency (ms)at 4 kHz were 2.02±0.09,2.88±0.16,3.77±0.25,4.69±0.29,and 5.78± 0.41,respectively.At 8 kHz they were 1.76±0.07,2.28±0.10,2.63±0.16,3.49±0.21,4.44±0.28,and 5.48±0.43;while at 12 kHz were1.76±0.08,2.24±0.12,2.61±0.25,3.53±0.25,4.46±0.32,and 5.52± 0.45;at 16 kHz were 1.79±0.10,2.25±0.12,2.70±0.18,3.62±0.27,4.52±0.37,and 5.61±0.49;at 24 kHz were 1.75±0.09,2.27±0.11,2.67±0.16,3.60±0.27,4.52±0.38,and 5.60±0.51;at 32 kHz were 1.77±0.10,2.24±0.12,2.64±0.20,3.59±0.34,4.52±0.40,and 5.61±0.52,respectively.Conclusion Wave Ⅳ was the best wave to determine threshold of click and tone burst evoked auditory brainstem response in rat.

To analyze the molecular mechanisms of cross-host transmission of the Aleutian mink disease vi rus (ADV), the hypervariable region fragment of the VP2 gene of the ADV in Jilin Province (China) was amplified. Sequencing analyses showed diversity at residue 174 by comparison with other VP2 genes in GenBank. The phylogenetic tree indicated that the ADV-JL strain had a close relationship with the highly pathogenic strain from Denmark: ADV-K. Results implied that residue 174 may be associated with ADV infectivity.
Aleutian Mink Disease ; virology ; Aleutian Mink Disease Virus ; chemistry ; classification ; genetics ; isolation & purification ; Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; China ; Mink ; Molecular Sequence Data ; Phylogeny ; Sequence Alignment ; Sequence Analysis

OBJECTIVE: To analysis the characteristics of sudden sensorineural hearing loss (SSHL) patients with tinnitus, and explore the relationship of characteristics of tinnitus and audiology. METHOD: Patients diagnosed as SSHL with tinnitus were studied in the research. All patients' clinical features were analyzed, such as tinnitus frequency, pure tone audiometry, tinnitus, hearing loss degree, results of residual inhibition test. RESULT: Thirty cases were identified as mild degree hearing loss, 13 cases as moderate degree, 28 cases as severe degree and 34 cases as profound degree. And hearing impaired frequency of 13 cases was ascertained at low-frequency, 39 cases at middle-high-frequency, and 53 cases at full-range-frequency. The incidence of patients with low-frequency was about 41. 9% (44/105), and it was about 21. 9% (23/105) in those with middle-frequency. And it was 36. 2% (38/105) in cases of high-frequency tinnitus. The chi-square test show statistically significant differences between patients with the low-frequency, middle-frequency and high-frequency of the hearing loss (P<0. 05). In tinnitus residual inhibition test, positive rate of convergence type masking curve was about 72.0%, tinnitus separated type masking curve 20.0%, overlapping type was 57.9%, and the spacing type was 43.5%. There was a statistically significant difference among cases with different type masking curve (P<0. 05)with the spacing residual inhibition test positive rate. CONCLUSION There are individual differences of clinical characteristics among SSHL patients with tinnitus. Tinnitus frequency is consistent with the frequency of hearing loss. Patients had the more serious the degree of hearing loss, who had more serious tinnitus grading. Cases with the converged type curve will be fit for tinnitus masking. Therefore, combining the tinnitus detection with the audiological tests, we could obtain the clinical characteristics of SSHL patients with tinnitus.
Audiometry, Pure-Tone ; Deafness ; Hearing Loss ; Hearing Loss, Sensorineural ; complications ; pathology ; Humans ; Tinnitus ; complications ; pathology

OBJECTIVE: The aim of this retrospective study was to analyze the recovery rate of sudden sensorineural hearing loss in children, and explore the prognostic factors in order to guide the clinical diagnosis and treatment. METHOD: A retrospective review was conducted for the prognosis of children with sudden sensorineural hearing loss during the past 5 years (from November 2010 to May 2015) in Chinese PLA General Hospital. This paper have a complete clinical data of 101 patients (113 ears)with sudden hearing loss, ranging from 0 to 18 years old Patients were divided into four groups according to hearing recovery and eight putative prognostic factors were analyzed. RESULT: Among 101 patients (113 ears), the ratio of male and female was 60:53. Treatment was initiated from 1 to 183 days after disease onset, with an average of (18.5 ± 22.1) d. Bilateral and unilateral hearing loss were 24 ears and 89 ears, respectively. The proportion of mild hearing loss, moderate hearing loss, severe hearing loss and profound hearing loss were 7.1%, 6.2%, 23.9% and 62.8%, respectively. Vertigo and tinnitus occurred in 54.9% and 77.9% of the patients, respectively. After the treatment, the complete recovery rate was 9.7% and the overall recovery rate was 36.3%. The degree of hearing loss, earlier treatment onset, sex and bilateral involvement were significantly associated with hearing recovery (P < 0.05). CONCLUSION Sudden sensorineural hearing loss in children was generally identified as severe and profound hearing loss, but after positive and timely treatment, it can be improved or even cured. The mild hearing loss, earlier treatment onset, unilateral hearing loss and female were positive prognostic factors. The concurrence of tinnitus or vertigo, the results of ABR and DPOAE had no significant influence on prognosis.
Adolescent ; Child ; Child, Preschool ; Female ; Hearing Loss, Sensorineural ; diagnosis ; Hearing Loss, Sudden ; diagnosis ; Hearing Loss, Unilateral ; diagnosis ; Hearing Tests ; Humans ; Infant ; Infant, Newborn ; Male ; Prognosis ; Retrospective Studies ; Tinnitus ; complications ; Vertigo ; complications

Hearing Loss, Noise-Induced ; physiopathology ; Humans

OBJECTIVE: To investigate the meaning of the mutation screening, prevalence, inheritance and the intervention or the prevention for the specific drugs in 10 families with non-syndrome hearing loss in Yunnan Province, China. METHOD: To do a questionnaire about the cases of ten families with non-syndrome hearing loss and to draw a detailed matriarchal family tree detailed. Following that, the A1555G mutation-positive individuals were detected and confirmed using DNA extracting, PCR amplification and sequencing for family volunteer. RESULT: There are 96 members have attended the blood collection in these ten families. Thirty-six of them had the normal hearing and 60 of them had the sensory neural hearing loss. However, 4 out of those had no A1555G point mutation, and 92 had A1555G point mutation (95.8%). While 7 of those were Heterogeneity, the rest were all homogeneous mutation. There were also 73 patients who had amino glycoside antibiotic medication history. However all the rest cases had a history of amino glycoside antibiotic medication were not clear yet. CONCLUSION The proportion of patients with drug-induced deafness is high in Yunnan province and the mutation rate of mitochondrial DNA A1555G is also high. It is worthy to do DNA 12SrRNA A1555G mutation screening for drug intervention and prevention.
Adolescent ; Adult ; Aged ; Child ; China ; epidemiology ; DNA Mutational Analysis ; DNA, Mitochondrial ; genetics ; Deafness ; epidemiology ; genetics ; Female ; Humans ; Male ; Middle Aged ; Pedigree ; Point Mutation ; RNA, Ribosomal ; genetics ; Young Adult

OBJECTIVE: To study the clinical and sequence character of the entire mitochondrial genome in five subjects with mitochondrial 12SrRNA T1095C mutation, and to analyze its relationship with the military noise-induced hearing loss (NIHL). METHOD: Three hundreds and four soldiers exposed to military noise were selected in Yunan and Beijing, including susceptible (experimental) and tolerance (control) groups. Mitochondrial 12SrRNA T1095C mutation were found in 5 subjects. Then the complete nucleotide sequence of five subjects were sequenced and its clinical character were analyzed. RESULT: m12SrRNA T1095C mutation were identified in 5 subjects of experimental group,and none were found in control group. There was significant difference between them (P < 0.05). All five soldiers had the history of military noise exposure and showed sensorineural deafness of different degrees. Sequence analysis of the complete mitochondrial genomes showed the distinct sets of mtDNA polymorphism besides T1095C mutation in five subjects. CONCLUSION The T1095C mutation in hearing loss subjects with various genetic background and history of military noise exposure, is involved in the pathogenesis of hearing impairment. It indicates that the T1095C mutation do relate well with military noise induced-hearing loss.
Adult ; Base Sequence ; DNA, Mitochondrial ; genetics ; Hearing Loss, Noise-Induced ; genetics ; Humans ; Male ; Military Personnel ; Mutation ; Young Adult

OBJECTIVE: To explore the expressions of thymidine phosphorylase (TP), midkine (MK) and MVD marked with CD105 antibody in laryngeal squamous cell carcinoma (LSCC) and their clinical significance. METHOD: The expressions of TP, MK and CD105 in LSCC tissues of 43 cases were studied by immunohistochemical staining. RESULT: The positive expression rates of TP and MK in LSCC were 67.4% and 60.5% respectively, the mean value of MVD was 6.01 +/- 1.78. MVD was significantly higher in tumor tissue with both positive TP and MK than in that with both negative TP and MK (7.07 +/- 3.26 vs. 4.03 +/- 1.90, P < 0.05). The expression of TP, MK and CD105 were all correlated with T-stage and lymph node metastasis. Positive TP, MK expression and high MVD were all associated with a poor survival, and positive expression of both TP and MK in tumors conferred a poorer prognosis than negative expression of those factors in tumors, but only the lymph node metastasis and MVD were independent prognostic factors on multivariate analysis. CONCLUSION Both TP and MK are important for angiogenesis in LSCC. TP, MK and angiogenesis are all closely correlated with the progress of LSCC and the lymph node metastasis. The lymph node metastasis and MVD marked with CD105 antibody were independent prognostic factors. TP and MK may affect the progression and prognosis of tumor by promotion of angiogenesis. A combinative detection of TP, MK and CD105 can be as valuable tumor marker and prognostic factor for LSCC.
Adult ; Aged ; Antigens, CD ; metabolism ; Carcinoma, Squamous Cell ; blood supply ; metabolism ; pathology ; Endoglin ; Female ; Humans ; Laryngeal Neoplasms ; blood supply ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Midkine ; Neoplasm Staging ; Neovascularization, Pathologic ; Nerve Growth Factors ; metabolism ; Prognosis ; Receptors, Cell Surface ; metabolism ; Thymidine Phosphorylase ; metabolism