Foot-and-mouth disease (FMD) is a highly contagious epidemic disease threatening the cattle industry since the sixteenth century. In recent years, the development of diagnostic assays for FMD has benefited considerably from the advances of recombinant DNA technology. In this study, the immunodominant region of the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) was fused to the T7 bacteriophage and expressed on the surface of the bacteriophage capsid protein. The recombinant protein of about 42 kDa was detected by the anti-T7 tag monoclonal antibody in Western blot analysis. Phage ELISA showed that both the vaccinated and positive infected bovine sera reacted significantly with the recombinant T7 particle. This study demonstrated the potential of the T7 phage displaying the VP1 epitope as a diagnostic reagent.
The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples.
Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100xTCID(50) of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6xTCID(50) resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100xTCID(50) of B/Malaysia/2506/04 virus. In this manuscript the critical factors described above were optimized and the results indicate that the described biotin-streptavidin conjugated influenza microneutralization assay is a rapid and robust method for detecting the presence of functional, influenza virus-neutralizing antibodies.
The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (Mr) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 degrees C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.
A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P<0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV.
Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.
Foot-and-mouth disease (FMD) is a highly infectious disease of cattle and other cloven-hoofed animals, causing huge economic losses annually worldwide. This disease is endemic in Pakistan where the serotypes of the foot-and-mouth disease virus (FMDV) are A, O and ASIA-1. At present, trivalent FMDV vaccines are being used to prevent FMD but the current production process is laborious and is unable to fulfill the needs of the meat and dairy industries. To meet the vaccine needs of Pakistan, the conventional method of using adherent cell lines to produce the vaccine could be replaced by suspension cell cultures which produce higher yields in less time and less volume. Therefore, the aim of this study was to investigate and optimize some of the factors that affect viable cell density and subsequent virus yield. The relationship between the yield of the 146S fraction and the TCID50 of the virus preparations obtained was also evaluated as a mean to control and check the quality of the vaccine product. The results provided optimized conditions for vaccine production using cell suspensions and showed that there was a linear relationship between TCID50 and 146S fraction yield. Either TCID50 or the 146S fraction yield, or both could be used as parameters for quality monitoring during vaccine production. Using TCID50 reduced the number of steps involved in virus production while measuring 146S fraction yield was useful for quality control. However, more studies are required to evaluate the relative effectiveness of vaccines produced by virus cultures using either TCID50 or 146S fraction as quality monitoring tools.