Sumalee Pruksakorn

Construction of infectious dengue 2 virus cDNA clones using high copy number plasmid

Procedures for cloning entire dengue serotype 2 virus genome in the multiple cloning site of a commercially available high copy number plasmid are described. The 10.7 kb viral RNA genome was reverse transcribed, amplified as three overlapping DNA fragments and successively ligated into pBluescript II KS, which contains the colE1 origin of replication. When propagated at room temperature (20-25 degrees C) under low level of antibiotic selection, the full-length recombinant plasmid was stable upon serial passages in two common Escherichia coli strains employed. Under the same culture conditions the whole dengue cDNA sequence was transferred successfully to another high copy number plasmid, pGem 3Z. Following in vitro transcription and lipofectin-mediated transfection, capped RNA transcripts derived from the plasmid initiated virus replication in C6/36 mosquito cells and BHK-21 cells within 3-4 days of transfection. Upon subsequent expansion in C6/36 cells, dengue viruses derived from the first- and eighth-plasmid passages achieved similar titers as the parent virus. They were also indistinguishable from the parent virus by the criteria of replication kinetics in mosquito and mammalian cell lines, and size and reactivity of selected viral proteins as detected with polyclonal and monoclonal antibodies. The cloning scheme and resultant recombinant plasmids based on high copy number cloning vectors allows greater flexibility in manipulation of dengue viral genome when compared with previous attempts employing low-copy number counterparts.

M protein typing of Thai group A streptococcal isolates by PCR-Restriction fragment length polymorphism analysis

BackgroundGroup A streptococcal (GAS) infections can lead to the development of severe post-infectious sequelae, such as rheumatic fever (RF) and rheumatic heart disease (RHD). RF and RHD are a major health concern in developing countries, and in indigenous populations of developed nations. The majority of GAS isolates are M protein-nontypeable (MNT) by standard serotyping. However, GAS typing is a necessary tool in the epidemiologically analysis of GAS and provides useful information for vaccine development. Although DNA sequencing is the most conclusive method for M protein typing, this is not a feasible approach especially in developing countries. To overcome this problem, we have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assay for molecular typing the M protein gene (emm) of GAS.ResultsUsing one pair of primers, 13 known GAS M types showed one to four bands of PCR products and after digestion with Alu I, they gave different RFLP patterns. Of 106 GAS isolates examined from the normal Thai population and from patients with GAS-associated complications including RHD, 95 isolates gave RFLP patterns that corresponded to the 13 known M types. Only 11 isolates gave RFLP patterns that differed from the 13 known M types. These were then analyzed by DNA sequencing and six additional M types were identified. In addition, we found that M93 GAS was the most common M type in the population studied, and is consistent with a previous study of Thai GAS isolates.ConclusionPCR-RFLP analysis has the potential for the rapid screening of different GAS M types and is therefore considerably advantageous as an alternative M typing approach in developing countries in which GAS is endemic.

Correlation between PFGE Groups and mrp/epf/sly Genotypes of Human Streptococcus suis Serotype 2 in Northern Thailand

Streptococcus suis infection is a severe zoonotic disease commonly found in Northern Thailand where people often consume raw pork and/or pigs blood. The most frequent clinical presentations are meningitis, sepsis, and endocarditis with higher rate of mortality and hearing loss sequelae. To clarify the correlation between pulsed-field gel electrophoresis (PFGE) groups and mrp/epf/sly genotypes of S. suis serotype 2, 62 patient and 4 healthy pig isolates from Northern Thailand were studied. By PFGE analysis, at 66% homology, most human isolates (69.4%) and 1 pig isolate were in group A, whereas 14.5% of human isolates and 3 out of 4 pig isolates were in group D. According to mrp/epf/sly genotypes, 80.6% of human isolates were identified in mrp + epf − sly − and only 12.9% were in mrp − epf − sly + genotypes; in contrast, 1 and 3 pig isolates were detected in these two genotypes, respectively. Interestingly, all isolates of S. suis serotype 2 classified in PFGE groups A, B, and E were set in mrp + epf − sly − genotypes. These data show a close correlation between PFGE groups and mrp/epf/sly genotypes of human S. suis serotype 2.

Can class I epitope of M protein be a diagnostic marker for rheumatic fever in populations endemic for group A streptococci

difference in antibody levels to the class I peptide was observed between Australian whites and other groups (p>0·05). Our results indicate that in populations of high group A streptococci endemicity, nearly all individuals, including controls, have similar antibody levels to the class I epitope, and suggest that in highly endemic populations, the detection of class I antibodies as a serological diagnostic for ARF would not be suitable as it may reflect intense streptotoccal exposure rather than ARF.

Ocular Bacterial Flora in HIV-Positive Patients and Their Sensitivity to Gentamicin

Normal ocular flora comprise a heterogenous collection of bacteria. The natural immunity, natural resistance, and specific immunologic phenomena that regulate them derive from the host and microbes. However, the natural defense is a combination of innate immunity and specific acquired immunity. Acquired immunodeficiency syndrome (AIDS) is caused by infection with the human immunodeficiency virus (HIV). This retrovirus infects and destroys T-helper cells, resulting in a profound deficiency of natural immunity. The purpose of this study was to investigate the ocular bacterial flora of patients who had HIV infection and their sensitivity to gentamicin.