ABSTRACT
Objective:To learn about the genotyping of human Brucella isolated from Sichuan Province. Methods:BCSP31-PCR and AMOS-PCR were used to identify the genus and biotype of the 66 strains isolated from confirmed human brucellosis cases in Sichuan Province from 2014 to 2020, respectively. The isolated strains were genotyped by multi-locus sequence typing (MLST)-9. The sequence type (ST) was compared trough the online MLST database. A minimum spanning tree (MST) was constructed to cluster the newly discovered and known ST using the BioNumerics software version 7.6.Results:The 66 strains isolated from human cases of brucellosis in Sichuan Province from 2014 to 2020 were Brucella, and 65 of them were Brucella melitensis while one strain was Brucella abortus. The MLST method identified three known STs (ST-8, ST-39 and ST-2) and one newly type (ST-101). Among them, ST-8 was the main ST in Sichuan Province (90.91%, 60/66), another 4 strains of Brucella melitensis were ST-39, and 1 strain of Brucella abortus was ST-2. The newly type ST-101 was isolated from Leshan City in 2019, belonging to the Brucella melitensis and closely related to the evolution of ST-8. Conclusion:Brucella melitensis is the main epidemic Brucella strain in Sichuan Province, ST-8 is predominant genotype, with a small amount of ST-39, ST-101 and ST-2.
ABSTRACT
A double-copy Moloney leukemia virus-based retroviral vector containing both the Neo~(R) gene and a mutant human dihydrofolate re-ductase(S31 mutation) cDNA was packaged into the Amphotropic packaging cell hne GP-EAM12( AM12), and a Amphotropic producer cell hne (named AM12-S31)was obtained. In this study, we investigated its drug resistant characteristics, viral titer and for murine hematopoietic progenitor cells transduction as well. MTT assay verified that the AM12-S31 cells were resistant to G418 and methotrexate(MTX), the IC50 were more than 800 ?g/ml and 100 ?M respectively while the control cell line AM12 was sensitive to both drugs, the IC50 were 180 ?g/ml and 10 ?M, respectively. The viral titer for this cell line was approximately 7.8? 104~4.2? 105 G418-resistant colony forming units/ml. The replication-competent virus can not be detected in this producer cell line. We also use the AM12-S31 cells to transfect murine hematopoietic cells (By coculture) . The positive colonies were found in all the G418 concentrations using CFU-GM assay. No G418-resistant colony was found using AM12 transfection. The infected murine marrow cells were returned to lethally irradiated(900rad)recipients. The murine transplanted with AM12-S31 infected marrow cells showed protection from lethal MTX toxicity as compared with AM12 infected animals. Evidence for integration and the proviral DNA was obtained by PCR amplification of proviral DNA. These results indicated this producer cell hne could produce high titer, high-efficiency and non-replcational competent virus. The murine marrow cells could be transfected successfully using this system, and express the foreign gene. The lethal irradiated murine marrow function could be reinstitution by infusing the hematopoietic progenitor cells tranducted with human mutant dihydrofolate reductase. In my opinion, this system would play an important role in research the long-term protection of murine marrow hematopoietic function and drug resistant gene therapy.