Evaluation of Antiviral Activities of Four Local Malaysian Phyllanthus Species against Herpes Simplex Viruses and Possible Antiviral Target

Nucleoside analogues such as acyclovir are effective antiviral drugs against herpes simplex virus infections since its introduction. However, with the emergence of acyclovir-resistant HSV strains particularly in immunocompromised patients, there is a need to develop an alternative antiherpetic drug and plants could be the potential lead. In this study, the antiviral activity of the aqueous extract of four Phyllanthus species were evaluated against herpes simplex virus type-1 (HSV-1) and HSV-2 in Vero cells by quantitative PCR. The protein expressions of untreated and treated infected Vero cells were studied by 2D-gel electrophoresis and Western blot. This is the first study that reported the antiviral activity of P. watsonii. P. urinaria was shown to demonstrate the strongest antiviral activity against HSV-1 and HSV-2, with SI >33.6. Time-of-addition studies suggested that the extract may act against the early infection stage and the replication stage. Protein expression studies indicated that cellular proteins that are involved in maintaining cytoskeletal structure could be potential target for development of antiviral drugs. Preliminary findings indicated that P. urinaria demonstrated potent inhibitory activity against HSV. Hence, further studies such as in vivo evaluation are required for the development of effective antiherpetic drug.     

Dengue virus infection and immune response in humanized RAG2(-/-)gamma(c)(-/-) (RAG-hu) mice

Dengue viral (DENV) pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive human immune response. Although currently available animal models have been very useful in dissecting some key aspects of disease pathogenesis, a major limitation with these is the lack of a human immune response. In this study, we sought to overcome this difficulty by utilizing a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immunocompetent humanized mice, neonatal RAG2(-/-)gamma(c)(-/-) mice were xenografted with human CD34+ hematopoietic stem cells, resulting in de novo development of major functional cells of the human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with DEN-2 serotype. Viremia lasting up to 3 weeks was detected in infected mice with viral titers reaching up to 10(6.3) RNA copies/ml. Fever characteristic of dengue was also noted in infected mice. Presence of human anti-dengue antibodies was evaluated using an antibody capture ELISA. Anti-dengue IgM was first detected by 2 weeks post-infection followed by IgG at 6 weeks. Sera from some of the infected mice were also found to be capable of dengue virus neutralization. Infected mouse sera showed reactivity with the viral envelope and capsid proteins in immunoprecipitation assay. These results demonstrate for the first time that humanized mice are capable of dengue viral primary human immune responses thus paving the way for new dengue immunopathogenesis and vaccine studies.     

Antibacterial effects of 18 medicinal plants used by the Khyang tribe in Bangladesh

Context: The resistance of bacteria to antibiotics is raising serious concern globally. Asian medicinal plants could improve the current treatment strategies for bacterial infections. The antibacterial properties of medicinal plants used by the Khyang tribe in Bangladesh have not been investigated.

Objective: The present study examines the antibacterial properties of 18 medicinal plants used by the Khyang tribe in day-to-day practice against human pathogenic bacteria.

Materials and methods: Leaves, bark, fruits, seeds, roots and rhizomes from collected plants were successively extracted with hexane, ethyl acetate and ethanol. The corresponding 54 extracts were tested against six human pathogenic bacteria by broth microdilution assay. The antibacterial mode of actions of phytoconstituents and their synergistic effect with vancomycin and cefotaxime towards MRSA was determined by time-killing assay and synergistic interaction assay, respectively.

Results and discussion: Hexane extract of bark of Cinnamomum cassia (L.) J. Presl. (Lauraceae) inhibited the growth of MRSA, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii with MIC values below 100 µg/mL. From this plant, cinnamaldehyde evoked at 4?×?MIC in 1?h an irreversible decrease of MRSA count Log10 (CFU/mL) from 6 to 0, and was synergistic with vancomycin for MRSA with fractional inhibitory concentration index of 0.3.

Conclusions: Our study provides evidence that the medicinal plants in Bangladesh have high potential to improve the current treatment strategies for bacterial infection.     

Detection of mecA and ermA genes and simultaneous identification of Staphylococcus aureus using triplex real-time PCR from Malaysian S. aureus strain collections

A triplex real-time polymerase chain reaction (PCR) assay was used for the simultaneous detection of mecA (methicillin resistance), ermA (erythromycin resistance) and femA (Staphylococcus aureus identification) genes in a single assay. Among 93 clinical S. aureus hospital isolates, there were 48 methicillin-resistant S. aureus (MRSA) and 45 methicillin-sensitive S. aureus (MSSA) isolates. Screening the isolates using the triplex real-time PCR assay, the mecA, ermA and femA genes were detected in all MRSA isolates. The triplex real-time PCR assay was completed within 3h and is a useful genotypic method for detecting the resistance determinants as well as for the identification of S. aureus isolates. These findings will assist the clinical laboratory in identifying these resistance genes and S. aureus rapidly, thus benefiting patient therapy. This study represents a valuable source of information for researchers to study the local antibiotic resistance pattern, which can increase our knowledge of the antibiotic resistance profile, using real-time PCR technology.     

Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay

For rapid identification of methicillin-resistant Staphylococcus aureus, molecular methods are generally targeting mecA and species-specific genes. Sa442 DNA fragment is a popular species-specific target. However, recently, there have been few reports on S. aureus isolates that are negative for Sa442 fragment; therefore, use of single gene or DNA-fragment-specific polymerase chain reaction (PCR) for identification of microbial isolate may result in misidentification. This study includes CoA gene in parallel with Sa442 marker for identification of S. aureus. This further improves the specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and can prevent misidentification of S. aureus isolates lacking Sa442 DNA fragment. In this study, the newly developed triplex real-time PCR assay was compared with a quadruplex conventional gel-based PCR assay using the same primer sets in both assays. The dual-labeled TaqMan probes (ProOligo, France) for these primers were specifically designed and used in a real-time PCR assay. The clinical isolates (n = 152) were subjected to both PCR assays. The results obtained from both assays proved that the primer and probe sets were 100% sensitive and 100% specific for identification of S. aureus and detection of methicillin resistance. This triplex real-time PCR assay represents a rapid and powerful method for S. aureus identification and detection of methicillin resistance.     

Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.     

Viral morphogenesis and morphological changes in human neuronal cells following Tioman and Menangle virus infection

Tioman virus (TioPV) and Menangle virus (MenPV) are two antigenically and genetically related paramyxoviruses (genus: Rubulavirus, family: Paramyxoviridae) isolated from Peninsular Malaysia (2001) and Australia (1997), respectively. Both viruses are potential zoonotic agents. In the present study, the infectivity, growth kinetics, morphology and morphogenesis of these two paramyxoviruses in a human neuronal cell (SK-N-SH) line were investigated. Sub-confluent SK-N-SH cells were infected with TioPV and MenPV at similar multiplicity of infection. These cells were examined by conventional and immunoelectron microscopy, and virus titres in the supernatants were assayed. Syncytia were observed for both infections in SK-N-SH cells and were more pronounced during the early stages of TioPV infection. The TioPV titre increased consistently (10¹) every 12 h after infection. In MenPV-infected cells, cellular material was frequently observed within budding virions, and microfilaments and microtubules were abundant. Viral budding was common, and extracellular MenPVs tended to be more pleomorphic compared to TioPVs, which appeared to be more spherical in appearance. The MenPV cytoplasmic viral inclusion appeared to be comparatively smaller, loose and interspersed with randomly scattered circle-like particles, whereas huge tubule-like cytoplasmic inclusions were observed in TioPV-infected cells. Both viruses also displayed different cellular pathology in the SK-N-SH cells. The intracellular ultrastructural characteristics of these two viruses in infected neuronal cells may allow them to be differentiated by electron microscopy.     

Oral mucosal lichen planus in nine-year-old child

Lichen planus (LP) is a fairly distinctive mucocutaneous disease. The etiology of the condition appears to be complex and multifactorial, with unique histopathological features. Immunofluorescence studies have provided some insight into a proposed immunopathogenesis. LP is seen frequently in the middle-aged and elderly population. The female-to-male ratio is approximately 2:1. Children are rarely affected. The incidence of oral LP in children is reportedly high among Asians. We present a 9-year-old Indian child with the documented clinical aspects, histopathology and immunofluorescence studies.