METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.
RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P
RESULTS: In this study, several genetically modified sub-genotype B4 EV-A71 mutants were constructed by site-directed mutations at positions 158, 475, 486, 487 and 5262 or through partial deletion of the 5'-NTR region (∆ 11 bp from nt 475 to 486) to generate a deletion mutant (PD). EV-A71 mutants 475 and PD caused minimal cytopathic effects, produced lowest viral RNA copy number, viral particles as well as minimal amount of viral protein (VP1) in RD cells when compared to mutants 158, 486, 487 and 5262.
CONCLUSIONS: The molecular determinants of virulent phenotypes of EV-A71 sub-genotype B4 strain 41 (5865/Sin/000009) were found to differ from the C158 molecular determinant reported for the fatal EV-A71 sub-genotype B1 strain (clinical isolate 237). The site-directed mutations (SDM) introduced at various sites of the cDNA affected growth of the various mutants when compared to the wild type. Lowest viral RNA copy number, minimal number of plaques formed, higher infectious doses required for 50% lethality of RD cells and much reduced VP1 of the EV-A71 sub-genotype B4 strain 41 genome was attained in mutants carrying SDM at position 475 and through partial deletion of 11 bp at the 5'-NTR region.
RESULTS: During infection experiment in a series of colorectal cancer cell lines, we adventitiously observed a development of persistent infection by NDV in SW480 cells, but not in other cell lines tested. This cell population, designated as SW480P, showed resistancy towards NDV killing in a re-infection experiment. The SW480P cells retained NDV genome and produced virus progeny with reduced plaque forming ability.
CONCLUSION: These observations showed that NDV could develop persistent infection in cancer cells and this factor needs to be taken into consideration when using NDV in clinical settings.
CASE PRESENTATION: The first calf died within 24 h of the onset of clinical signs; the second calf died within 12 h of the onset of clinical signs. The third calf succumbed within 72 h. Necropsies revealed that all 3 calves had similar presentations of EEHV HD but in the third calf with less severity. We conducted conventional polymerase chain reaction (cPCR) assays and found EEHV DNA at all 7 loci in the 3 calves; it was identified as EEHV1A, the virus type that has been found in most other reported cases.
CONCLUSION: Typical EEHV HD clinical signs and the molecular confirmation of EEHV by cPCR and sequencing point to EEHV as the cause of death. Further genetic investigation of the strain is in progress.
METHODS: The effect of geraniin on DENV-2 RNA synthesis in infected Vero cells was tested using quantitative RT-PCR. The in vivo efficacy of geraniin in inhibiting DENV-2 infection was then tested using BALB/c mice with geraniin administered at three different times. The differences in spleen to body weight ratio, DENV-2 RNA load and liver damage between the three treatment groups as compared to DENV-2 infected mice without geraniin administration were determined on day eight post-infection.
RESULTS: Quantitative RT-PCR confirmed the decrease in viral RNA synthesis of infected Vero cells when treated with geraniin. Geraniin seemed to provide a protective effect on infected BALB/c mice liver when given at 24 h pre- and 24 h post-infection as liver damage was observed to be very mild even though a significant reduction of DENV-2 RNA load in serum was not observed in these two treatment groups. However, when administered at 72 h post-infection, severe liver damage in the form of necrosis and haemorrhage had prevailed despite a substantial reduction of DENV-2 RNA load in serum.
CONCLUSIONS: Geraniin was found to be effective in reducing DENV-2 RNA load when administered at 72 h post-infection while earlier administration could prevent severe liver damage caused by DENV-2 infection. These results provide evidence that geraniin is a potential candidate for the development of anti-dengue drug.
METHODS: Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay.
RESULTS: Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity.
CONCLUSIONS: Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.
FINDINGS: Of the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA.
CONCLUSIONS: This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.
METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.
RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.
CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.