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Japanese encephalitis virus (JEV) causes severe viral encephalitis in humans and some other mammalian animals. Highly efficient culture of the virus is critical for antigen preparation, vaccine production and other basic researches. We have investigated the influence of a number of variables such as the strain virulence, the state of the host cells, medium composition, infection method and others on the proliferation of distinct JEV strains in BHK-21 cells. The results showed that two distinct strategies are needed for the propagation of virulent or attenuated JEV strains. The most critical variables were the method of infection, and especially the density of the host cell. Our studies indicate the general approaches to the in vitro culture of other JEV strains using BHK-21 cell line.

Marek's disease virus (MDV) is a long-recognized oncogenic herpesvirus, which induces lymphoma in its natural host that can be prevented by vaccination. MDV infection provides an excellent biological model for investigating the biology, genetics and immunology of viral oncogenesis. Recently discovered microRNAs (miRNAs) in the MDV genome have been suggested to have regulatory roles during MDV oncogenesis. We have examined the expression profiles of all 22 previously reported miRNAs encoded by MDV-1 in chickens artificially challenged with MDV-GX0101. We found that a subset of the miRNAs was differentially expressed during different phases of the developing disease. These miRNAs show early or late expression during disease progression, accompanied by obvious tissue-specific and differential expression patterns. This temporal and differential tissue distribution suggest that these miRNAs may perform different regulatory roles in switching from latency to lytic replication, immunosuppression, neoplastic transformation or other aspects of lymphoma formation. These reported in vivo expression profiles indicate the potentially functional MDV-1-encoded miRNAs that should be selected for further investigation of their functions in MDV oncogenesis.

MicroRNAs (miRNAs) are the recently discovered small non-coding RNA molecules that have post-transcriptional regulatory functions in many important biological processes. A large number of miRNAs have been found to be encoded by viral genomes, especially in herpesviruses. Previous research regarding miRNAs encoded by herpesviruses, including Marek's disease virus (MDV), has demonstrated their involvement in lytic replication, latent infection, T-lymphocyte transformation and tumorigenesis. MDV is an oncogenic alphaherpesvirus, with the ability to induce tumors in natural hosts; however, formation of these tumors can be prevented by immunization with attenuated or nonpathogenic forms of the virus. Marek's disease is considered to be a good biomedical model for investigating the biology, genetics, and immunology of tumorigenesis. In this paper, we review the discovery and identification of MDV-encoded miRNAs, along with their genomics, expression profiles, and currently known functions. We also discuss the prospects and techniques possibly applicable to the further investigation of the biological roles of MDV-encoded miRNAs.

An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken beta-actin/rabbit beta-globin (CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells. The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3'-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient, especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression in different mammalian cells was obtained than the reported vectors using the CMV promoter.

In this study, four strains of Japanese Encephalitis Virus (JEV) were isolated from the cerebrospinal fluid of aborted fetuses or stillborn piglets collected randomly from a number of piggeries in central China. The E genes were cloned by RT-PCR and sequenced. Phylogenetic analysis was performed with 48 JEV isolates previously reported in China and other countries, and showed that all four isolates can be classified into the subcluster of genotype III. The results strongly suggest that the genotype III of JEV is the major variant currently circulating in the swinery of central China.

The evolutionary position of Giardia lamblia has been challenged in recent years. Comparison of the snoRNAs identified from Archaea and unicellular eukaryotes shows that G. lamblia snoRNAs are more similar in most respects to the counterparts identified from Dictyostelium discoideum, Plasmodium falciparum, fungi, and some metazoans, remarkably differently from those of Euglenozoa protozoans. We propose that G. lamblia emerged somewhat later than Trypanosoma and Euglena during the early evolution of "excavata" eukaryotes.

In this paper, the infectivity and propagation of two attenuated infectious bursal disease virus (IBDV) strains in DT40 cells were investigated. The results showed that both of the tested strains, TAD and HN(3), directly infect and proliferate in DT40 cells, requiring no adaptive passages. Unexpectedly, IBDV can be rapidly propagated and continuously harvested at high titers for a long time, accompanied by the rapid growth of host cells and showing no increase in pathogenicity. Our results provide further support to suggest that DT40 cells can be used as an ideal model for studying IBDV pathogenesis. Additionally, the DT40 cell line could also serve as a potential system for commercial IBDV vaccine preparation.

Aqueous solubility is a critical physicochemical property and must be addressed early during drug discovery research. Due to the difficulty in accurately predicting aqueous solubility in silico, high throughput experimental determination of aqueous solubility is in great demand. This study evaluates a method using a multi-wavelength UV plate reader and disposable 96-well UV plates for fast solubility determination. It was demonstrated that this method has the sensitivity and reproducibility to effectively determine solubility as low as 1 micro M. Excellent correlation (R>0.97) was observed between the solubility determined using the UV reader method and the HPLC method over the range of 1-1000 micro M for a diverse set of pharmaceutical compounds. In addition to excellent sensitivity and reproducibility, the UV plate reader method also offers the flexibility of being able to determine thermodynamic solubility in the presence or absence of dimethyl sulfoxide, which is a solvent widely used for combinatorial compounds during high throughput screening.

Artificial membrane permeability measurement is a potentially high throughput and low cost alternative for in vitro assessment of drug absorption potential. It will be an ideal screening/profiling tool in the lead generation program of drug discovery research if it is proven to be generally applicable for classifying drug absorption potential and is advantageous over other in vitro or in silico methods. This study provides an in-depth evaluation of the method in close comparison to Caco-2, LogD, LogP, polar surface area (PSA), and quantitative structure-property relationship (QSPR) predictions using a large and diverse compound set. It showed that the accuracy of using artificial membrane permeability in assessing drug absorption is comparable to Caco-2, but significantly better than LogP, LogD, PSA, and QSPR predictions. This study also explored the artificial membrane composition by adopting a hydrophilic filter membrane for artificial membrane (lecithin-dodecane) support. The use of hydrophilic filter membrane increased the rate of permeation significantly and reduced the transport time to 2 h or less as compared with over 10 h when a hydrophobic filter membrane is used.