Identification of five novel MHC class II alleles in cynomolgus macaques of Vietnamese origin.

We report here the identification of one Mafa-DPA1 and four Mafa-DQB1 novel alleles of Vietnamese cynomolgus macaques.

Separation and confirmation of nine Enterobacteriaceae strains that carry the blaNDM-1 gene.

The aim of the present study was to confirm the existence of carbapenem-resistant Enterobacteriaceae carrying the blaNDM-1 gene in clinics in Hainan province, China. Collected clinical bacterial isolates that were Enterobacteriaceae strains suspected of producing carbapenemase were used as experimental strains. Drug resistance to imipenem, meropenem and other antibacterial agents was tested. Imipenem/imipenem inhibitor (IP/IPI) E-testing was conducted to identify the bacterial strains that produced metallo-ß-lactamases. The blaNDM-1 drug resistance gene was amplified by polymerase chain reaction (PCR), and agarose gel electrophoresis (AGE) and sequencing were conducted to identify the products. The species of the strains carrying the blaNDM-1 gene were determined using a biochemical identification system. Through the IP/IPI E-test, 21 of the 30 collected Enterobacteriaceae strains were found to be positive, indicating that 70% of the strains produced metallo-ß-lactamases. Following blaNDM-1 gene PCR amplification, AGE and sequencing tests confirmed that nine of the strains carried the blaNDM-1 drug resistance gene. The biochemical identification system indicated that four of the strains were Klebsiella pneumoniae, two were Escherichia coli, two were Enterobacter cloacae and one was Enterobacter aerogenes. Drug susceptibility testing in vitro demonstrated that the strains were 100% resistant to a broad spectrum antibiotic plus lactamase inhibitor, cephalosporins and carbapenems. However, they had high sensitivity rates to polymyxin B and tigecycline of 100 and 88.9%, respectively. The sensitivity rate to amikacin was also high at 77.8%, whereas sensitivity to ciprofloxacin and gentamicin was moderate at rates of 44.4 and 33.3% respectively. This clinical study of Enterobacteriaceae strains that carry the blaNDM-1 gene in Hainan shows a bacterial tolerance that is different from that in previous studies, which requires further in-depth study.

External quality assessment of prenatal diagnosis of a rare and subtle chromosomal structural abnormality.

PURPOSE OF THE STUDY: To explore the misdiagnosis probability of subtle chromosomal structural abnormalities and find proper strategy to improve the accuracy of prenatal genetic diagnosis, we carried out a preliminary external quality assessment of prenatal detection of a rare case. PATIENTS AND METHODS: Three karyograms of a rare case of cri du chat syndrome associated with t(11;22) translocation [46,XY, del(5)(p15.2), t(11;22)(q23;q11.2)] were chosen. The patient's information and karyograms were emailed to 21 laboratories simulating the scenarios of prenatal diagnosis. The laboratories were required to provide a report using current nomenclature. RESULTS: Seven laboratories sent results for evaluation (response rate: 33.33%). For t(11;22), Two labs incorrectly reported that chromosome 22 was deletion. 5 of 7 labs reported t(11;22) translocation consistent with the actual karyotype. Among them, lab 6 suspected the abnormal 5q and lab 7 incorrectly considered chromosome 22 was deletion or reduplication. All laboratories missed to report the karyotype of del(5). CONCLUSIONS: Conventional cytogenetic analysis couldn't always detect subtle chromosomal structure abnormalities correctly during prenatal diagnosis. To improve the quality of prenatal genetic diagnosis, an excellent external quality assessment (EQA) scheme is currently imperative in China.

In vivo generation and characterization of a soluble form of the Semliki forest virus fusion protein.

During infection of host cells, a number of enveloped animal viruses are known to produce soluble forms of viral membrane glycoproteins lacking the transmembrane domain. The roles of such soluble glycoproteins in viral life cycles are incompletely understood, but in several cases they are believed to modulate host immune response and viral pathogenesis. Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells through low-pH-dependent fusion and buds from the plasma membrane. Fusion is mediated by the E1 subunit of the SFV spike protein. Previous studies described the in vivo generation of E1s, a truncated soluble form of E1, under conditions in which budding is inhibited in mammalian host cells. We have here examined the properties of E1s generation and the biological activity of E1s. E1s cleavage required spike protein transport out of the endoplasmic reticulum and was independent of virus infection. Cell surface E1 efficiently acted as a precursor for E1s. E1s generation was strongly pH dependent in BHK cells, with optimal cleavage at a pH of < or =7.0, conditions that inhibited the budding of SFV but not the budding of the rhabdovirus vesicular stomatitis virus. The pH dependence of E1s production and SFV budding was unaffected by the stability of the spike protein dimer but was a function of the host cell. Similar to the intact virus and in vitro-generated E1 ectodomain, treatment of E1s at low pH in the presence of target membranes triggered specific acid-dependent conformational changes. Thus, under a variety of conditions, SFV-infected cells can produce a soluble form of E1 that is biologically active.

Semliki forest virus budding: assay, mechanisms, and cholesterol requirement.

All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells.

The cholesterol requirement for sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence.

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV. Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of approximately 100-fold in infection and approximately 250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.