Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2

Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.     

Mutagenesis of the NS3 protease of dengue virus type 2

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.     

A simple reverse transcription-polymerase chain reaction for dengue type 2 virus identification

A morphofunctional changes of peritoneal and mesenteric mast cell populations in rats from first minutes up to 10 day after irradiation in dose 5.5 Gr were studied. It is shown that the increased mast cell degranulation is of the first reactions of organism on irradiation. Mast cell reaction on irradiation is biphasic; the quick, short first phase is observed approximately during 0.5 h after irradiation, the gradual prolonged second phase reaches its peak about 5-12 h and continues at least during 10 days.     

Culture-amplified detection of dengue virus from serum in an outbreak of dengue fever

PURPOSE: To evaluate the effect of laser thermokeratoplasty (LTK) in eyes that previously had a lamellar corneal cut. SETTING: University of Al-Azhar, Cairo, Egypt, and Instituto Oftalmológico de Alicante, Spain. METHODS: In 15 eyes (10 patients), noncontact LTK was applied 6 to 8 weeks after a lamellar corneal cut had been made. Central pachymetry, keratometry, and videokeratography were performed and uncorrected visual acuity, best spectacle-corrected visual acuity (BSCVA), and manifest and cycloplegic refractions measured before and 1, 6, 12, and 18 months after LTK. RESULTS: Mean follow-up was 19.13 months. Mean refraction was +5.93 diopters (D) +/- 1.9 (SD) before LTK and -0.43 +/- 1.5 D at 1 month, +1.63 +/- 1.6 D at 6 months, 1.91 +/- 1.41 at 12 months, and +2.01 +/- 1.5 D at the end of the study. Total regression did not occur in any case. Mean BSCVA before LTK was 0.66 +/- 0.2, and spontaneous visual acuity at the end of the study was 0.58 +/- 0.18. No patient lost any lines of preoperative BSCVA. There was no significant difference between the results at 12 months and at the end of the study. CONCLUSION: Corneal lamellar cutting appeared to improve the magnitude of the refractive effect of noncontact LTK and to decrease the amount of regression.     

Functions of conserved motifs in the RNA-dependent RNA polymerase of a yeast double-stranded RNA virus

At least eight conserved motifs are visible in the totivirus RNA-dependent RNA polymerase (RDRP). We have systematically altered each of these in the Saccharomyces cerevisiae double-stranded RNA virus ScVL1 by substituting the conserved motifs from a giardiavirus. The results help define the conserved regions of the RDRP involved in polymerase function and those essential for other reasons.     

Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets

The GK (Goto-Kakizaki) rat is a lean model of type 2 diabetes in which the diabetic state was spontaneously induced. We recently demonstrated the presence in GK rats of two functional point mutations in the promoter region of the type 3 adenylyl cyclase (AC3) gene that resulted in overexpression of AC3 mRNA associated with increased cAMP generation. The AC3 gene promoter mutations are the first molecular changes to be described in any specific gene in the GK rat. Here we report cloning of a full-length cDNA encoding human AC3 from a human fetal brain cDNA library using a PCR-based screening method. This 4142-bp cDNA predicts an open reading frame encoding 1144 amino acids containing putative 12 transmembrane-spanning domains which are typically found in other mammalian AC isoforms. Comparison of the translated amino acid sequence of the AC3 gene between human and rat shows 95% homology. Using RT-PCR, clear AC3 expression was detected in isolated human islets as well as a cDNA panel containing templates from eight different tissues (brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle). This wide distribution of AC3 expression may involve a number of physiological and pathophysiological metabolic processes.     

Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein

BACKGROUND: Neutrophil apoptosis is crucial in the resolution of inflammation. The role of interleukin (IL)-8 in neutrophil apoptosis has not been previously studied; we hypothesized that in addition to its role as a chemoattractant, IL-8 would regulate polymorphonuclear leukocyte (PMN) apoptosis. METHODS: PMNs were adhered to plastic during hypoxia or normoxia and treated with IL-8 dosages of 0 to 1000 ng/mL. Apoptosis was assessed by cellular histology and the TUNEL assay. For receptor inhibition, blocking antibodies to IL-8 receptors in the presence of IL-8 were added. Apoptosis of PMNs treated with anti-Fas antibody +/- IL-8 was also analyzed. RESULTS: After treatment with 100 ng/mL IL-8 apoptosis was decreased from an average of 39.1% 9.3%. Inhibition of IL-8RA was able to restore apoptosis to 59.4%. Western analysis showed that with IL-8, there was a marginal decrease of total Fas protein, whereas Fas ligand was increased. After incubation with an apoptosis inducing-Fas antibody plus IL-8 reduced apoptosis to 9.5%. CONCLUSIONS: IL-8 not only promotes the inflammatory response by recruiting PMNs but also acts to suppress apoptosis mainly through the IL-8RA in an oxygen tension independent manner. The reduction in apoptosis is associated with changes in Fas and FasL where the presence of IL-8 suppresses the proapoptotic function of Fas-FasL interactions.     

Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system

We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.     

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.     

登革2型病毒B株E基因部分序列原核蛋白表达及单克隆抗体制备

目的 通过构建登革2型病毒B株E基因区1～476 bp的原核表达载体,进行原核表达,并制备单克隆抗体,为研制登革病毒诊断试剂奠定基础.方法 将登革2型病毒B株E基因序列克隆入原核表达载体pET28a(+),构建原核表达重组体pET28a(+)-E,将重组表达载体转化BL21(DE3)菌株进行原核表达.纯化后的蛋白免疫BALB/e小鼠,经融合、筛选制备特异性单克隆抗体.结果 成功构建了pET28a(+)-E原核表达重组质粒,SDS-PAGE分析表明,E基因区部分序列获得高效表达,获得了1株持续分泌抗E蛋白抗体的杂交瘤细胞株8B3,该单抗为IgG2a亚类.结论 成功制备了抗E蛋白mAb,为对登革病毒引起感染的早期诊断提供了有力的工具.     

Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice

1. The influence of Ca2+ on the effects of glucagon on glycolysis was investigated in the isolated perfused rat liver. Livers from fed rats were perfused in an open system with Krebs/Henseleit-bicarbonate buffer (pH 7.4). Glucose release, lactate plus pyruvate production (glycolysis) and oxygen uptake were measured. The following results were obtained: 2. In livers perfused with Ca(2+)-free Krebs/Henseleit-bicarbonate buffer and after depletion of the intracellular pools, the initial and transient stimulation of glycolysis, which is normally observed shortly after the onset of glucagon infusion, was more pronounced when compared to livers perfused with normal perfusion fluid (2.5 mM Ca2+) and without previous depletion of the intracellular pools (controls); the subsequent inhibition of glycolysis was delayed in Ca(2+)-free perfused livers and was less pronounced in comparison with the controls at the end of the glucagon infusion period (20 min). 3. Perfusion with a Ca(2+)-free medium supplemented with EDTA, without previous depletion of the intracellular pools, also produced a substantial reduction in the effects of glucagon on glycolysis. 4. Ca(2+)-free perfusion did not affect the stimulative action of glucagon on glucose release (glycogenolysis) and oxygen uptake. 5. Glycolysis inhibition by cAMP also was abolished in Ca(2+)-free perfused livers, and the initial stimulation was enhanced. 6. Mn2+, a metal ion known as a competitor of Ca2+, considerably reduced the action of glucagon on glycolysis; Mn2+ did not affect the basal rates of glycolysis. 7. Sr2+, a metal ion that is often recognized as Ca2+ by several biological structures and processes, increased the inhibitory action of glucagon on glycolysis. 8. Several organic compounds, which directly or indirectly take part in Ca2+ fluxes, were also able to diminish (e.g., verapamil) or even to abolish (carbenoxolone) the inhibitory action of glucagon on glycolysis. 9. It was concluded that, under the conditions of the living cell, Ca2+ is important for glycolysis inhibition by glucagon. In principle at least, the results can be explained in terms of the known Ca2+ dependencies of several protein kinases and protein phosphatases.     

Three cases of dengue 1 virus infection from islands in the Gulf of Thailand

Three Australian tourists who recently travelled to islands in the Gulf of Thailand developed febrile illnesses associated with myalgias, thrombocytopenia, and atypical lymphocytosis. Dengue 1 virus was isolated from all three patients. The patients' clinical features and serological and virological investigations are presented. These cases highlight the need for awareness of dengue amongst travellers and the preventive precautions required when visiting endemic regions. After the urgent exclusion of malaria, dengue should be considered in the differential diagnosis of febrile persons who have recently returned from endemic regions.