[Emerging of Japanese encephalitis virus and Getah virus from specimen of mosquitoes in Inner Mongolia Autonomous Region].

Objective: To investigate the types and distribution of blood-sucking insects and arboviruses in Inner Mongolia autonomous region, and provide basic data for the prevention of arbovirus transmitted disease. Methods: Blood-sucking insects were collected by lamp trapping method in nature. Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen. Viruses were isolated in cell culture and characterized, using molecular biological methods. Results: A total of 24 240 mosquitoes and 17 110 aphids were collected from 2 sites of 5 counties (Flags) in Inner Mongolia in 2014 and during 2017-2018. Among them, Japanese encephalitis virus gene was detected in Culex pipiens pallens, and 4 virus strains isolates which could be stably passaged. The isolates were identified as Getah virus and densonucleosis virus by molecular biology identification. Phylogenetic analysis on the E2 gene of the Getah virus (NMDK1813-1) showed that it belonged to the same evolutionary branch of the Gansu isolates (GS10-2) and having six common amino acid variation sites. Conclusions: The emergence of Japanese encephalitis virus and Getah virus from specimen of mosquitoes in Inner Mongolia indicated the new challenges on the prevention and control of arbovirus and related diseases. The results pf this study provided basic data for the prevention and control stretagies of arbovirus transmitted diseases in Inner Mongolia.

[Isolation and identification of Arbovirus in Hainan province, 2017-2018].

Objective: To understand the types and distribution of Arboviruses in Hainan province. Methods: Blood-sucking insects were collected in Hainan province from 2017 to 2018. After laboratory treatment, BHK-21 cells and C6/36 cells were inoculated with grinding supernatant of all blood-sucking insects to isolate all of involving virus. Arbovirus genes in blood-sucking insects were detected in parallel by RT-PCR method. Results: A total of 15 062 mosquitoes were classified into four genera (Culex, Armigeres, Aedes, Anopheles) and 11 360 midges were collected. Culex tritaeniorhynchus was in the majority and accounted for 92.88% (13 990/15 062) of all the mosquitoes collected. Four strains of virus isolates were notified by tissue culture method. Three strains of viruses belonged to Japanese encephalitis virus (JEV), with the other one as Getah virus (GETV). Five pools of JEV gene amplification were positive, from Culex tritaeniorhynchus. Results from the phylogenetic analysis showed that they belonged to genotype JEV-Ⅰ. The minimum infection rate of JEV was 0.57‰ (8/13 990). A total of 5 pools of Akabane virus (AKV) gene amplification were positive. The minimum infection rate of AKV was 0.44‰ (5/11 360). Based on the S gene and M gene sequences of the virus, data from the phylogenetic analysis showed that the five AKV strains carried by midges in Hainan province were in a separate evolutionary branch and with formed unique geographical distribution. Conclusions: JEV and GETV had been isolated again from the mosquito specimens in this survey, since the 1980s. AKV was detected from the midge specimens in Hainan province. These results showed the needs of strengthening the programs on detection and monitor of JEV, GETV and AKV that were related to animal and human diseases in order to reduce the risks of related diseases in this area.

[Mosquitoes, midges and related arboviruses in southeast Sichuan province].

Objective: To investigate the distribution patterns of mosquitoes, midges and related arboviruses in Sichuan province. Methods: Blood-sucking insects were collected from houses and pens, using the ultraviolet lights. Mosquito samples were classified according to morphologic characteristics and then stored at liquid nitrogen. All samples were incubated with BHK-21 and C6/36 cells for virus isolation and then detected for their viral genes. Sequences of the virus were identified and analyzed by molecular biological software, such as BioEdit 7.0.5.3, MEGA 6.0. Results: In total, 17 019 mosquitoes from 3 genera and 4 species and 12 700 midges were collected from the southeast regions of Sichuan province in 2016 and 2017. Among them, 79.4% (13 519/17 019) belonged to Culex tritaeniorhynchus with 11.1% (1 897/17 019) as Armigeres subalbatus, 5.5% (930/17 019) were Anopheles sinensis and 4.0% (673/17 019) were Anopheles sinensis 3 virus strains that isolated from Culex tritaeniorhynchus were identified as typeⅠ Japanese encephalitis virus. Seven pools of mosquitoes isolated from Hejiang county were identified Japanese encephalitis virus gene positive through PCR amplification. With 4 pool midges were detected positive for Akabane virus through PCR gene amplification while midges samples didn't have virus isolates. Conclusions: Culex tritaeniorhynchus appeared the predominant species in the southeast regions of Sichuan. Japanese encephalitis virus transmitted by mosquitoes and Akabane virus by midges were prevalent in southeast Sichuan province.

[Analysis on molecular genetic evolution of Banna virus based on the 12th segment].

Objective: To understand the evolution characteristics of Banna viruses (BAVs) isolated worldwide from 1980 to 2012. Methods: In this study, a phylogenetic analysis using Bayesian Markov Chain Monte Carlo simulations was conducted on all available 12th segment of genes of BAVs isolated worldwide from 1980 to 2012 to investigate the evolutionary and epidemiologic dynamics of BAVs. Results: The Bayesian phylogenetic analysis of BAVs revealed that the common ancestor of BAVs appeared 315 (95%HPD: 63-619) years ago. The evolutionary rate of BAV based on the 12th segment gene was estimated to be 2.33×10-3 (95%HPD: 2.84× 10-4-8.52×10-3) substitution per site per year, indicating BAV belong to an emerging arbovirus with rapid evolution. Conclusion: The evolution of emerging BAVs is rapid and the distribution of BAVs has expanded with new variant being detected, so it is necessary to enhance the surveillance to fully understand the natural distribution and pathogenicity of BAVs.

Specific nucleotide changes in the subgenomic promoter region influence infectivity of the sindbis virus.

Transcription of the subgenomic mRNA of Sindbis virus (SINV) is initiated at a subgenomic promoter (SP). Alignment of SINV sequences identified a 68-nucleotide conserved domain spanning -19 to +49 relative to the subgenomic mRNA start site. Nucleotide T or C is present at -18 or +49 in all known SINVs while a Sindbis-like virus XJ-160 has an A or T at a corresponding position. Our results indicate that deletion or substitution of the T at +49 decreased the activity of SP, while substituting T for A at -18 did not decrease the activity of SP or genetic stability of recombinant SINV.

A specific and sensitive antigen capture assay for NS1 protein quantitation in Japanese encephalitis virus infection.

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml(-1) NS1. Up to 1µgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV.

[Molecular characterization of full-length genome of Japanese encephalitis virus (02-76) newly isolated in China.].

BACKGROUND: To sequence and analyze the complete nucleotide sequence of the Japanese encephalitis virus (JEV) strain 02-76, newly isolated in 2002 in China and to provide information for the genomic structure of JEV and the characteristics of virulence. METHODS: Overlapping primers were designed according to the full-length genomes from GenBank. RT-PCR was used to amplify the fragments, sequencing was performed and all the nucleotides were connected to acquire the full-length genome. Computer software was used to analyze the nucleic acid data, deduced amino acid sequence and phylogenetic trees including Clustal X(1.8), DNASTAR, GENEDOC(3.2). RESULTS: The result of sequence analysis showed that the genome of 02-76 strain was 10,977 nucleotides long. An open reading frame from 95 to 10,391 including 10,296 bases was found capable of coding for a 3432 amino acid polyprotein. Compared with the Beijing 1 strains isolated in 1949 in China, there was a 248 nucleotide divergence and 16 amino acid divergence. Comparison of the complete genome sequences of different JEV isolates showed a 0.6%-15.1% nucleotide sequence divergence among them, which resulted in 0.2%-4.6% amino acid sequence divergence. Phylogenetic analysis through PrM/C,E,3'NTR and full-length genome showed that the 02-76 strain belonged to genotype 3. CONCLUSION: Analysis based on the complete genome sequences of different JEV isolates showed that the 02-76 isolate in 2002 belonged to genotype 3 and was close to the old Chinese isolates SA-14.

[Preparation and characterization of the monoclonal antibodies against Japanese encephalitis virus.].

BACKGROUND: To prepare mouse monoclonal antibodies (McAbs) against Japanese encephalitis virus (JEV)and evaluate their biological characteristics. METHODS: McAbs against JEV were prepared by immunizing, fusing, cloning and screening. Their sensitivity, specificity, universality and neutralizing function were analyzed with ELISA, IFA, NT and Western blot. RESULTS: Titers of three McAbs against JEV were higher than 106. Three McAbs only reacted with JEV and not with other nine arboviruses. F12.37 could react with ten strains of JEV and sensitively detected ten replicating strains of viruses in BHK cell. The strains P3 and SH03-103 of JEV were neutralized by F12.37, its titers of protecting 50% cell were 3.2x105 and 105. Western blot showed that F12.37 reacted with envelop(E)protein of JEV. CONCLUSION: Three McAbs against JEV had high titer and good specificity. And F12.37 was very sensitive and universal in reacting with JEV, and neutralized JEV of Genotype I and Genotype .The binding site of F12.37 lays in E protein of JEV.

[Production of pharmaceutical proteins with mammary gland bioreactor].

Mammary gland bioreactor is a useful biological system which expresses foreign genes in the mammary gland and produces functional pharmaceutical proteins in milk. This production route is appealing for it's advantages, such as the simplicity of access to the expressed protein, the high production of the mammary gland, the capabilities to perform translational modifications. As an alternative of cell culture systems, it is a new biotechnology. The article reviews some aspects on generation and characterization of mammary gland bioreactor, separation and purification of foreign protein from milk and some questions that need to be answered on the route.

Isolation and complete nucleotide sequence of a Chinese Sindbis-like virus.

Infection with alphaviruses is common in the Chinese population. Here we report the isolation of a Sindbis-like virus from a pool of Anopheles mosquitoes collected in Xinjiang, China during an arbovirus survey. This virus, designated XJ-160, rapidly produced cytopathic effects on mosquito and hamster cells. In addition, it was lethal to neonatal mice if inoculated intracerebrally. Serologically, XJ-160 reacted with and was neutralized by an anti-Sindbis antibody. Anti-XJ-160 antibodies were found in several cohorts of Chinese subjects. The complete 11626-base nucleotide sequence of XJ-160 was determined. XJ-160 has diverged significantly from the prototype Sindbis virus, with an 18% difference in nucleotide sequence and an 8.6% difference in amino acids; there are 11 deletions and 2 insertions, involving 99 nucleotides in total. XJ-160 is most closely linked to Kyzylagach virus isolated in Azerbaijan. Both belong to the African/European genetic lineage of Sindbis virus, albeit more distantly related to other members.

Radioimmunodetection of prostatic cancer: in vivo use of antibody against r-seminoprotein for diagnosis of prostatic cancer.

Forty patients (25 cases of prostatic cancer and 15 cases of benign prostatic hyperplasia) were examined with radioimmunodetection (RAID) using antibody against r-Seminoprotein (r-Sm). The images of malignant tumor sites were revealed from the scan of single photon emission computerized tomography with the tracing of dual radionuclide and computer digital subtraction technique. Of the 25 patients with prostatic cancer tested, twenty-four were demonstrated in RAID with a positive rate of 96%. The ratio of taking in nuclide radioactivity between the tumor tissue and normal tissue (T/N) was 6.9 and the best showing time was the 96th hr from injection of radioactive antibody against r-Sm. The minimum diameter of the tumor detected in RAID was 0.5 cm. All of the 8 cases of metastatic prostatic cancer in pelvic or bone location were detected, with a rate of 100%. Of the 15 cases of benign prostatic hyperplasia, only one showed mild positive result. B-ultrasonography and CT showed a positive detective rate of 72.7% and 65%, respectively. Our results have indicated that RAID using 131I-labeled antibody against r-Sm possesses more advantages in specificity, because RAID not only defines the involved sites, but also shows the sites of the original tumors, and the metastatic location as well as the relationship between them.

Curative effects of highly active atrial natriuretic peptide on severe pregnancy-induced hypertension.

A new highly active atrial natriuretic peptide (haANP), synthesized by a solid phase technique, was given by intravenous infusion to 20 patients with severe pregnancy-induced hypertension (PIH) and the curative result of haANP was observed. Compared with basal values, supine systolic and diastolic BP was lowered significantly (P < 0.01), which may be related to the specific receptor of hANP and inhibition of renin-angiotensin-aldosterone system (RAAS). The haANP was found to possess significant effects of antispasm, detumescence and reducing proteinuria, probably by repairing mildly injured glomerulae, strong effects of diuresis and improving heart function with no side effects. Auto-antibody of hANP was found in patients with severe PIH, which affected the function of target cells of highly concentrated endogenous hANP. This auto-antibody might be one of the causes for PIH.

Distribution and characteristics of placental ANP receptors in normal and hypertensive pregnancy.

The ANP (atrial natriuretic peptide) receptor binding site was studied in human placentas of normal and hypertensive pregnancy. The results showed there were specific high affinity ANP receptors in the nonbrush border (fetal side), and their affinity to ANP was higher than that in the microvillous membrane (meternal side). The ANP receptor affinity in the nonbrush border and microvillous membrane of normal pregnancy was higher than that of hypertensive pregnancy. Though the weight of placentas of hypertensive pregnancy was lower than that of normal pregnancy, high ANP concentrations in the placental tissues, umbilical and maternal blood were found in hypertensive pregnancy. It is believed that the distribution of ANP receptors in the placentas is related to hemodynamics, maternal exchange and fluid and electrolyte balance. The decrease of ANP receptors and lowering of affinity in hypertensive pregnancy may influence the the target cell effect of ANP, especially in the fetal side. This may be related to the pathogenesis of hypertensive pregnancy.

Significance of changes in serum superoxide dismutase level in hypertensive syndrome of pregnancy.

The concentration of human serum superoxide dismutase-1 (hSOD-1) containing copper and zinc ions were measured by radioimmunoassay healthy nonpregnant women, 15 normal pregnant women, 15 patients with mild to moderate hypertension (MMHSP) and 15 with severe hypertensive syndrome of pregnancy (SHSP). The mean serum hSOD-1 concentration in nonpregnant women was 148.84 +/- 60.53 (x +/- s) micrograms/L; while in the other 3 groups it was 394.19 +/- 122.21 micrograms/L, 377.12 +/- 173.45 micrograms/L and 581.15 +/- 118.50 micrograms/L. The results suggest that harmful free radicals increase gradually and a strong body defence system against oxidation damage of tissue cells is produced in the course of normal pregnancy and MMHSP. With cardionatrin treatment serum hSOD-1 concentrations of patients ameliorated returned to the level of normal pregnancy. The results indicate that there is a positive correlation between cardionatrin and hSOD-1 levels (r = 0.569, P less than 0.05), and a physiological regulation of the defence system exists, which may be related to the white blood cells. Hence, hSOD-1 probably plays a significant role in defence during normal pregnancy and hypertensive syndrome of pregnancy (HSP).